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Beta-Cell Replacement by Transplantation in Diabetes Mellitus: When Pancreas, When Islets, and How To Allocate the Pancreas? David E.R. Sutherland and Dixon B. Kaufmann 99 Cytomegalovirus and Development of Cardiac Allograft Vasculopathy: Evidences and Therapeutic Implications Luciano Potena and Hannah A. Valantine 108 Calcineurin Inhibitor-Free Maintenance Therapy After Liver Transplantation I: Mycophenolate Mofetil and Renal Function Lydia Barrera-Pulido, José María Álamo-Martínez, Miguel Ángel Gómez-Bravo, Carmen Bernal-Bellido, Luis Miguel Marín-Gómez, Gonzalo Suárez-Artacho, Juan Serrano-Díez Canedo and Francisco Javier Padillo-Ruiz 117 Kidney Transplantation from Donors with a Positive Serology for Hepatitis C: The Facts and the Challenges Beatriz Domínguez-Gil, Nuria Esforzado, Amado Andrés, Jose M Campistol and Jose M Morales 129 Living Donor Liver Transplantation Juan Carlos García-Valdecasas, Itxarone Bilbao Aguirre, Ramón Charco Torra, Constantino Fondevila Campo, Josep Fuster Obregón, Juan Carlos García-Valdecasas, Paloma Jara Vega, Rafael López Andújar, Pedro López Cillero, Juan Carlos Meneu-Díaz, Miguel Navasa Anadón and Fernando Pardo Sánchez 138

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Volume IV • Number 3 • September-December 2010 ISSN: 1887-455X www.trendsintransplantation.com

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Trends David E.R. in Transplant. Sutherland2010;4:99-107 and Dixon B. Kaufmann: Beta-Cell Replacement by Transplantation in Diabetes Mellitus

Beta-Cell Replacement by Transplantation in Diabetes Mellitus: When Pancreas, When Islets, and How To Allocate the Pancreas? David E.R. Sutherland1 and Dixon B. Kaufmann2 1

Division of Transplantation, Schulz Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, USA; 2Division of Transplantation, Department of Surgery, Comprehensive Transplant Center Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA

Abstract A successful pancreas or islet transplantation produces an insulin-independent, euglycemic state that normalizes hemoglobin A1C levels for as long as the graft functions. Pancreas transplantation has been shown to definitely influence the progression of many secondary complications of diabetes. Islet transplants, even if they do not function well enough to induce insulin independence, still improve quality of life and reduce the frequency of hypoglycemic episodes in those with unawareness. Currently, approximately 1,300 pancreas transplants are performed annually in the USA, a frequency about 20-times that of islet allotransplants. The insulin-independence rates over time are definitely higher in the simultaneous pancreas/kidney transplantation category than in any islet recipient category. However, the insulin-independence rates are somewhat closer when comparing solitary pancreas transplants to recent islet transplant, a consequence of improved immunosuppression and islet isolation. Currently, five-year solitary pancreas graft survival rates are approximately 55% versus at best approximately 40% for islets. However, islet transplantation has a low morbidity and thus remains attractive as a minimally invasive procedure. For widespread application it needs to be made more efficient. The attrition of viable islets during isolation and engraftment is the main problem. The failure to obtain a high yield of viable islets from some donor pancreases creates the need to use more than one donor to provide a sufficient beta-cell mass to achieve insulin-independence in many recipients. Efforts are being made to increase the efficiency of islet isolation and engraftment so islet transplantation can become the form of beta-cell replacement therapy used in the majority of candidates. A deceased donor pancreas allocation policy for beta-cell replacement should be designed to foster efficiency and access to the most candidates for pancreas or islet transplantation. Current United Network for Organ Sharing policy in the USA on pancreas and islet transplants reflects the efficiency and durability of pancreas transplants, so candidates for a whole organ are given preference for donors < 50 years old and with a body

Correspondence to: David E.R. Sutherland Division of Transplantation Department of Surgery University of Minnesota 420 Delaware St. Minneapolis, MN 55455, USA E-mail:[email protected]

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mass index < 30. The United Network for Organ Sharing is now in the process of revising pancreas allocation for both solid organ and islet transplantation. The new allocation system will reduce the geographic inequities related to pancreas utilization, access to transplantation, and how long the candidates wait. It will maximize capacity by improving the opportunity for pancreas and islet candidates to receive a transplant. It will enhance efficiency and minimize the complexity of implementing and maintaining the operational requirements of a new allocation system. However, no matter how much the deceased donor organ allocation system is refined, there will never be enough human pancreases to provide beta-cell replacement therapy for all who could benefit. To do so will require the use of xenografts or insulin-producing, expanded autologous or allogeneic cell lines. These new modalities hold great promise for clinical application because of the unlimited supply and modifiable or intrinsic potential to escape some of the immunological consequences that plague solid organ xenografts as well as conventional allogeneic beta-cell replacement therapies. (Trends in Transplant. 2010;4:99-107) Corresponding author: David E.R. Sutherland, [email protected]

Key words Pancreas and islet transplantation. Pancreas allocation. Diabetes.

Hyperglycemia is the most important factor in the development and progression of secondary complications of diabetes. The Diabetes Control and Complication Trial (DCCT) demonstrated that the microvascular and possibly macrovascular complications of diabetes may be prevented by maintaining euglycemia1,2. This study gave further support to the application of beta-cell replacement as an alternative to exogenous insulin administration in efforts to achieve optimal glycemic control so that the progression of long-term complications can be altered without the risk of hypoglycemia. The only treatments other than intensive insulin therapy that can influence the progression of secondary complications is beta-cell replacement by either pancreas or islet transplantation. Since diabetes is not a rapidly fatal disease, and because transplant procedures require the patient to receive life-long immunosuppression, the results of islet or pancreas transplantation must be sufficiently efficacious and safe to warrant their application in place of standard medical management of the primary disease. Even though pancreas transplantation has a high 100

success rate, it is associated with surgical morbidity. Pancreas transplantation is a proven therapeutic treatment option for diabetes and is superior to manual intensive insulin therapy with regard to the efficacy of achieving glycemic control and beneficial effects on diabetic secondary complications. Islet transplantation is an alternative method of beta-cell replacement therapy. Currently, islet transplantation is an investigational procedure for highly selective cases. An obvious advantage of islet transplantation is that it is minimally invasive for the recipient, but logistically it is more difficult3-5. A successful pancreas or islet transplant produces a euglycemic, insulin-independent state that normalizes hemoglobin A1C levels for as long as the graft functions. Transplantation also has the added physiological properties of pro-insulin and C-peptide release, not possible with intensive insulin therapy6. Through improved metabolic control by pancreas transplantation, many secondary complications of diabetes, including diabetic

David E.R. Sutherland and Dixon B. Kaufmann: Beta-Cell Replacement by Transplantation in Diabetes Mellitus

neuropathy7, autonomic neuropathy-associated sudden death8, and diabetic nephropathy, in both uremic and nonuremic patients9,10, may be markedly improved. A successful pancreas transplant significantly improves quality of life11 and life expectancy12,13. The effect of islet transplantation on secondary complications has not undergone as rigorous a study on secondary complications as pancreas transplantation, but preliminary studies suggest the same effect14. Islet transplantation improves quality of life and has a significant ameliorating effect on the frequency of episodes of hypoglycemia15. One prospective study showed that islet transplantation not only reduced HbA1c levels more than intensive medical therapy, but was also associated with less progression of retinopathy during three years of follow-up16. Approximately 1,300 pancreas transplants are performed annually in the USA, about 20-times more than islet allotransplants. Of the pancreas transplants, 65-70% involves a simultaneous pancreas and kidney (SPK) transplant for patients with type 1 diabetes and chronic renal failure. These individuals are excellent candidates for an SPK transplant from the same donor because the immunosuppressive medications that are needed are similar to those for a kidney transplant alone and the surgical risk of adding the pancreas is low. The benefits of adding a pancreas transplant to ameliorate diabetes are profound–transplantation saves lives12,13,17. Simultaneous islet/kidney transplants are rarely performed, in part because of difficult logistics and because a successful islet isolation occurs at best in 50% of cases. The second category for pancreas and islet transplantation consists of patients with type 1 diabetes who have received a previous kidney transplant from either a living or deceased donor18-20. This pancreas after kidney transplant category accounts for approximately 20% of patients receiving pancreas transplants, while approximately 15% of islet transplantations

are done after a kidney transplant. The important consideration is that of technical risk21, since the risk of immunosuppression has already been assumed for both groups22. The third category for pancreas and islet transplantation is composed of non-uremic patients with type 1 diabetes23. Candidates are those in whom the risk of immunosuppression is judged to be less than the risk of remaining diabetic on exogenous insulin therapy. Most of the candidates for a pancreas transplant alone have extremely labile diabetes and have difficulty managing day-to-day, with frequent emergency room visits or inpatient hospitalizations for hypoglycemia or diabetes. Other patients have significant difficulty with hypoglycemic unawareness that results in unconsciousness without warning, need assistance from those around them, and never should be left alone. For select patients, this state can be a devastating problem that affects their employment and their ability to keep a driver’s license and creates concern about lethal hypoglycemia while asleep, as well as imposing an emotional toll on family members. Pretransplant evaluation often incorporates an assessment of the Clarke Score24 to semi-quantitatively determine the severity of hypoglycemic complications in an effort to more fully understand the risk/benefit relationship for undergoing a pancreas or islet transplantation. Only about 15% of pancreas transplants are performed for this scenario (because so many pancreas transplants are done in renal allograft recipients who are already obligated to immunosuppression and thus the indications for beta-cell replacement are much more liberal), whereas for islet transplant, it accounts for about 85% of cases since almost all to date are in nonrenal allograft recipients. The outcomes currently favor pancreas transplants in the SPK transplant group over any islet group. However, the results are much closer when solitary pancreas transplants are compared to islet transplants as immunosuppression 101

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protocols have improved for islet transplantation. Currently, five-year solitary pancreas graft survival rates are approximately 55% in this group and at best approximately 40% for islets25. Importantly, islet transplantation has a low morbidity. Unfortunately, it is also inefficient because of the attrition of viable islets during isolation and engraftment. This problem results in failed attempts to obtain a sufficient yield of islets for transplants from some donor pancreases, and creates the need for more than one donor (retransplantation) to achieve a sufficient beta-cell mass in many recipients. Ideally, beta-cell replacement should be done by the least invasive means possible. However, if one is to maximize the number of recipients in the face of a scarce resource, deceased donor organ allocation for pancreas and islet allotransplantation has to be integrated in a way that balances the two objectives: treating as many as possible and minimizing morbidity26. The decision as to whether a beta-cell replacement candidate should receive an immediately vascularized solitary pancreas graft or an injection of isolated islets can be dictated, in part, by recipient characteristics27, to circumvent the limitations of islet graft inefficiency. Exogenous insulin requirements are a rough guide to the number of beta-cells required to induce insulin-independence; the lower the requirement, the fewer islets that will be needed. Thus, beta-cell replacement candidates with high insulin requirements would be better suited for a pancreas transplant, while those with low insulin requirements might get by with a single-donor islet transplant. A deceased donor pancreas allocation policy for beta-cell replacement should be designed to foster efficiency, and thus minimize the use of multiple islet donors (euphemism for retransplantation) for a single recipient. In the USA, organ allocation policies are set by the United Network for Organ Sharing (UNOS). 102

The current pancreas allocation is complicated by the need for two lists: one for those only in need of beta-cell replacement, and one for those who also need a kidney. Currently, the priority varies according to the policies of local organ procurement organizations. In some organizations, uremic diabetic patients waiting for a kidney/pancreas transplant have no priority over uremic patients waiting for a kidney alone; a kidney/pancreas is allocated only when a uremic diabetic is at the top of the list. In other organ procurement organizations, the highest ranked uremic diabetic gets priority for a kidney/pancreas, no matter what the rank; in others, however, a level of rank is specified above which the highest ranked kidney/pancreas gets priority. In all organ procurement organizations, if no kidney/pancreas candidate is ranked high enough for an offer of both organs, the pancreas is offered to the highest ranked candidate for solitary pancreas or islet transplantation. In regard to UNOS policy on pancreas and islet transplants, organs from donors < 50 years old are first offered to pancreas candidates, and those from donors > 50 first to islet candidates, primarily because intact pancreas transplants from donors over 50 years of age have increased technical complication rates at most pancreas programs. Pancreases from obese donors (BMI > 30) are also preferentially for islets, both because of the increased technical complication rate with pancreas transplants from such donors and the fact that the absolute number of islets isolated is proportional to donor size28. We know that nondiabetic, obese individuals have more islets than lean individuals because the beta-cell mass increases to cope with the increased insulin needs associated with obesity. This means that pancreases from obese donors could be assigned preferentially to recipients suitable to receive islet transplants because of their low insulin requirements, while pancreases from lean donors would be used for whole pancreas transplants to recipients with high insulin requirements.

David E.R. Sutherland and Dixon B. Kaufmann: Beta-Cell Replacement by Transplantation in Diabetes Mellitus

Currently, UNOS is in the process of designing the pancreas allocation for both solid organ and islet transplantation29. UNOS is interested in a new national pancreas allocation system that will better address the needs of patients with diabetes with and without concurrent renal failure. There are several concerns with the way pancreases are currently allocated. First, there is no nationally established allocation practice for patients with diabetes and renal failure. Current pancreas allocation policy allows organ procurement organizations several choices on pancreas (pancreas alone) allocation practice. The candidates can be listed on separate or combined SPK/pancreas-alone waiting lists. The kidney may be allocated to SPK candidates based upon the kidney/pancreas match run, the kidney-alone match run, or a combination of match runs. Consequently, waiting times for SPK transplants vary widely across the country because of local or regional allocation decisions. Furthermore, current practice does not seek to maximize the utilization of the pancreas. Simultaneous pancreas and kidney transplants receive offers after other renal/extrarenal multiorgan transplants, kidney paybacks, and zero mismatch kidneyalone candidates. This allocation order leads to discarding of grafts that would likely be used if offered in the context of SPK transplantation but are declined for pancreas-alone transplants. Under the current system, 66% of pancreases are used for SPK transplant candidates. However, there are no specific listing criteria for SPK transplants with respect to the degree of pancreas dysfunction necessary to qualify to receive waiting time for an SPK transplant, it is only the criteria for a kidney that is used: glomerular filtration rate (GFR) or creatinine clearance (CrCl) of 20 ml/min or less. A revised system is needed to improve the current pancreas allocation process. It should be consistent with the Organ Procurement Transplantation Network’s long-range strategic goals and priorities: geographic equity in

access and waiting time to deceased donor organs for transplantation; maximizing capacity of deceased donor organ transplantation; achieving operational efficiency and cost-effectiveness in implementing and maintaining the organ allocation system. Depending on where a transplant candidate lives, some candidates may have to wait longer than others for a pancreas transplant. The first goal of the proposed pancreas allocation system reduces the geographic inequities related to deceased donor pancreas utilization, access to transplantation, and how long the candidates wait. Accomplishing these goals would mean instituting a consistent national system. Under this system, if a diabetic, uremic candidate on the list for an SPK transplant is allocated a pancreas from a local deceased donor and accepts it, then that candidate would also receive a kidney from the same deceased donor. The second goal is to maximize capacity by improving the opportunity for pancreas candidates to receive a transplant. This would be accomplished by combining SPK and pancreas-alone candidates onto a single match run list. On a single list, candidates for both categories of pancreas transplants would have an equal opportunity to receive offers of high quality organs. A single list for all pancreas candidates would be operationally efficient for organ procurement organizations. It would also retain some high quality kidneys for the kidney allocation system in the situations in which a pancreas graft is allocated for pancreas-alone transplantation. Right now, diabetic, uremic candidates are not fully incentivized to receive a kidney from a living donor if they will subsequently be put on a wait list for a solitary pancreas in a donation service area that allocates organs to SPK candidates before allocating them to pancreasalone candidates. In this situation, if a candidate chooses to take a living donor kidney and then wait on the list for a pancreas, that 103

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candidate would receive a local pancreas offer only if all the local SPK candidates had turned down that pancreas. This process results in additional waiting time for pancreas after kidney transplantation compared to declining a living donor kidney and continuing to wait for the SPK transplant. Also, in many circumstances these pancreases that are refused by all the SPK candidates are of lower quality than the pancreases the candidate would be able to receive if he or she had waited for an SPK rather than taking the living donor kidney. This situation might discourage candidates who need both a kidney and a pancreas from taking a living donor kidney followed by a deceased donor pancreas. The proposed allocation change would mean that candidates may be more inclined to accept a kidney from a living donor, knowing they subsequently will get a good quality solitary pancreas offer. On the other hand, for pancreas transplant programs that readily accept regional and national solitary pancreas offers, the waiting time for a pancreas after kidney transplant may be relatively short. Thus, in these programs or donation service areas, a living donor kidney followed by a solitary deceased donor pancreas transplantation can actually reduce the time of being both dialysis-free and insulin-independent for uremic diabetics over those on the SPK waiting list. Thus, the value on survival of preempting dialysis with a living donor kidney transplant offsets the negative aspects of waiting for a solitary pancreas transplant and having two operations18-20. And of course, there is the option of a living donor SPK transplant30,31. The third goal is to enhance efficiency and minimize the complexity of implementing and maintaining the operational requirements of a new pancreas allocation system. The proposed method would allocate deceased donor pancreases separately from the current kidney allocation system. This method would effectively disentangle the system of 104

a pancreas allocation from kidney allocation. There appears to be enough deceased donor kidneys (both standard and expanded criteria) available to accommodate this allocation change without adversely affecting pediatric or adult kidney transplant activity. Importantly, this process would result in a faster and more efficient method of allocating organs. It would also be less costly to implement and maintain. The fourth goal is to optimize pancreas transplant access without adversely affecting kidney transplantation. Specifically, a new pancreas allocation system would not affect transplant volume for adult and pediatric kidney recipients as well as ethnicity, age, and gender of recipients. This goal would be accomplished by instituting objective medical qualifying criteria relating to renal dysfunction and diabetes for SPK candidates. These candidates would be eligible to accrue SPK waiting time only if they meet qualifying criteria based on renal and metabolic function. The kidney function criteria for qualifying includes either being on dialysis, having a GFR or CrCl ≤ 20 ml/min. Qualifying pancreas function criteria includes either being on insulin and having a C-peptide value ≤ 2 ng/ml or being on insulin with glycemic intolerance with a C-peptide value > 2 ng/ml and a body mass index (BMI) ≤ 30 kg/m2. In addition, a single list for all pancreas transplant candidates would retain some high quality kidneys for the kidney allocation system. Finally, the proposal includes a system to monitor allocation of standard criteria deceased donor kidneys for pediatric and adult kidney alone recipients and SPK recipients with respect to donor ages ≤ 35 and > 35 years. It should be noted that pancreas transplantation is effective in inducing insulin-independence in diabetic patients (including type 2), regardless of the presence or absence of C-peptide or the levels32; nevertheless, C-peptide is being used in the policy formulations. Advances are being made in the isolation of islets to increase the proportion of islets

David E.R. Sutherland and Dixon B. Kaufmann: Beta-Cell Replacement by Transplantation in Diabetes Mellitus

that remain viable for transplantation; for example, the use of agents that prevent apoptosis after isolation33. As these advances are applied clinically, the insulin requirement threshold below which a single-donor islet transplant would be sufficient to induce insulin independence could be raised, increasing the proportion of beta-cell replacements done by the minimally invasive technique. Conversely, the BMI requirements to be an islet donor could be progressively lowered as the islet isolation efficiency increases in terms of viability. Ultimately, such improvements would result in a fully integrated list of pancreas and islet candidates, each being able to accept nearly all donors regardless of characteristics, and most beta-cell replacement therapy would be done by islet transplantation. We are, of course, not at that point yet.

If one deceased donor pancreas could consistently yield enough islets to induce insulin independence in a diabetic recipient, regardless of donor characteristics or recipient’s exogenous insulin requirements, islet transplantation would largely replace pancreas transplantation. An exception would be candidates who also have exocrine deficiency, for example, those who became diabetic as a result of pancreatectomy for benign disease39. Islet autotransplantation performed at the time of pancreatectomy for chronic pancreatitis can preserve insulin independence in some patients40,41, but for those in whom it does not, or in whom it was never attempted, it makes sense to transplant a pancreas (rather than simply islets) with enteric drainage of the exocrine secretions so that normal intestinal absorption can also be restored39.

Besides improving the efficiency of islet isolation, other alterations in strategies may allow a lower number of isolated islets to induce insulin-independence in recipients than is currently the case, for example, using a truly nondiabetogenic immunosuppressive regime (even the Edmonton protocol is diabetogenic with its inclusion of a calcineurin inhibitor). In many pancreas transplant programs, the immunosuppressive regimen is free of steroids22,34.

The shortage of deceased donors for those in need of organ replacement therapy of all kinds has led to the use of living donors, including for the pancreas. Segmental pancreas transplants from living donors have been done since 1979 at the University of Minnesota31, and this institution had an even earlier experience with two cases of islet allografts from living donors42. In countries where deceased organ donors are in even shorter supply, the incentive to use living donors is particularly strong. For example, in Japan the number of living donor liver transplants greatly exceeds that from deceased donors, and the transplant group in Kyoto also did a living donor islet transplantation a few years ago43,44. However, it is unlikely that the use of both deceased and living donors can meet the demand for beta-cell replacement therapy any better than it has for any other organ, even in countries with relatively high numbers of both types. Thus, in the long term, either islet xenografts or induction of endogenous beta-cell regeneration will be the answer26,38.

By combining several aspects of the recipient and donor selection criteria, technical improvements in islet isolation and immunosuppressant selection, as outlined above, has allowed insulin independence to be achieved with islets from a single donor35. There are several recent reviews on islet transplantation that show the promise of islet transplantation to eventually be the nearly sole method of beta-cell replacement therapy14,36,37. However, to be truly a treatment for all diabetics, an unlimited source of islets is needed, and this could only be met by islet xenotransplantation, as recently reviewed38, or through development of glucose-responsive, insulin-producing expanded cell line.

Meanwhile, we must use the resource at hand: a limited number of allogeneic donor 105

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pancreases for beta-cell replacement therapy. To answer the questions posed in the title, beta-cell replacement therapy should be done in insulin-dependent diabetic patients who are either obligated to immunosuppression (nearly all diabetic renal allograft recipients should be candidates) or in those whose problems with achieving sufficient metabolic control of diabetes using exogenous insulin (primarily those with hypoglycemic unawareness) exceed the potential side effects of immunosuppression. For candidates in whom the insulin requirements are low enough so that islets isolated from a single donor would predictably induce insulin independence, perform an islet transplant; in those whose requirements are so high that more than one donor would be needed for the islet approach, and who are not at high risk for surgical complications, carry out a pancreas transplant. If the surgical risk is unacceptable in high insulin-requiring candidates, then perform an islet transplant, with the exception that islet retransplantation can be done over time to eventually achieve insulin independence. With this approach, beta-cell replacement can be done in the most patients with the highest insulin-independent rate possible, while allowing minimally invasive surgery to be done in some candidates at no expense to those who require more (a solid organ). Finally, a word about the mortality risk of beta-cell replacement therapy versus remaining on insulin for a diabetic patient. The mortality risk of pancreas transplantation, in absolute terms, is very low, with patient survival rates at one year ranging from 95 to 98% in all three (simultaneous pancreas/kidney, pancreas after kidney, pancreas alone) recipient categories45. However, Venstrom, et al., in an analysis of UNOS data from 1995 through 2000, found that the posttransplant mortality rate of solitary pancreas transplant recipients (pancreas alone or pancreas after kidney transplant) was higher than for candidates who remained on the waiting list46. Analyses of this type are very difficult to perform, and in a separate analysis by 106

Gruessner, et al.47, but this time counting patients only once that were multiply listed or changed centers, the mortality rate for solitary pancreas transplant recipients was not higher than for wait-listed patients. Thus, it appears that pancreas transplantation and exogenous insulin treatment are at least equal in survival probabilities for the diabetic patients accepted as transplant candidates. As reviewed by Robertson48, every study that has been done on quality of life favors pancreas transplantation over exogenous insulin for such patients, most of whom have significant problems with the latter (such as hypoglycemic unawareness). What is needed now is to increase the efficiency of islet isolation and engraftment so beta-cell replacement therapy can be done by the minimally invasive technique in the majority, rather than the minority, of candidates.

References

1. Epidemiology of Diabetes Interventions and Complications (EDIC) Research Group. Effect of intensive diabetes treatment on carotid artery wall thickness in the epidemiology of diabetes interventions and complications. Epidemiology of Diabetes Interventions and Complications (EDIC) Research Group. Diabetes. 1999;48:383-90. 2. DCCT/EDIC Research Group. Effect of intensive therapy on the microvascular complications of type 1 diabetes mellitus. JAMA. 2002;287:2563-9. 3. Stock P. Beta-cell replacement for type 1 diabetes mellitus - islet versus solid organ pancreas. Curr Opin Organ Transplant. 2005;10:70-4. 4. Deng S, Markmann JF, Rickels M, et al. Islet alone versus islet after kidney transplantation: metabolic outcomes and islet graft survival. Transplantation 2009;88:820-5. 5. Markmann JF, Kaufman DB, Ricordi C, Schwab PM, Stock PG. Financial issues constraining the use of pancreata recovered for islet transplantation: a white paper. Am J Transplant. 2008;8:1588-92. 6. Morel P, Goetz F, Moudry-Munns KC, et al. Long term metabolic control in patients with pancreatic transplants. Ann Intern Med. 1991;115:694-9. 7. Navarro X, Kennedy WR, Loewenson RB, et al. Influence of pancreas transplantation on cardiorespiratory reflexes, nerve conduction, and mortality in diabetes mellitus. Diabetes. 1990;39:802-6. 8. Kennedy WR, Navarro X, Goetz FC, et al. Effects of pancreatic transplantation on diabetic neuropathy. N Engl J Med. 1990;322:1031-7. *This study was the first to definitively show that a pancreas transplant could reverse clinical manifestations of neuropathy or prevent progression in diabetic patients. 9. Fioretto P, Mauer SM, Bilous RW, et al. Effects of pancreas transplantation on glomerular structure in insulin-dependent diabetic patients with their own kidneys. Lancet. 1993;342:1193-6. *This study was the first to definitively show that a pancreas transplant alone could reverse histological manifestations of nephropathy in native kidneys of diabetic patients, though renal function may not improve because of the nephrotoxic effect of calcineurin-inhibitor immunosuppression.

David E.R. Sutherland and Dixon B. Kaufmann: Beta-Cell Replacement by Transplantation in Diabetes Mellitus 10. Bilous RW, Mauer SM, Sutherland DE, et al. The effects of pancreas transplantation on the glomerular structure of renal allografts in patients with insulin-dependent diabetes. N Engl J Med. 1989;321:80-5. 11. Zehr PS, Milde FK, Hart LK, et al. Pancreas transplantation: assessing secondary complications and life quality. Diabetologia. 1991;34(Suppl 1):S138-40. 12. Ojo AO, Meier-Kriesche HU, Hanson JA, et al. Impact of simultaneous pancreas-kidney transplantation on long-term patient survival. Transplantation. 2001;71:82-90. 13. Mohan P, Safi K, Little DM, et al. Improved patient survival in recipients of simultaneous pancreas-kidney transplant compared with kidney transplant alone in patients with type 1 diabetes mellitus and end-stage renal disease. Br J Surg. 2003;90:1137-41. 14. Fioriana P, Shapiro AM, Ricordi C, Secchi A. The clinical impact of islet transplantation. Am J Transplant. 2008;8:1990-7. *Excellent review on every aspect of clinical islet transplantation. 15. Tharavanij T, Betancourt A, Messinger S, et al. Improved long-term health-related quality of life after islet transplantation. Transplantation. 2008;86:1161-7. 16. Warnock GL, Thompson DM, Meloche RM, et al. A multi-year analysis of islet transplantation compared with intensive medical therapy on progression of complications in type 1 diabetes. Transplantation. 2008;86:1762-6. 17. Sollinger HW, Odorico JS, Becker YT, et al. One thousand simultaneous pancreas-kidney transplants at a single center with 22-year follow-up. Ann Surg. 2009;250:618-30. *Largest series of SPK transplants from a single institution. 18. Sutherland DE, Gruessner AC, Radosevich DM. Transplantation: kidney or kidney-pancreas transplant for the uremic diabetic? Nat Rev Nephrol. 2009;5:554-6. 19. Kleinclauss F, Fauda M, Sutherland DE, et al. Pancreas after living donor kidney transplants in diabetic patients: impact on long-term kidney graft function. Clin Transplant. 2009;23:437-46. *This article shows the attractiveness of doing a LD kidney transplant to preempt dialysis followed by a deceased donor pancreas transplant in uremic diabetics. 20. Kaufmann D. Pancreas-after-kidney transplantation: to have and to have not. Clin Transplant. 2009;23:435-6. 21. Boggi U, Vistoli F, DelChiaro M, et al. Surgical techniques for pancreas transplantation. Curr Opin Organ Transplant. 2005;10:75-87. 22. Kaufmann D, Salvalaggio PR. Immunosuppression for pancreas transplantation. Curr Opin Organ Transplant. 2005;10:88-94. 23. Gruessner R, Sutherland DE, Kandaswamy R, Gruessner A. Over 500 solitary pancreas transplants in nonuremic patients with brittle diabetes mellitus. Transplantation. 2008;85:42-7. *Largest series of pancreas transplants alone from a single institution. 24. Clarke WL, Cox DJ, Gonder-Frederick LA, et al. Reduced awareness of hypoglycemia in adults with IDDM. A prospective study of hypoglycemic frequency and associated symptoms. Diabetes Care. 1995;18:517-22. 25. White SA, Shaw JA, Sutherland DE. Pancreas transplantation. Lancet. 2009;373:1808-17. *Most up to date review on current status of pancreas transplantation. 26. Sutherland DE, Gruessner A, Hering J. Beta-cell replacement therapy (pancreas and islet transplantation): an integrated approach. Endocr Metab Clin N Amer. 2004;33:135-48. 27. Sutherland DER. Pancreas and Islet Transplant Population. Transplantation of the Pancreas. Gruessner RW, Sutherland DE (eds). New York: Springer-Verlag, 2004:91-2. 28. Matsumoto I, Sawada T, Nakano M, et al. Improvements in islet yield from obese donors for human islet transplants. Transplantation. 2004;78:880-5. 29. http://www.unos.org/CommitteeReports/board_main_PancreasTransplantationCommittee_6_24_2010_9_59.pdf - application/pdf 30. Sutherland D, Najarian J. Living Donor Pancreas Transplantation. In Living Related Transplantation. Hakim NS, Canelo R, Papalois V (eds). Imperial College Press, London. 2010:95-117. 31. Sutherland DE, Gruessner RW, Dunn DL, et al. Lessons learned from more than 1,000 pancreas transplants at a single institution. Ann Surg. 2001;233:463-501. 32. Nath DS, Gruessner A, Kandaswamy R, Gruessner R, Sutherland DER, Humar A. Outcomes of pancreas trans-

















plants for patients with type 2 diabetes mellitus. Clin Transplant. 2005;19:792-7. 33. Nakano M, Matsumoto I, Sawada T, et al. Capsase-3 inhibitor prevents apoptosis of human islets immediately after isolation and improves graft function. Pancreas. 2004;29:104-9. 34. Sutherland DE, Kandaswamy R, Humar A, Gruessner RW. Calcineurin-inhibitor-free protocols: Use of the anti-T-cell agent Campath H-1 for maintenance immunosuppression in pancreas and pancreas/kidney recipients. Clin Transplant. 2010;18:14-15. 35. Hering BJ, Kandaswamy R, Ansite J, et al. Single-donor, marginal-dose islet transplantation in patients with type 1 diabetes mellitus. J Am Med Assoc. 2005;293:830-5. 36. Korsgren O, Nilsson B. Improving islet transplantation: a road map for a widespread application for the cure of persons with type I diabetes. Curr Opin Organ Transplant. 2009;14:683-7. *Most up to date review on current status of islet transplantation. 37. Robertson RP. Islet transplantation a decade later and strategies for filling a half-full glass. Diabetes. 2010;59:1285-91. 38. Hering BJ, Walawalkar N. Pig-to-nonhuman primate islet xenotransplantation. Transpl Immunol. 2009;21:81-6. *This article reviews the promise of islet xenotransplantation for solving the human organ shortage problem, and how close this is to clinical reality. 39. Gruessner RW, Sutherland DE, Dunn DL, et al. Transplant options for patients undergoing total pancreatectomy. J Am Coll Surg. 2004;198:559-67. 40. Blondet J, Carlson A, Kobayashi T, et al. The role of total pancreatectomy and islet autotransplantation for chronic pancreatitis. Surg Clin North Am. 2007;87:1477-501. 41. Sutherland DE, Gruessner AC, Carlson AM, et al. Islet autotransplant outcomes after total pancreatectomy: A contrast to islet allograft outcomes. Transplantation. 2008;86:1799-802. 42. Sutherland DE, Gores PF, Farney AC, et al. Evolution of kidney, pancreas, and islet transplantation for patients with diabetes at the University of Minnesota. Am J Surg. 1993;166:456-91. 43. Matsumoto S, Okitsu T, Iwanaga Y, et al. Insulin independence after living-donor distal pancreatectomy and islet allotransplantation. Lancet. 2005;365:1642-4. 44. Matsumoto S, Okitsu T, Iwanaga Y, et al. Insulin independence of unstable diabetic patient after single living donor islet transplantation. Transplant Proc. 2005;37:3427-9. 45. Gruessner AC, Sutherland DE. Pancreas transplant outcomes for United States (US) and non-US cases as reported to the United Network for Organ Sharing (UNOS) and the International Pancreas Transplant Registry (IPTR) as of June 2004. Clin Transplant. 2005;19:433-55. 46. Venstrom JM, McBride MA, Rother KI, Hirshberg B, Orchard TJ, Harlan DM. Survival after pancreas transplantation in patients with diabetes and preserved kidney function. J Am Med Assoc. 2003;290:2817-23. 47. Gruessner RD, Sutherland DE, Gruessner AC. Mortality assessment for pancreas transplants. Am J Transplant. 2004;4:201826. *This article and the one preceding both definitively show the great beneficial effect of kidney-pancreas transplant on survival of uremic diabetics over those who remain on the wait list. What is controversial is whether the mortality risk of a solitary pancreas transplant exceeds that of not being transplanted and in the article by Gruessner, et al. the answer is no. 48. Robertson RP. Impact of pancreas and islet transplantation on acute and chronic complications of diabetes. Curr Opin Organ Transplant. 2005;10:95-9.

Suggested Readings

Sutherland DER. Beta-cell replacement by transplantation in diabetes mellitus: which patients at what risk, which way (when pancreas, when islets), and how to allocate deceased donor pancreases. Curr Opin Organ Transplant. 2005;10:147-9. *This issue has several articles, besides the one cited, on all aspects of pancreas and islet transplantation. The article cited has formed the basis of the current discussion adding Dr. Kaufman’s perspective as current Chairman of the UNOS Pancreas Allocation Committee.

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Trends in Transplant. Transplantation 2010;4:108-16 2010;4

Cytomegalovirus and Development of Cardiac Allograft Vasculopathy: Evidences and Therapeutic Implications Luciano Potena1 and Hannah A. Valantine2 1 Cardiovascular Department of the University of Bologna, Bologna, Italy; 2Division of Cardiovascular Medicine, Stanford University, Palo Alto, USA

Abstract Cardiac allograft vasculopathy remains the major cause of long-term failure of heart transplantation. Cytomegalovirus infection was identified as a major risk factor for cardiac allograft vasculopathy development in pioneering studies, even though the possibility that the virus is only an innocent bystander was not completely excluded. Only recently, convincing clinical and experimental evidences support the hypothesis of a direct involvement of cytomegalovirus in cardiac allograft vasculopathy pathogenesis. In this article, we review the mechanisms and clinical evidences supporting the hypothesis that subclinical cytomegalovirus infection leads to adverse long-term graft outcome by favoring cardiac allograft vasculopathy development. In addition, we discuss data pointing to the need for antiviral approaches designed to suppress subclinical cytomegalovirus activation as a long-term strategy to prevent cardiac allograft vasculopathy and chronic allograft damage. (Trends in Transplant. 2010;4:108-16) Corresponding author: Luciano Potena, [email protected]

Key words Heart transplant. Cytomegalovirus. Cardiac allograft vasculopathy.

Introduction Short-term survival after heart transplantation has greatly improved over the last three decades as a consequence of advances in immunosuppressive therapy and perioperative management. However, improvement

Correspondence to: Luciano Potena Cardiovascular Department Heart Failure and Heart Transplant Program University of Bologna Padiglione 21 Via Massarenti, 9 40138 Bologna, Italy E-mail: [email protected]

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in long-term outcome is still significantly impeded by the consequences of chronic allograft vasculopathy (CAV), the major cause of late failure of the transplanted heart1. Although numerous immune-mediated and metabolic risk factors have been identified for CAV progression2, to date no effective treatment is available to fully eliminate its related adverse outcomes. Therefore, the main therapeutic strategy against CAV is the prevention and treatment of the factors known to trigger or accelerate the disease3. Among these known risk factors is cytomegalovirus (CMV) infection, which plays a key role in CAV progression, possibly through its complex interaction with the host immune system4,5. Importantly, strategies that target CMV offer the

Luciano Potena and Hannah A. Valantine: CMV and Cardiac Allograft Vasculopathy

possibility of effective prevention of CAV while also advancing our understanding of its pathogenesis. However, the efficacy of distinct antiCMV strategies in limiting CAV requires further evaluation, in concert with studies that identify the specific mechanisms by which CMV mediates graft injury. This knowledge is necessary to advance the field and settle the much-debated controversy of whether CMV is an innocent bystander or directly involved in CAV pathogenesis6. This article reviews recent studies that provide evidence in support of the involvement of CMV in the pathogenesis of CAV. Specifically discussed are the implication of new clinical data and mechanistic pathways potentially implicated in CMV-induced allograft damage. Also discussed are the implications of these data that point to the need for chronic suppression of subclinical viral activation as a long-term strategy to prevent CAV and chronic allograft damage.

Relevance of cytomegalovirus infection in heart transplantation Cytomegalovirus is a member of the β-Herpesviridae family that includes human herpesvirus-6 (HHV-6) and HHV-77. In the general population, CMV is present in peripheral blood monocytes of 50-90% of individuals, but does not normally cause symptomatic disease. In contrast, CMV is the most clinically relevant posttransplant infectious agent, affecting up to 80% of heart transplant recipients, depending on donor-recipient serostatus, intensity of the immunosuppressive regimen, and the diagnostic system used to assay CMV replication8,9. Following transplantation, immunosuppression causes the reactivation of latent virus, or allows de novo transmission of CMV from a seropositive donor to a seronegative recipient (D+/R–)10. Bidirectional interaction between the suppressed host immune system

and the immune modulating virus itself increases the risk of infection and CMV disease11. Cytomegalovirus infection has both direct and indirect effects. Direct effects, attributed to the CMV syndrome, typically present as prolonged high fever, fatigue, malaise, anorexia, arthralgias and myalgias, and the leukopenia and thrombocytopenia symptomatic of myelosuppression12. Tissue-invasive disease manifests as nephritis, hepatitis, carditis, pneumonitis, pancreatitis, colitis, or rarely as retinitis, seen more frequently among patients coinfected with HIV13. Indirect effects of CMV include allograft injury and rejection, and increased risk for the development of EpsteinBarr virus-associated posttransplant lymphoproliferative disorder12. Viral disruption of immune responses and damage to endothelial cells are believed to be responsible for many of the indirect effects of CMV14,15. Transplant recipients who develop CMV infection are at increased risk of mortality. Of note, increased risk for overall mortality appears associated not only with tissue-invasive CMV disease, but also with asymptomatic infection, as detected by pp65 antigenemia16, supporting the concept that CMV is capable to indirectly promote graft dysfunction. Taken together, the evidence supports the conclusion that acute CMV disease following transplantation is a predictor of acute morbidity, and is likely to predispose to chronic long-term graft dysfunction. Thus, strategies directed to prevent CMV disease and to balance the burden of immunosuppressive therapy are mandatory for an optimal posttransplant care.

Clinical evidence associating cytomegalovirus with chronic allograft vasculopathy The association of CMV infection with CAV was first reported over two decades ago, 109

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in the pre-ganciclovir era17-19. These early observational studies were conducted at a time when the methods for detection of CMV infection were relatively insensitive, relying predominantly on clinical manifestations of viral disease and confirmation by histology or viral culture. From these observations developed the concept that CMV may not only cause an organ-specific or systemic disease due to direct viral damage, but is also capable of inducing immune activation that targets the allograft and thus indirectly results in acute rejection and CAV20. The advent of antiviral drugs, particularly ganciclovir, led to effective therapeutic strategies for preventing CMV disease and to a reduction in virus-related graft failure, both in experimental models and in heart transplant recipients21-23. These advances in antiviral therapy were paralleled by the development of highly sensitive diagnostic tools to detect CMV infection, enabling the identification of a large number of patients who developed subclinical viral infection, hitherto unrecognized by prior methods24,25. By monitoring asymptomatic CMV activation in peripheral blood, Emery, et al. reported that the risk of overt CMV disease is proportional to the level of viral DNA detected26. These observations linking viral load with acute disease raised the question of whether asymptomatic viral replication may also predict the long-term consequences of CMV infection. Several studies, summarized below, provide confirmatory evidence for this link. In a large retrospective analysis including more than 400 kidney recipients16, Sagedal, et al. showed that in absence of either prophylaxis or preemptive strategies, CMV infection at a subclinical level as detected by pp65 antigenemia was associated with increased risk for overall and cardiovascular mortality. The authors additionally showed that subclinical CMV disease during the first 110

100 days after transplantation increased the risk for subsequent rejection27. As opposed to a universal prophylaxis strategy, the preemptive strategy involves the administration of antiviral drugs only in those patients who reach a certain threshold level of viral activity28. Thus, patients managed by a preemptive strategy develop significantly higher levels of subclinical CMV replication compared to those managed by a prophylaxis strategy. These two distinct approaches have allowed for the comparison of outcomes with respect to asymptomatic infection29. We have shown that in heart transplant recipients managed by a preemptive strategy, asymptomatic CMV infection was associated with increased risk of developing CAV, defined as abnormal coronary remodeling one year after transplantation30. In this study, antiviral treatment was administered only to patients who developed > 30 pp65 positive cells per 105 polymorphonuclear cells, consistent with the preemptive approach. In a subsequent prospective study undertaken at Stanford, despite universal antiviral prophylaxis with ganciclovir, CMV DNA indicating active infection was detected in over 90% of the patients, who however remained asymptomatic. In the majority of patients developing CMV infection (80%), CMV was detected only after discontinuing prophylaxis, raising the question of the importance of the duration and type of CMV prophylaxis. To address this question, we compared the outcomes in patients receiving a “standard regimen” of intravenous ganciclovir for 28-days, compared to D+/R– patients who received a more aggressive regimen consisting in three months of (val-)ganciclovir and CMV hyperimmune serum (CMVIG)31. Despite being at higher risk for CMV activation because of the serological mismatch, recipients treated with the aggressive regimen showed delayed and reduced CMV infection rates and, most importantly, a reduced risk of acute rejection and CAV as

Luciano Potena and Hannah A. Valantine: CMV and Cardiac Allograft Vasculopathy

compared to patients treated with the standard regimen. 0.9

Taken together, the findings of these studies suggest a pathophysiological role of CMV in chronic graft failure, limiting long-term outcome in heart transplant recipients. Most important, the recent data suggest that such chronic damage to the graft may progress unabated, even in the absence of overt clinical CMV disease32. Randomized clinical trials are required to confirm these observations.

Possible mechanisms of cytomegalovirus-mediated injury Cytomegalovirus drives a complex interaction with the recipient immune system that, under conditions of iatrogenic immunosuppression, can promote a local proinflammatory milieu, disrupt tolerogenic mechanisms, and exert immunosuppressive effects. These consequences of CMV infection directly influence the alloimmune response in the transplant recipient and may explain the pathogenesis of CMV-induced CAV. Interaction of CMV with the host inflammatory response sets the stage for viral

0.8 0.7 Change in MIT (mm)

More recently, pursuing the hypothesis that an aggressive anti-CMV strategy could reduce CAV development in CMV-positive recipients, we compared the outcomes of patients managed with a preemptive strategy with a consecutive cohort receiving a 40-day course of valganciclovir, followed by CMV monitoring and additional treatment when patients developed > 30 pp65 positive cells per 105 polymorphonuclear cells. In this study, the aggressive strategy led to a delayed and reduced magnitude of CMV infection. Most importantly, the prophylaxis-based strategy was associated with a reduced increase in coronary maximal intimal thickness one year after transplantation (Fig. 1)29.

p = 0.001

0.6 0.5 0.4 0.3 0.2 0.1 0.0 –0.1 Preemptive group (n = 21)

Prophylaxis group (n = 19)

MIT: maximal intimal thickness

Figure 1. Changes in coronary maximal intimal thickness in patients treated with valganciclovir prophylaxis and in those followed by preemptive approach (reproduced with permission from Elsevier)29.

replication and active CMV infection. Viral glycoprotein B-mediated virion entry and interaction with host leukocyte toll-like receptors leads to activation of the transcription factor nuclear factor kappa B (NFκB), required for CMV transcription, even in the absence of complete viral particle33,34. The NFκB regulates many inflammatory cytokine genes and adhesion molecules and has recognition sites for the CMV major immediate early promoter35. Virus-cell interaction is sufficient for activation of NFκB, which is required to initiate the CMV transcription machinery33,35. In addition, triggering of NFκB by immuno-inflammatory stimuli (e.g. infections or allograft recognition) contributes to CMV activation in cells harboring latent infection, including monocytes differentiating into macrophages36. Of note, allograft transplantation, but not isograft transplantation, induces CMV reactivation in a murine model of latent infection37, and pharmacologic inhibition of NFκB may reduce endothelial cell replication of CMV in vitro38. Activation of NFκB stimulates endothelial cell 111

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replication of CMV. Cytokines and chemokines induced by CMV and released over the course of the immune response activate endothelial cells, resulting in upregulation of adhesion molecules, increased class II major histocompatibility complex protein expression, and the production of cytokines exacerbating allograft inflammation39-43. In turn, these events are associated with the development of allograft vascular disease20,44. In addition to stimulating the synthesis of inflammatory mediators by host cells, CMV encodes for chemokine and cytokine homologs. Cytokine homolog genes are believed to favor viral spreading by inducing cell migration and cell proliferation4. Particular importance has been ascribed to US28, a viral gene encoding a G-protein-coupled chemokine receptor homolog that induces smooth muscle cell proliferation45. In a rat model, deletion of the functional homolog of US28 rCMV led to reduced CMV-dependent transplant vasculopathy46. It must be noted, however, that these CMV-dependent inflammatory mechanisms require allogeneic responses to accelerate graft rejection because infected animals receiving syngeneic organs do not develop disease47. Therefore, interplay between CMV and the host immune system appears to be a crucial factor in CMV-associated graft injury. A direct vascular effect of the activation of inflammatory mediators in the graft vascular system is reduced nitric oxide (NO) synthesis by endothelial layer, which rapidly becomes dysfunctional, impairing NO-mediated vasodilation48. Endothelial dysfunction is a mechanism preceding and associated with systemic and graft atherosclerosis49,50. Of note, CMV infection has been linked to endothelial dysfunction of both graft coronary arteries and systemic vasculature in heart recipients51,52. Indeed, chronic endothelial dysfunction, and thus abnormal vascular response to injury, may explain the consistent finding of coronary lumen loss associated with negative remodeling, 112

instead of intimal hyperplasia, shown by CMVinfected patients53. A possible process involved in the induction of endothelial dysfunction by CMV takes into account asymmetric dimethylarginine (ADMA)53,54. This ADMA is an endogenous inhibitor of endothelial NO synthesis, increases in conditions of intracellular oxidative stress, amplifies the disruption of endothelial homeostasis and thus may be regarded as a systemic marker of impaired endothelial function48,55,56. Cytomegalovirus infection is capable of increasing ADMA in cultured endothelial cells, and patients with CMV DNA detected in peripheral blood were shown to have higher ADMA plasma concentrations and were more likely to develop CAV than recipients with no CMV detection53,54. In addition to being associated with CAV pathogenesis, further negative effects of asymptomatic CMV infection on the peripheral vascular system have been hypothesized by the group from the Great Ormond Street Children’s Hospital in London. In pediatric heart transplant recipients, these investigators show that children who experience asymptomatic CMV infection develop chronic endothelial dysfunction in the systemic circulation. These data suggest that the consequences of CMV infection after transplantation may not only be limited to the allograft, and reinforce the concept that subclinical CMV replication may negatively influence later vascular health globally, even when it is no longer detectable in the circulation51. The negative effect of CMV infection on graft tolerance has been elegantly investigated in a recent study by Cook, et al.37. In a murine model of heart transplantation where tolerance may be effectively achieved with gallium nitrate treatment, recipients harboring latent CMV not only reactivate the virus, but also develop graft rejection leading to 80% of graft loss, as opposed to the 8% graft loss in CMV-negative recipients. Interestingly, while infiltrating the graft, CMV does not disrupt

Luciano Potena and Hannah A. Valantine: CMV and Cardiac Allograft Vasculopathy

graft expression of regulatory genes, nor stimulate allograft-specific immunity, but induces intra-graft inflammatory response mediated by type I interferon upregulation, ultimately leading to graft rejection37. In addition to mechanisms inducing inflammation, CMV exerts several effects relevant to immune-system escape and suppression of cellular immunity4, which, paradoxically, may be involved in acute rejection and CAV pathogenesis. In particular, the lack of CMV-specific CD4-positive immunity in CMV-seropositive heart recipients appears to favor earlier onset and magnitude of CMV infection57-59. Furthermore, recipients with delayed CMV-specific immunity had also an increased incidence of acute rejection and a more accelerated progression of CAV detected by intravascular ultrasound (IVUS), as compared with those with early CMV-specific immunity57. Interestingly, similar findings have been reported also in kidney transplant recipients60. Taken together, these data suggest that CMV-specific immunity is protective for preserving graft function, and not induce allograft cross-reactivity. Moreover, we may speculate that the lack of CD4-positive activation is the consequence of a successful CMV strategy of immune system escape that indirectly favors graft injury.

Antiviral strategies to prevent transplant atherosclerosis Two strategies are commonly recommended for the prevention of CMV infection and disease: universal prophylaxis and preemptive therapy28. Their rationales are based on two different preventive assumptions. Prophylaxis almost abolishes viral replication during the first weeks/months after transplantation, when the burden of immunosuppression is higher, thereby delaying the eventual appearance of the infection until a later phase of follow-up, by which time the immunosuppressive burden and risk of rejection is expected

to be lower. As opposed to prophylaxis, a preemptive strategy permits early low-grade viral replication in the belief that it may stimulate the host’s own immune response against the virus and will reduce the number of patients needing anti-CMV drugs61. A key issue in identifying the optimal strategy for prevention of CMV infection is the choice to limit or to allow asymptomatic CMV replication. In this paragraph, we discuss evidences supporting the graft-related benefit of approaches designed to suppress and prevent asymptomatic CMV replication. A large meta-analysis of randomized trials evaluating the effect of CMV prophylaxis in solid organ recipients showed that in addition to a clear prevention of CMV disease, the universal prophylaxis strategy was associated to superior survival and lower rejection episodes as compared to placebo62. This contrasted with the observations from a metaanalysis of preemptive strategy trials in which the preemptive approach was shown to be effective in preventing CMV disease, but failed to demonstrate any effect on survival or graftrelated endpoints63. Although it must be noted that in this systematic review, the small sample size of preemptive studies may have hidden its long-term efficacy; in two recent randomized studies, secondary analyses of graft-related endpoints suggested prolonged graft survival in kidney recipients receiving prophylaxis as compared with those followed with a preemptive approach64,65. In heart transplant recipients, with a non-randomized design, we have shown that aggressive antiCMV approaches – based either on a prolonged (val)ganciclovir and CMVIG regimen, or on valganciclovir prophylaxis followed by CMV monitoring and adjunctive treatment – are associated with lower progression of CAV as reflected by vascular remodeling66 and of intimal hyperplasia29. In addition to antiviral drugs, new lines of evidence regarding the anti-CMV effect of 113

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inhibitors of the mammalian target of rapamycin (mTOR), such as sirolimus and everolimus, offer new strategies to limit the impact of CMV infection on graft function. These two drugs, approved for prevention of acute rejection, have both been shown to reduce the occurrence of CMV infection in solid organ transplant recipients when compared with tacrolimus or mycophenolate67-70. Most importantly, both drugs also reduce the progression of CAV by IVUS measurement of coronary artery intimal hyperplasia71,72. Of note, the anti-CMV effect of mTOR inhibitors depends on their ability to inhibit the cell proliferation machinery, and not on a direct effect on CMV proteins. Nevertheless, the magnitude of mTOR inhibitors’ action in limiting CMV infection appears to exceed the protective effect of valganciclovir prophylaxis. Indeed, in a preliminary observational study73 including patients receiving valganciclovir prophylaxis or followed by preemptive strategy, we found that those receiving a maintenance immunosuppression regimen that included everolimus developed less CMV infection than those receiving mycophenolate. Importantly, while prophylaxis was effective in reducing CMV infection in mycophenolate-treated patients, patients receiving everolimus had such a low incidence of CMV infection that the advantage of prophylaxis over a preemptive strategy was no longer apparent. Thus, the use of mTOR inhibitors may represent a potent approach to minimize the risk of CMV reactivation and to limit CAV progression. Immunomodulatory agents provide yet another strategy for preventing CMV infection, reducing its effects on allograft injury. We and others have shown that the combination of ganciclovir with CMVIG appears superior to ganciclovir alone in preventing acute rejection and CAV31,74,75. Although we cannot exclude that different durations of prophylaxis may be even more important, there is evidence that hyperimmune sera can provide an additional beneficial immunomodulation of host responses, 114

thereby reducing the risk of both acute CMV disease and rejection76,77. In addition to the effects of improved humoral immunomodulation associated with CMVIG therapy, modulation of CMV-specific cellular immunity appears to play a role in preventing CMV reactivation and CMV-mediated graft injury57. These data raise the hypothesis that interventions designed to augment increasing CMV-specific immunity (e.g. by development of a vaccine) may provide yet another strategy for protection from CMV infection and CAV. Taken together, these studies underscore the concept that aggressive limitation of even subclinical CMV infection with strategies based on prophylaxis with antiviral agents and/or hyperimmune sera and on maintenance immunosuppression regimens may effectively protect long-term graft function.

Conclusions Although modern antiviral strategies significantly limit the immediate negative impact of CMV disease in heart transplant recipients, a growing body of evidence suggests that subclinical CMV infection leads to adverse long-term graft outcome. Several experimental studies support the hypothesis that the mechanism of CMV-dependent graft dysfunction is mediated by an active disruption of the interplay between graft and host’s immune system. Anti-CMV strategies aggressively targeting subclinical infection may effectively limit these effects by means of prophylaxis with antiviral drugs, immune-system reconstitution approaches, or even by the selection of maintenance immunosuppression. However, well designed randomized controlled studies are needed to confirm observational data regarding the benefits of aggressive anti-CMV approaches and to ascertain whether such expected benefits outweigh cost and toxicity.

Luciano Potena and Hannah A. Valantine: CMV and Cardiac Allograft Vasculopathy

References 1. Taylor DO, Edwards LB, Boucek MM, et al. Registry of the International Society for Heart and Lung Transplantation: twenty-third official adult heart transplantation report–2006. J Heart Lung Transplant. 2006;25:869-79. 2. Valantine HA. Cardiac allograft vasculopathy: central role of endothelial injury leading to transplant “atheroma”. Transplantation. 2003;76:891-9. 3. Mehra M. Contemporary concepts in prevention and treatment of cardiac allograft vasculopathy. Am J Transplant. 2006;6:1248-56. 4. Mocarski E. Immune escape and exploitation strategies of cytomegaloviruses: impact on and imitation of the major histocompatibility system. Cell Microbiol. 2004;6:707-17. 5. Abate D, Watanabe S, Mocarski E. Major human cytomegalovirus structural protein pp65 (ppUL83) prevents interferon response factor 3 activation in the interferon response. J Virol. 2004;78:10995-1006. *This paper dissects one of the mechanisms through which CMV induces immune evasion by modulating cellular gene-expression. 6. Vamvakopoulos J, Hayry P. Cytomegalovirus and transplant arteriopathy: evidence for a link is mounting, but the jury is still out. Transplantation. 2003;75:742-3. 7. Naraqi S. Cytomegalovirus. In: Textbook of Human Virology. Edited by: Mosby-Year Book inc. 1991;2:889-924. 8. Pescovitz MD. Is low-dose valganciclovir the same as appropriate-dose valganciclovir? Transplantation. 2007;84:126. 9. Potena L, Holweg CT, Vana ML, et al. Frequent occult infection with Cytomegalovirus in cardiac transplant recipients despite antiviral prophylaxis. J Clin Microbiol. 2007;45:1804-10. 10. Rowshani AT, Bemelman FJ, van Leeuwen EM, van Lier RA, ten Berge IJ. Clinical and immunologic aspects of cytomegalovirus infection in solid organ transplant recipients. Transplantation. 2005;79:381-6. 11. Steininger C. Clinical relevance of cytomegalovirus infection in patients with disorders of the immune system. Clin Microbiol Infect. 2007;13:953-63. 12. Fishman JA. Infection in solid-organ transplant recipients. N Engl J Med. 2007;357:2601-14. 13. Yust I, Fox Z, Burke M, et al. Retinal and extraocular cytomegalovirus end-organ disease in HIV-infected patients in Europe: a EuroSIDA study, 1994-2001. Eur J Clin Microbiol Infect Dis. 2004;23:550-9. 14. Kas-Deelen AM, de Maar EF, Harmsen MC, Driessen C, van Son WJ, The TH. Uninfected and cytomegalic endothelial cells in blood during cytomegalovirus infection: effect of acute rejection. J Infect Dis. 2000;181:721-4. 15. Grefte A, van der Giessen M, van Son W, The TH. Circulating cytomegalovirus (CMV)-infected endothelial cells in patients with an active CMV infection. J Infect Dis. 1993;167: 270-7. 16. Sagedal S, Hartmann A, Nordal KP, et al. Impact of early cytomegalovirus infection and disease on long-term recipient and kidney graft survival. Kidney Int. 2004;66:329-37. *This is the first paper raising the hypothesis of adverse outcome caused by subclinical CMV infection. 17. Grattan M, Moreno-Cabral C, Starnes V, Oyer P, Stinson E, Shumway N. Cytomegalovirus infection is associated with cardiac allograft rejection and atherosclerosis. JAMA. 1989;261:3561-6. 18. Loebe M, Schuler S, Zais O, Warnecke H, Fleck E, Hetzer R. Role of cytomegalovirus infection in the development of coronary artery disease in the transplanted heart. J Heart Lung Transplant. 1990;9:707-11. 19. Mc Donald K, Rector S, Braunlin E, Kubo S, Olivari M. Association of coronary artery disease in cardiac transplant recipients with cytomegalovirus infection. Am J Cardiol. 1989;64:359-62. 20. Koskinen P, Lemstrom K, Bruggeman C, Lautenschlager I, Hayry P. Acute cytomegalovirus infection induces a subendothelial inflammation (endothelialitis) in the allograft vascular wall. Am J Pathol. 1994;144:41-50. 21. Merigan T, Renlund D, Keay S, et al. A controlled trial of ganciclovir to prevent cytomegalovirus disease after heart transplantation. N Engl J Med. 1992;326:1182-6. 22. Lemstrom K, Sihvola R, Bruggeman C, Hayry P, Koskinen P. Cytomegalovirus infection-enhanced cardiac allograft































vasculopathy is abolished by DHPG prophylaxis in the rat. Circulation. 1997;95:2614-16. 23. Valantine H, Gao S, Menon S, et al. Impact of prophylactic immediate posttransplant ganciclovir on development of transplant atherosclerosis. A post hoc analysis of a randomized, placebo-controlled study. Circulation. 1999;100:61-6. 24. Gerna G, Percivalle E, Baldanti F, et al. Diagnostic significance and clinical impact of quantitative assays for diagnosis of human cytomegalovirus infection/disease in immunocompromised patients. New Microbiol. 1998;21:293-308. 25. Boeckh M, Huang M, Ferrenberg J, et al. Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR. J Clin Microbiol. 2004;42:1142-8. 26. Emery VC, Sabin CA, Cope AV, Gor D, Hassan-Walker AF, Griffiths PD. Application of viral-load kinetics to identify patients who develop cytomegalovirus disease after transplantation. Lancet. 2000;355:2032-6. 27. Sageda S, Nordal KP, Hartmann A, et al. The impact of cytomegalovirus infection and disease on rejection episodes in renal allograft recipients. Am J Transplant. 2002;2:850-6. 28. Preiksaitis JK, Brennan DC, Fishman J, Allen U. Canadian society of transplantation consensus workshop on cytomegalovirus management in solid organ transplantation final report. Am J Transplant. 2005;5:218-27. 29. Potena L, Grigioni F, Magnani G, et al. Prophylaxis versus preemptive anti-cytomegalovirus approach for prevention of allograft vasculopathy in heart transplant recipients. J Heart Lung Transplant. 2009;28:461-7. *This is the first study comparing prophylaxis with preemptive approach in heart transplant recipients towards a graft-related outcome. 30. Potena L, Grigioni F, Ortolani P, et al. Relevance of cytomegalovirus infection and coronary-artery remodeling in the first year after heart transplantation: a prospective three-dimensional intravascular ultrasound study. Transplantation. 2003;75:839-43. 31. Potena L, Holweg C, Chin C, et al. Acute rejection and cardiac allograft vascular disease is reduced by suppression of subclinical cytomegalovirus infection. Transplantation. 2006;82:398-405. *This study shows that suppression of subclinical CMV infection is associated with a slower CAV progression. 32. Thomas LD, Milstone AP, Miller GG, Loyd JE, Stephen Dummer J. Long-term outcomes of cytomegalovirus infection and disease after lung or heart-lung transplantation with a delayed ganciclovir regimen. Clin Transplant. 2009; 23:476-83. 33. Compton T, Kurt-Jones EA, Boehme KW, et al. Human cytomegalovirus activates inflammatory cytokine responses via CD14 and Toll-like receptor 2. J Virol. 2003;77:4588-96. 34. Stassen FR, Vega-Cordova X, Vliegen I, Bruggeman CA. Immune activation following cytomegalovirus infection: more important than direct viral effects in cardiovascular disease? J Clin Virol. 2006;35:349-53. 35. Kowalik T, Wing B, Haskill J, Azizkhan J, Baldwin A, Huang E. Multiple mechanisms are implicated in the regulation of NFκB activity during human cytomegalovirus infection. Proc Natl Acad Sci USA. 1992;90:1107-11. 36. Laegreid A, Medvedev A, Nonstad U, et al. Tumor necrosis factor receptor p75 mediates cell-specific activation of nuclear factor kappa B and induction of human cytomegalovirus enhancer. J Biol Chem. 1994;269:7785-91. 37. Cook CH, Bickerstaff AA, Wang JJ, et al. Disruption of murine cardiac allograft acceptance by latent cytomegalovirus. Am J Transplant. 2009;9:42-53. *This paper shows an elegant experimental demonstration of a possible mechanism implicated in CMV-dependent graft injury. 38. Potena L, Frascaroli G, Grigioni F, et al. Hydroxymethylglutaryl coenzyme a reductase inhibition limits cytomegalovirus infection in human endothelial cells. Circulation. 2004;109:532-6. 39. Sedmak DD, Knight DA, Vook NC, Waldman JW. Divergent patterns of ELAM-1, ICAM-1, and VCAM-1 expression on cytomegalovirus-infected endothelial cells. Transplantation. 1994;58:1379-85. 40. Knight DA, Waldman WJ, Sedmak DD. Cytomegalovirusmediated modulation of adhesion molecule expression by human arterial and microvascular endothelial cells. Transplantation. 1999;68:1814-18.

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patients receiving solid organ transplantation. Am J Transplant. 2006;6:2356-64. 60. Nickel P, Bold G, Presber F, et al. High levels of CMV-IE-1specific memory T cells are associated with less alloimmunity and improved renal allograft function. Transpl Immunol. 2009;20:238-42. 61. Emery VC. Prophylaxis for CMV should not now replace pre-emptive therapy in solid organ transplantation. Rev Med Virol. 2001;11:83-6. 62. Hodson EM, Jones CA, Webster AC, et al. Antiviral medications to prevent cytomegalovirus disease and early death in recipients of solid-organ transplants: a systematic review of randomised controlled trials. Lancet. 2005;365:2105-15. 63. Strippoli GF, Hodson EM, Jones C, Craig JC. Preemptive treatment for cytomegalovirus viremia to prevent cytomegalovirus disease in solid organ transplant recipients. Transplantation. 2006;81:139-45. 64. Kliem V, Fricke L, Wollbrink T, Burg M, Radermacher J, Rohde F. Improvement in long-term renal graft survival due to CMV prophylaxis with oral ganciclovir: results of a randomized clinical trial. Am J Transplant. 2008;8:975-83. 65. Reischig T, Jindra P, Hes O, Svecova M, Klaboch J, Treska V. Valacyclovir prophylaxis versus preemptive valganciclovir therapy to prevent cytomegalovirus disease after renal transplantation. Am J Transplant. 2008;8:69-77. 66. Potena L, Holweg CT, Chin C, et al. Acute rejection and cardiac allograft vascular disease is reduced by suppression of subclinical cytomegalovirus infection. Transplantation. 2006;82:398-405. 67. Hill JA, Hummel M, Starling RC, et al. A lower incidence of cytomegalovirus infection in de novo heart transplant recipients randomized to everolimus. Transplantation. 2007;84: 1436-42. 68. Vigano M, Dengler T, Mattei MF, et al. Lower incidence of cytomegalovirus infection with everolimus versus mycophenolate mofetil in de novo cardiac transplant recipients: a randomized, multicenter study. Transpl Infect Dis 2009 [Epub ahead of print]. 69. Demopoulos L, Polinsky M, Steele G, et al. Reduced risk of cytomegalovirus infection in solid organ transplant recipients treated with sirolimus: a pooled analysis of clinical trials. Transplant Proc. 2008;40:1407-10. 70. Haririan A, Morawski K, West MS, et al. Sirolimus exposure during the early post-transplant period reduces the risk of CMV infection relative to tacrolimus in renal allograft recipients. Clin Transplant. 2007;21:466-71. 71. Eisen HJ, Tuzcu EM, Dorent R, et al. Everolimus for the prevention of allograft rejection and vasculopathy in cardiactransplant recipients. N Engl J Med. 2003;349:847-58. 72. Keogh A, Richardson M, Ruygrok P, et al. Sirolimus in de novo heart transplant recipients reduces acute rejection and prevents coronary artery disease at 2 years: a randomized clinical trial. Circulation. 2004;110:2694-700. 73. Potena L, D’Agostino C, Abate D, et al. Interaction of CMV prophylaxis and preemptive strategies with immunosuppressive therapy: potential antiviral effect of everolimus. J Heart Lung Transplant. 2010;29:S155-6. 74. Valantine H, Luikart H, Doyle R, et al. Impact of cytomegalovirus hyperimmune globulin on outcome after cardiothoracic transplantation. Transplantation. 2001;72:1647-52. 75. Ruttmann E, Geltner C, Bucher B, et al. Combined CMV prophylaxis improves outcome and reduces the risk for bronchiolitis obliterans syndrome (BOS) after lung transplantation. Transplantation. 2006;81:1415-20. 76. Toyoda M, Petrosyan A, Pao A, Jordan SC. Immunomodulatory effects of combination of pooled human gammaglobulin and rapamycin on cell proliferation and apoptosis in the mixed lymphocyte reaction. Transplantation. 2004;78: 1134-8. 77. Jordan SC, Vo A, Bunnapradist S, et al. Intravenous immune globulin treatment inhibits crossmatch positivity and allows for successful transplantation of incompatible organs in living-donor and cadaver recipients. Transplantation. 2003;76: 631-6.

Trends in Transplant. 2010;4:117-28 Lydia Barrera Pulido, et al.: Calcineurin Inhibitor-Free Maintenance with MMF

Calcineurin Inhibitor-Free Maintenance Therapy After Liver Transplantation I: Mycophenolate Mofetil and Renal Function Lydia Barrera-Pulido, José María Álamo-Martínez, Miguel Ángel Gómez-Bravo, Carmen Bernal-Bellido, Luis Miguel Marín-Gómez, Gonzalo Suárez-Artacho, Juan Serrano-Díez Canedo and Francisco Javier Padillo-Ruiz Hepatobiliopancreatic Surgery and Liver Transplant Unit, Virgen del Rocio University Hospital, Seville, Spain

Abstract It has been widely reported that continued therapy with calcineurin inhibitors can cause an up to fourfold increase in morbidity and mortality in long-term liver transplant patients due to the development of chronic renal failure as well as neurotoxicity, arterial hypertension, hyperglycemia, hyperlipidemia, and increased risk of de novo tumors. These side effects have led to the development of other treatment options that allow these drugs to be minimized or withdrawn. Mycophenolate mofetil is one of the immunosuppressive drugs that has made it possible to discontinue calcineurin inhibitors in liver transplantation. Its side effects are mainly related to the gastrointestinal tract and bone marrow. Furthermore, it lacks nephrotoxic, metabolic, and neurological effects. In the last decade numerous papers have been published, based on the study of liver transplant patients treated with mycophenolate mofetil monotherapy in different countries. They analyzed the safety and efficacy of this therapy, focusing primarily on its effect on chronic renal failure, metabolic complications, and incidence of graft rejection. After reviewing all these works we know that mycophenolate mofetil therapy reduces calcineurin inhibitor-induced renal damage by allowing minimization of the doses of these drugs and their subsequent withdrawal. It has been widely shown that the switch to mycophenolate mofetil monotherapy improves and maintains stable serum creatinine and creatinine clearance values as well as improving hypertension and hyperlipidemia in the long term. On the other hand, most studies found that the improvement in the clinical variables analyzed occurred in the first three months after conversion, so it is clear that a large part of the renal damage and other side effects are induced by the calcineurin inhibitors because it is in that period when the largest reduction is made in the dose of these drugs until their complete withdrawal.

Lydia Barrera Pulido Unidad de Cirugía Hepato Bilio Pancreática y Trasplantes Hospital Universitario Virgen del Rocío Av. Manuel Siurot s/n, 41013 Sevilla, España E-mail: [email protected]

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However, these variables continue to improve after withdrawal so we should consider that the long-term effect of mycophenolate mofetil monotherapy is beneficial. The disparity in the incidence of rejection in the different studies presented should be highlighted, but nevertheless, they all have in common the fact that rejections occurred in the majority of cases in the first three months after the start of conversion. The side effects of mycophenolate mofetil, such as gastrointestinal complications and hematological problems, were reversed in most cases simply by a temporary reduction in the drug dose, so we can consider that the benefits outweigh the risks in this regard. Based on all the studies analyzed, we can infer that the ideal patient for long-term withdrawal of calcineurin inhibitors is a patient who clearly has calcineurin inhibitor-induced chronic renal failure and is not on dialysis, who has not suffered severe acute rejection episodes in the last year, and who has not shown intolerance to mycophenolate mofetil previously. (Trends in Transplant. 2010;4:117-28) Corresponding author: Lydia Barrera Pulido, [email protected]

Key words Calcineurin inhibitor. Chronic renal failure. Mycophenolate mofetil. Monotherapy. Acute rejection.

Introduction

is multifactorial, in over 70% of cases renal damage is directly related to the CNI dose.

In 1980, a new class of immunosuppressive agents called calcineurin inhibitors (CNI) was developed. This allowed the safety of the immunosuppressive regimens used in liver transplantation (LTx) to be improved, since the use of these CNI provided a considerable reduction in the risk of suffering rejection and also increased short-term survival1. As a result, CNI, either tacrolimus or cyclosporine, are a key element in all baseline immunosuppression therapies.

It is also known that hemodialysis and even renal transplantation is required in nearly 10% of patients with end-stage renal disease; this was analyzed in detail in a study of 834 patients with 13 years of post-LTx follow-up5.

However, it has been widely reported that continued use of these drugs can cause up to a fourfold increase in morbidity and mortality in long-term liver transplant patients due to the development of chronic renal failure (CRF), as well as neurotoxicity, arterial hypertension, hyperglycemia, hyperlipidemia and increased risk of de novo tumors2-4. The incidence of CRF at five years posttransplantation is high, and although its origin 118

The nephrotoxic impact, among others, caused by these CNI has led to the development of other treatment options that allow these drugs to be minimized or withdrawn, mainly in the maintenance phase, and thus reduce the incidence and prevalence of CRF. One of the immunosuppressive drugs that has made it possible to discontinue these CNI in LTx is mycophenolate mofetil (MMF). This is a semi-synthetic ester of mycophenolic acid, which acts as a potent inhibitor of the proliferation of B and T lymphocytes6. Its side effects are mainly related to the gastrointestinal tract (diarrhea, nausea, abdominal pain, etc.)

Lydia Barrera Pulido, et al.: Calcineurin Inhibitor-Free Maintenance with MMF

and bone marrow (leukopenia, thrombopenia, and anemia). Furthermore, it lacks nephrotoxic, metabolic, and neurological effects. The first steps towards MMF monotherapy were made in studies where MMF was introduced due to CNI-induced renal toxicity, with the consequent reduction in the doses of these CNI7-9. Pfitzmann, et al. conducted a study in a series of 101 patients receiving both tacrolimus and cyclosporine as CNI, who developed CNIinduced CRF and were treated by reducing the dose of CNI and adding MMF to treatment. The results obtained were a reduction in serum creatinine (SCr 0.4 mg/dl; p < 0.001) after a mean follow-up of 40 months. Of these 101 patients, 56 also had graft dysfunction, and it was found that they also showed improvements versus baseline in bilirubin (p < 0.019) and alkaline phosphatase (p < 0.002) from 2.9 ± 0.8 to 1.3 ± 0.3 mg/dl and from 321 ± 41 to 208 ± 18 UI/l, respectively. It should be noted that there were two patient deaths from sepsis and renal dysfunction and that MMF therapy was associated with a high rate of side effects (37 patients): gastrointestinal (n = 26), bone marrow toxicity (n = 9), and infections (n = 2). However, the rate of acute rejection did not increase with respect to standard full-dose CNI therapy7. On the other hand, Cantarovich, et al. analyzed 19 LTx patients receiving cyclosporine who developed posttransplant renal dysfunction induced by this drug. They reported a clear improvement in renal function evaluation parameters with the introduction of MMF and reduction in cyclosporine dose, since mean creatinine clearance (CrCl) increased by 18 ml/min (p < 0.02) and mean glomerular filtration rate by 24 ml/min (p = 0.002); in addition, 71% of patients who were receiving antihypertensive therapy were able to discontinue it. However, the rate of

acute rejection as a result of the treatment change was high (29%)8. A third study on CNI dose reduction for CRF by the introduction of MMF was published by Beckebaum, et al.9. It was randomized study (2:1) in which all patients were diagnosed with CRF and it compared the changes in different clinical variables (mainly related to renal function) between the control group, which continued with normal doses of CNI monotherapy, versus the case group in which MMF was introduced and CNI doses were minimized. After three months of follow-up, significant improvements were observed in the group receiving low-dose CNI but not in the group receiving standard therapy. Mean values of SCr decreased from 1.88 ± 0.36 to 1.58 ± 0.33 mg/dl (p < 0.001) and CrCl increased from 51.4 ± 10.8 to 61.6 ± 14.1 ml/min (p < 0.001). The authors also suggested that despite the fact that the mean time from LTx to the start of treatment was 5.6 ± 3.6 years, CNI-induced renal damage appeared to be partially reversible. They also found that the group of patients with low-dose CNI and MMF improved their lipid profile and blood pressures at three months, and more importantly, they found that transaminases were significantly reduced. The efficacy of MMF therapy in improving CRF and its safety on liver graft function was thus demonstrated in patients with CNI-induced toxicity.

Mycophenolate mofetil monotherapy Since numerous studies have shown that use of MMF allows CNI doses to be minimized 119

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safely and effectively in LTx, the next step to achieve CNI-free therapies would be to consider immunosuppressive regimens without them7-9. A long-term treatment option could be MMF monotherapy, as this would largely avoid the side effects of CNI, mainly chronic renal disease, arterial hypertension, hyperlipidemia, hyperglycemia, and de novo tumors. In the last decade, a considerable number of studies have been generated on this topic, where it can be seen that there are some authors with results favorable to MMF monotherapy as a safe, long-term treatment in LTx, while others found that the risk was greater than the benefit (Table 1). One of the first published studies on this problem was that of Herrero, et al., who attempted conversion to MMF monotherapy in a group of 11 patients with CRF (SCr > 1.5 mg/dl), stable liver function, and no episodes of acute rejection within one year before the treatment change. All patients were started on full doses of MMF (2 g/day), simultaneously slowly reducing the dose of cyclosporine. After a mean time of 15 months, seven patients had achieved CNI-free therapy with MMF, with SCr decreasing from 2.22 ± 0.13 mg/dl at baseline to 1.90 ± 0.19 mg/dl and CrCl increasing from 38.16 ± 5.60 to 47.01 ± 6.76 ml/min (p = 0.005). In addition, these patients experienced an improvement in control of arterial hypertension, with a reduction in the number of antihypertensive drugs, as only two of seven patients required antihypertensive treatment at the end of follow-up. The side effects observed were those expected for MMF, and in six patients the dose had to be reduced due to mild anemia. Complete conversion to MMF was not achieved in four of 11 patients as two patients were switched to tacrolimus due to acute re120

jection (18%) and another two continued with low-dose cyclosporine. The results seemed quite promising since the patients considerably improved renal function and tolerance of MMF was good. The incidence of rejection was also acceptable since it was easily reversed and no graft loss or patient death occurred10. However, several years later two studies were published with the same objectives of conversion to MMF monotherapy to minimize CRF from CNI, with very unpromising results since despite improving renal function the incidence of acute rejection increased alarmingly11,12. The first study was conducted by Stewart, et al. and consisted of a case-control study. The study enrolled patients with CRF who in some cases also had associated arterial hypertension. The initially estimated number of patients was 18, of which nine would be the control group (treated with azathioprine and CNI) and the case group would be composed of another nine patients treated with MMF monotherapy with slow CNI tapering. However, when five patients had already been enrolled, the study had to be discontinued because of the high rate of severe organ rejection (two chronic rejections and one acute rejection). The consequence was that two of the five patients had to be retransplanted soon (mean 4 months) after starting the study and one patient received steroid therapy for acute rejection. Therefore, the risk posed by this treatment was unacceptable, independently of whether it improved renal function parameters11. Schlitt, et al. obtained similar results to the previous group. They designed a casecontrol study with 28 patients; 14 patients continued with standard CNI therapy and the other 14 patients in the case group were con-

Lydia Barrera Pulido, et al.: Calcineurin Inhibitor-Free Maintenance with MMF

verted to MMF monotherapy. Eight patients from the latter group continued with steroids combined with MMF, so only six were treated with MMF monotherapy. Of these, three patients suffered recurrence of hepatitis C virus accompanied by mild rejection, one patient stopped treatment due to intolerable diarrhea, and the two remaining patients also developed moderate acute cellular rejection12. This discrepancy of results between the different groups led to the generation of more controlled studies with larger numbers of patients in which it was attempted to analyze long-term MMF monotherapy, taking special care due to the high risk of rejection and consequent organ loss. Fortunately, the studies that were published in successive years presented results of series with rejection incidence rates not superior, in the majority of cases, to 10-15%13-15,17-20. In 2003, the data from Raimondo, et al. were published. They conducted a study with 45 patients, all with CRF associated with CNI; one of the treatment arms was formed by patients on MMF monotherapy (n = 16) in doses of 2 g/day. The mean follow-up period was 24 months, SCr values improved in five of eight patients who completed the two years of treatment from 1.79 mg/dl (1.20-3.36) at baseline to 1.22 mg/dl (97-2.15) at the end of the study13. Four patients died from causes not directly related to immunosuppression with MMF monotherapy. Only one case of acute rejection (6%) was diagnosed in the 16 patients included, and interestingly, it was the only patient who had had rejection prior to inclusion in the study. The authors concluded that the presence of rejection episodes could be a risk factor to be considered when deciding on monotherapy. This may be what occurred in the studies by Stewart and Schlitt, as it is not defined

if the patients randomized in their studies had previously suffered any episode of acute rejection. Perhaps this, among other factors, explains their high rate of rejection. Therefore, the fact that MMF monotherapy is clearly beneficial in improving renal function in patients who only have CNI nephrotoxicity is unquestionable, even if therapy is started several years after LTx, because although in many cases normalized values of SCr or CrCl are not achieved, an improvement in renal function is obtained. One year later, three new studies were published that help to improve our learning about CNI-free therapy with MMF monotherapy through the experience of different centers performing LTx14-16. The first study carried out by Koch, et al. included 32 patients with CRF who were split into two groups according to time since LTx. Thus, one group was formed by patients less than six months posttransplantation (n = 14) and the other by patients who were transplanted more than six months previously (n = 18)14. In 88% of patients, there was a significant reduction in SCr values from 2.63 ± 0.39 to 1.74 ± 0.34 mg/dl. Furthermore, a higher proportion of patients normalized SCr values in the group with early MMF conversion: 64% versus 22% in the second group. As a negative point, it should be noted that three patients had to be entered in hemodialysis, but it should be clarified that none of them had diabetic nephropathy. As in previous studies, the rejection rate was minimal at 6% (2/32 patients), and may have been because patients with previous episodes of severe rejection were not excluded. Special mention should be made of the fact that five patients died in this study: two from cardiovascular problems, one from de novo 121

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Table 1. Studies about treatment in monotherapy with MMF in LTx with CRF induced by CNIs Author

Year of Type of study publication, city

Number of patients

Herrero, et al.10

1999, Pamplona

Prospective

11

Stewart, et al.11

2001, Newcastle

Prospective randomized case-control

5

Schlitt, et al.12

2001, Hannover

Prospective randomized case-control

14

Raimondo, et al.13

2003, London

Retrospective

Moreno-Planas, et al.15

2004, Madrid

Koch, et al.14

Follow-up from LTx to conversion (months)

Indication for conversion

Type of CNI

CRF

CsA

2

CRF Hypertension

CsA

2

76

CRF

CsA/Tac

2

16

45

CRF

CsA/Tac

2

Prospective

50

81

CRF Hypertension

CsA/Tac

2

2004, Innsbruck

Prospective

32

25.6

CRF

CsA/Tac

2

Fairbanks and Thuluvath16

2004, Baltimore

Retrospective

13

69

CRF Histoplasmosis

CsA/Tac

Not specified

Pierini, et al.17

2005, Turin

Retrospective

32

50

CRF De novo tumor

CsA/Tac

1.5

Orlando, et al.18

2007, Rome

Prospective

42

70.5

CRF CsA/Tac Hypertension Hyperlipidemia Hyperuricemia Gingival hyperplasia

1.5

Barrera-Pulido, et al.20

2008, Seville

Prospective

31

87

CRF

CsA/Tac

2

Ko, et al.19

2008, Vancouver

Retrospective

15

135

CRF

CsA/Tac

2

Kamphues, et al.21

2009, Berlin

Retrospective

123

91

CRF

CsA/Tac

2

32

Not specified

MMF daily dose (grams)

LTx: liver transplantation; CNI: calcineurin inhibitor; MMF: mycophenolate mofetil; CRF: chronic renal failure; CsA: cyclosporin A; Tac: tacrolimus; HCV: hepatitis C virus; HCC: hepatocellular carcinoma.

122

Lydia Barrera Pulido, et al.: Calcineurin Inhibitor-Free Maintenance with MMF

Patients on MMF monotherapy at the end of the study

Acute rejection

Follow-up time (months)

Adverse effects of MMF

Patients who developed intolerance to MMF

Improvement of renal function

Improvement of arterial hypertension

Death

6/11 (55%)

2/11 (18%)

15

6/11 (55%)

0/11 (0%)

10/11 (91%)

6/7 (86%)

0/11 (0%)

2/5 (40%)

3/5(60%)

0

–

–

–

–

0/5 (0%)

6/14 (42.8%)

5/14 (36%)

6

8/14 (57%)

0/14 (0%)

11/14 (79%)

14/14 (100%)

0/14 (0%)

8/16 (50%)

1/16 (6%)

33

2/16 (13%)

0/16 (0%)

5/8 (63%)

–

4/16 (25%): 3 recurrences, 1 alcoholic, 1 HCV and 1 HCC. 1 de novo tumor

39/50 (78%)

5/50 (10%)

18

26/50 (52%)

3/50 (6%)

32/40 (80%)

24/32 (75%)

2/50 (4%): alcoholic recurrence

9/32 (28%)

2/32 (6%)

57

17/32 (53%)

0/32 (0%)

8/9 (88%)

–

5/32 (16%): 2 cardiovascular problem, 1 de novo neoplasm, 1 tumor recurrence, 1 sepsis

11/13 (85%)

3/13 (23%)

22

Minimal

0/13 (0%)

No significant improvement

–

3/13 (23%): 2 severe liver failure due to alcoholic recurrence, 1 HCV recurrence

32/32 (100%)

1/32 (3%)

17.9

9/32 (28%)

0/32 (0%)

Significant improvement (% not specified)

–

0/32 (0%)

41/42 (98%)

9/42 (21%)

24

7/42 (17%)

0/42 (0%)

31/36 (89%)

4/5 (80%)

0/42 (0%)

31/31 (100%)

0/31 (0%)

12

5/31 (16%)

0/31 (0%)

21/31 (67.7%)

–

0/31 (0%)

12/15 (80%)

1/15 (7%)

10

5/15 (33%)

3/15 (20%)

13/15 (87%)

–

0/15 (0%)

123/123 (100%)

0/123 (0%)

12

Minimal

0/123 (0%)

Significant improvement (% not specified)

–

0/123 (0%)

123

Trends in Transplantation 2010;4

pancreatic neoplasm, one from recurrence of cholangiocarcinoma, and one from sepsis due to cholangitis. The second study, published in 2004 with optimum results in terms of rejection with MMF monotherapy, was that of Moreno, et al.15. Fifty patients were converted to this treatment because of CNI-associated toxicity: 45 had CRF (in 11 associated with arterial hypertension) and five had hypertension as the only complication.

These data led to the use of MMF monotherapy being questioned again as this study attributed a 19% risk of death to treatment with MMF alone. The results lead us to think that special care should be taken when selecting patients and the time of conversion to be sure that the benefit outweighs the risk associated with the use of this therapy.

At 18 months, 78% of patients were no longer receiving CNI as immunosuppressive treatment. The SCr values decreased from 1.81 to 1.49 mg/dl (p < 0.0001), CrCl increased from 44.7 to 55.1 ml/min (p < 0.0001); therefore, 80% of patients achieved an improvement in renal function.

Fortunately, in the previous year the results of the study by Italian group from Turin were published in which they retrospectively analyzed their experience with MMF monotherapy17.

An acute rejection rate seen was 10% (five patients). Side effects occurred in 52% of patients and consisted mainly of asthenia, diarrhea, and viral infections.

Conversion to MMF was at a median of 50 months post-LTx in 32 patients (for CRF in 30 and de novo tumors in two), and over 90% were receiving cyclosporine as the CNI. Unlike the regimens of the other centers, the mean dose of MMF administered was 1.5 g/day.

In conclusion, this study reinforces the idea that MMF monotherapy late after LTx is well tolerated and safe and clearly improves CNI-induced CRF and hypertension. In contrast to the two previous studies is a third retrospective study published in the same year and including 13 patients with CRF16. The results obtained for the incidence of rejection in this series were rather more dangerous at 28% (three of the 13 patients included). In addition to these three patients, two died due to rejection and another had to be re-transplanted. With regard to renal function, even though conversion to MMF was indicated for CRF, MMF therapy was not effective in some cases as four patients required dialysis. However, in those not requiring dialysis, SCr values were decreased from 2.51 ± 1.12 to 1.85 124

± 0.58 mg/dl (p = 0.01), as has been widely reported in the studies we are analyzing.

Once more, the positive effect of MMF monotherapy on renal function was confirmed, with baseline SCr values decreasing from 2.02 to 1.7 mg/dl (p = 0.0001). The rejection rate was also minimal as only one case was diagnosed among the 32 patients (3%). Obviously, the treatment change was not free of side effects, as was also reported by other authors, such as diarrhea (12.5%) and leukopenia (15.6%). Two years later, Orlando, et al. published their experience with 42 patients18. In this case, they attempted to optimize MMF monotherapy in order to avoid the high incidence of MMF-related side effects. Therefore, they converted all patients to MMF therapy at initial doses of 1.5 g/day instead of 2 g/day (standard therapy).

Lydia Barrera Pulido, et al.: Calcineurin Inhibitor-Free Maintenance with MMF

Another novel feature of this study is the expansion of the indications for monotherapy: only for CRF (n = 22), CRF associated with hyperlipidemia (n = 10), hypercholesterolemia (n = 4), CRF associated with hyperlipidemia and hypertension (n = 2), hypercholesterolemia associated with hypertension (n = 1) and gingival hyperplasia (n = 1). Calcineurin inhibitors were reduced by 25% monthly until permanent withdrawal (mean of 4.5 months). Of the 35 patients included for CRF, 31 improved their renal function at one year, as SCr decreased from 1.8 ± 0.4 to 1.56 ± 0.4 mg/dl and CrCl increased from 47.8 ± 10.4 to 57.6 ± 17 ml/min (p < 0.05). They also obtained considerable improvements in patients converted for hyperlipidemia, as triglycerides decreased in 14 of 17 patients (82%) and cholesterol in 12 of 13 patients (92%) at one year and the reductions were maintained at two years of follow-up. In addition, three of the five patients who were being treated with statins were able to discontinue this treatment. Conversion also allowed blood pressure to be controlled and improved (80%), as two of the four patients who were receiving antihypertensive treatment were able to discontinue it at five and seven months after conversion to MMF. However, it should be noted that there was a high incidence of suspected rejection episodes, all within the first six months after conversion, since they occurred in nine of the 42 patients studied (21%). In any case, the authors state that this does not represent an important clinical problem as no graft loss or untreatable rejection occurred. In fact, all rejections were reversed by increasing the MMF dose to 2 g/day and/or optimizing the CNI dose. A very positive finding of this study was the considerable reduction in side effects

related to MMF. Only seven of 42 patients (16%) experienced any side effect: nausea and vomiting in two patients, asthenia in two, leuko-thrombopenia in three, and herpes zoster skin infection in one patient. It should be stressed that no case required treatment discontinuation. This article demonstrated the efficacy of MMF monotherapy in doses of 1.5 g/day to improve renal function, dyslipidemia, and hypertension as well as its relative safety. However, the authors stress that it was at three months from the start of conversion when a frank improvement was observed in most patients, that is when CNI had been reduced by 75%. Therefore, they propose the idea that perhaps it is not necessary to completely withdraw the CNI, but rather to reduce them to a minimum and simultaneously administer MMF in doses of 1.5 g/day, with the consequent reduction in undesirable effects. Subsequently, in 2008, the experiences of another two centers were published who treated their long-term liver transplant patients with CNI-free therapies based on MMF19,20. The first study conducted by Ko, et al. in Vancouver has a clear limitation because the sample size is small (18 patients) and the median follow-up time is very short, in addition to being done retrospectively19. No dialysis patients were included who had suffered a rejection episode in the year previous to conversion, nor patients who had CRF induced by any other cause than CNI toxicity. Nevertheless, the results obtained provide quite a lot of information since they analyzed the effect of conversion to MMF monotherapy at three and six months post conversion and, like other authors, concluded that a significant improvement occurred in the different clinical variables during the 125

Trends in Transplantation 2010;4

first three months, with no differences between the third and sixth month. Median SCr values at baseline were 1.44 mg/dl: 1.29 mg/dl at three months (p = 0.001) and 1.39 mg/dl at six months (p = 0.008). Side effects were those usually seen with MMF. Three patients experienced gastrointestinal intolerance (one had to discontinue MMF), one had anemia (also discontinued MMF), and one had atrial fibrillation (despite being unrelated to MMF, it was discontinued as a precaution). In terms of graft function, only one patient experienced elevated liver enzymes, which was considered acute rejection (6.7%), although biopsy was not performed, and was treated by adding sirolimus to immunosuppressive treatment. The second published series was carried out prospectively at our institution, Virgen del Rocio University Hospital in Seville20. Like the other groups, we made the switch to MMF monotherapy in patients with CNI-induced CRF, slowly reducing the CNI dose by 25% every 2-3 months up to complete withdrawal. Unlike the experiences of other authors, our patients were not on CNI monotherapy and subsequently switched to MMF, but were already receiving this dual therapy previously. Like the previous authors, we excluded from the study patients who were on dialysis, patients with CRF not induced by CNI, patients with chronic rejection or any episode of acute rejection in the last year, and finally we excluded patients who were receiving dual immunosuppressive therapy (CNI plus MMF) and who had shown intolerance to MMF in full doses (2 g/day). The mean time from LTx to monotherapy was 87 months (range 14-186 months) and the minimum follow-up time post conversion was 12 months. 126

The different clinical variables analyzed improved significantly between three and six months posttransplantation and remained stable at 12 months. Thus, mean SCr values were reduced from 1.63 ± 0.47 mg/dl at baseline to 1.49 ± 0.33 mg/dl at six months (p < 0.05). No significant side effects were recorded, although we had to change the dose of MMF in three cases due to gastrointestinal disturbances and reduce the dose in two patients because of mild leukopenia. With regard to graft function, there was no case of graft loss or rejection. Therefore, we also concluded that this therapy based on MMF is effective and safe provided that patients are carefully selected and closely monitored. Nevertheless, we must continue longer-term evaluation of these patients because most currently continue on this immunosuppressive therapy. The last study published in 2009 on CNI-free therapy based on MMF was conducted by the group of Kamphues, et al. in Berlin21. It is a retrospective analysis of 123 liver transplant patients in whom MMF monotherapy was carried out effectively for CNI-induced CRF. They only included patients who completed conversion to MMF and did not suffer acute rejection episodes in the first three months after conversion from CNI to MMF. They present and analyze the experience of other groups in terms of the incidence of rejection and the results they obtained showed that most rejections occur at three months after the switch to monotherapy11,12,18; therefore, if they eliminate this group of patients from the start they can evaluate the real effect of treatment with MMF alone. They also included another novelty with respect to previous studies, since in 59 of the 123 patients they performed biopsies before

Lydia Barrera Pulido, et al.: Calcineurin Inhibitor-Free Maintenance with MMF

and after conversion (although not at a specific time pre- and post-LTx) to evaluate the histopathological changes that might be caused by the drug in the organ, including acute rejection, chronic rejection, fibrosis, steatosis, etc. The results obtained were very positive as no episode of chronic or acute rejection was recorded in 12 months of follow-up post conversion. Fibrosis was observed in eight of 59 patients (13%), a lower grade of fibrosis was detected in 14 patients (24%), and fibrosis remained stable versus the pre conversion MMF biopsy in 37 patients (63%). On the other hand, an increase in liver fat content was detected in 24 of the 59 patients (41%). In addition, mean fat content of all patients analyzed by biopsy (n = 59) was significantly increased from 9.8 ± 15.9% before conversion to MMF monotherapy to 16.1 ± 21.0% after conversion (p < 0.05). The authors were unable to explain this pathophysiological effect of increased fat in the liver, and were also unable to compare their experience with that of other groups because this was the first study in which biopsy was done before and after the start of treatment. As this effect of MMF on liver tissue has not been reported by other authors, it would be of great utility to design a prospective study with protocol biopsies before and after conversion to see if the results are repeated in patients from other groups; this would help us to further advance our knowledge on the safety of this CNI-free therapy. In their study, as in the rest of the previously mentioned studies, renal function was significantly improved from baseline SCr values of 1.54 ± 0.59 to 1.47 ± 0.61 mg/dl at 12 months.

Conclusions After reviewing all the above studies, we know that MMF therapy reduces CNI-induced renal damage by allowing minimization of the doses of these drugs and their subsequent withdrawal. It has been widely shown that the switch to MMF monotherapy improves and maintains stable SCr and CrCl values as well as improving hypertension and hyperlipidemia in the long term. On the other hand, most studies found that the improvement in the clinical variables analyzed occurred in the first three months after conversion, so it is clear that a large part of the renal damage and other side effects are induced by the CNI because it is in that period when the largest reduction is made in the dose of these drugs until their complete withdrawal. However, these variables continue to improve after withdrawal so we should consider that the long-term effect of MMF monotherapy is beneficial. The disparity in the incidence of rejection in the different studies presented should be highlighted, but, nevertheless, they all have in common that rejections occurred in the majority of cases in the first three months after the start of conversion. The side effects of MMF, such as gastrointestinal complications and hematological problems, were reversed in most cases simply by a temporary reduction in the drug dose so we can consider that the benefits outweigh the risks in this regard. Therefore, special care should be taken to have an adequate degree of immunosuppression, to analyze well as to when after LTx we should consider the switch to MMF monotherapy and when we should completely withdraw the CNI because we must select very carefully the patients who may benefit from this therapy. 127

Trends in Transplantation 2010;4

Based on all the studies analyzed, we can infer that the ideal patient for long-term withdrawal of CNI is a patient who clearly has CNI-induced CRF and is not on dialysis, who has not suffered severe acute rejection episodes in the last year, and who has not shown intolerance to MMF previously.

References

1. Olyaei AJ, De Mattos AM, Bwnnett WM. Nephrotoxicity of immunosuppressive drugs: new insight and preventive strategies. Curr Opin Crit Care. 2001;7:384. 2. Danovitch GM. Immunosuppressant-induced metabolic toxicities. Transplant Rev. 2000;14:65-81. 3. Monsour HP, Wood RP, Dyer CH, et al: Renal insufficiency and hypertension as long-term complications in liver transplantation. Semin Liver Dis. 1995;15:123. 4. Ojo AO, Held PJ, Port FK, et al. Chronic renal failure after transplantation of a nonrenal organ. N Engl J Med. 2003;349:931. **Interesting paper, with a very large cohort of nonrenal transplant patients, which concludes that the development of chronic renal failure increases the risk of death fourfold. 5. Gonwa TA, Mai ML, Melton LB, et al. End-stage renal disease (ESRD) after orthotopic liver transplantation (OLTX) using calcineurin-based immunotherapy: risk of development and treatment. Transplantation. 2001;72:1934-9. *Role of CNI in the long-term development of end-stage renal disease with required dialysis and renal transplantation. 6. Ascher NL. Immunosuppressant substitutes in liver transplantation. Lancet. 2001;357:571-2. 7. Pfitzmann R, Klupp J, Langrehr JM, et al. Mycophenolate mofetil reduces calcineurin inhibitor-induced side effects after liver transplantation. Transplant Proc. 2002;34: 2936. 8. Cantarovich M, Tzimas GN, Barkun J, Deschenes M, Alpert E, Tchervenkov J. Efficacy of mycophenolate mofetil combined with very low-dose cyclosporine microemulsion in long-term liver-transplant patients with renal dysfunction. Transplantation. 2003;76:98-102. 9. Beckebaum S, Cicinnati VR, Klein CG, et al. Impact of combined mycophenolate mofetil and low-dose calcineurin inhibitor therapy on renal function, cardiovascular risk factors, and graft function in liver transplant patients: preliminary results of an open prospective study. Transplant Proc. 2004;36:2671-4. *Suggests that the renal damage caused by CNI is partially reversible.

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10. Herrero JI, Quiroga J, Sangro B, et al. Conversion of liver transplant recipients on cyclosporine with renal impairment to mycophenolate mofetil. Liver Transpl Surg. 1999; 5:41420. *First study that demonstrates the efficacy and safety of the monotherapy with MMF in LTx patients. 11. Stewart SF, Hudson M, Talbot D, et al. Mycophenolate mofetil monotherapy in liver transplantation. Lancet. 2001;357:609-11. *This paper shows a high risk of rejection using MMF in monotherapy. 12. Schlitt HJ, Barkmann A, Böker KH, et al. Replacement of calcineurin inhibitors with mycophenolate mofetil in livertransplant patients with renal dysfunction: a randomised controlled study. Lancet. 2001;357:587-91. 13. Raimondo ML, Dagher L, Papatheodoridis GV, et al. Longterm mycophenolate mofetil in combination with calcineurin inhibitors for chronic renal dysfunction after liver transplantation. Transplantation. 2003;75:186-90. **Indicates that previous rejection events prior to monotherapy with MMF, increases the risk of other acute rejection. 14. Koch RO, Gaziadei IW, Schulz F, et al. Long-term efficacy and safety of mycophenolate mofetil in liver transplant recipients with calcineurin-induce renal dysfunction. Transplant Int. 2004;17:518-24. *Introduces the concept of early conversion to MMF monotherapy to reverse kidney failure. 15. Moreno JM, Cuervas-Mons V, Rubio E, et al. Mycophenolate mofetil can be used as monotherapy late after liver transplantation. Am J Transplant. 2004;4:1650-5. *Shows the success of monotherapy with MMF in improving CRF and hypertension induced by CNI. 16. Fairbanks KD, Thuluvath PJ. Mycophenolate mofetil monotherapy in liver transplant recipients: a single center experience. Liver Transpl. 2004;10:1189-94. 17. Pierini A, Mirabella S, Brunati A, Ricchiuti A, Franchello A, Salizzoni M. Mycophenolate mofetil monotherapy in liver transplantation. Transpl Proc. 2005;37:2614-15. 18. Orlando G, Baiocchi L, Cardillo A, et al. Switch to 1.5 grams MMF monotherapy for CNI-related toxicity in liver transplantation is safe and improves renal function, dyslipidemia and hypertension. Liver Transpl. 2007;13:46-54. **Extends the indication of the use of the monotherapy of MMF at doses of 1.5 g/day in case of metabolic syndrome. Furthermore, adverse drug effects were substantially reduced. 19. Ko HH, Greanya E, Lee TK, Steinbrecher UP, Erb SR, Yoshida EM. Mycophenolate mofetil in liver transplant patients with calcineurin-inhibitor-induced renal impairment. Ann Hepatol. 2008;7:376-80. 20. Barrera Pulido L, Alamo Martínez JM, Pareja Ciuró F, et al. Efficacy and safety of mycophenolate mofetil monotherapy in liver transplant patients with renal failure induced by calcineurin inhibitors. Transplant Proc. 2008;40:2985-7. 21. Kamphues C, Bova R, Röcken C, et al. Safety of mycophenolate mofetil monotherapy in patients after liver transplantation. Ann Transplant. 2009;14:40-6. *Confirms that most rejections occur three months after the change to monotherapy.

Trends BeatrizinDomínguez-Gil, Transplant. 2010;4:129-37 et al.: Kidney Transplantation from Donors with a Positive Serology for Hepatitis C

Kidney Transplantation from Donors with a Positive Serology for Hepatitis C: The Facts and the Challenges Beatriz Domínguez-Gil 1, Nuria Esforzado 2, Amado Andrés 3, Jose M Campistol 2 and Jose M Morales 3 1 National Transplant Organization, Madrid, Spain; 2Nephrology Service, Clinic i Provincial Hospital, Barcelona, Spain; 3Nephrology Service, 12 de Octubre Hospital, Madrid, Spain

Abstract The use of kidneys from donors with a positive serology for hepatitis C virus into recipients with anti-HCV-positive antibodies seems to be a safe approach in the long term. Results provided by center-based experiences show a favorable outcome of HCV-positive recipients in terms of graft survival, patient survival, and HCV-related liver disease with kidneys transplanted from HCV-positive donors. Registry studies have raised doubts on the safety of this approach, but do not represent a standardized policy. The safety of this policy can be improved by limiting the transplantation of these kidneys to patients with a positive HCV RNA before transplantation and, ideally, by matching donors and recipients according to the HCV genotype. Kidneys from HCV-positive donors are being lost today because of remaining doubts that seem to be reasonably overcome nowadays and by the lack of appropriate recipients. Organizational measures, such as devising preemptive transplantation for HCV RNA-positive recipients accepting to be transplanted with kidneys from HCV-positive donors, and international cooperation seem essential to avoid the loss of these organs at a moment of dramatic organ shortage. (Trends in Transplant. 2010;4:129-37) Corresponding author: Beatriz Domínguez-Gil, [email protected]

Key words Kidney transplantation. Hepatitis C. Interferon. Liver disease. Donors.

Introduction Organ transplantation has become a consolidated therapy which saves the lives or improves the qualities of life of about 100,000 patients worldwide every year1. However, one of

Correspondence to: Beatriz Domínguez-Gil Organización Nacional de Trasplantes C/ Sinesio Delgado 6, pabellón 3 28029 Madrid, España E-mail: [email protected]

the main obstacles that preclude the full development of transplantation is the shortage of organs to satisfy the need. At the end of 2009 there were 63,000 patients in the waiting list for an organ in the European Union, while only about 28,000 transplant procedures were performed during that entire year2. The UNOS registry shows a rather similar dramatic situation for the USA. In November 2010 more than 100,000 patients were registered in the waiting list, but the number of transplant procedures performed annually in that country is about 28,0003. As a consequence of shortage, patients with low survival expectancies 129

Trends in Transplantation 2010;4

might not be included in the lists and many will deteriorate or die while waiting to be transplanted. Added to the unequal distribution of wealth in the world, organ shortage is the root case for unacceptable practices such as organ trafficking and transplant tourism4. Different strategies have been devised to increase organ availability, including the use of organs from expanded criteria donors5 and from non standard risk donors. A hazard of a decreased graft survival is assumed in the first case and a hazard of donor-derived diseases in the second. Since hepatitis C virus (HCV) infection is transmitted through organ transplantation, donors with a positive serology for hepatitis C virus (HCVD+) are included in the latter group6-12. Controversies regarding the safety of transplanting kidneys from HCVD+ have been overcome at least partially in the last years through the evidence provided by center-based experiences. However, some centers do not accept kidneys from HCVD+ for transplantation yet. Moreover, there are countries with technical or legal provisions in place that preclude the transplantation of organs from these donors13. In parallel, progress in the therapeutic approach to endstage renal disease patients with an HCV infection raises doubts about the usefulness of policies for the transplantation of kidneys from HCVD+. This article intends to provide an update on the facts about the use of kidneys from these donors and the related challenges for the coming years.

Transmission of HCV infection through kidney transplantation Soon after the description of HCV in 1989 , several units published their experiences in the transplantation of kidneys from HCV RNA-positive donors6-12,15. The HCV infection was transmitted through kidney transplantation, although the rate of transmission ranged between 1410 and 100%8, depending 14

130

on the series. Moreover, the clinical consequences of the transmission of HCV infection were also variable. Pereira, et al.8 showed that 50% of the patients acquiring HCV infection through kidney transplantation developed criteria of chronic liver disease (CLD), something otherwise infrequent in the experience of the Columbus University12. Variability among the series with regards to the viral load in the transplanted organ, the infectivity of the HCV strain involved, the volume of the preservation solution, the preservation method used, and the diagnostic tests applied might justify these heterogeneous results9,16. These experiences lead to the general consensus that kidneys from HCVD+, regardless of HCV RNA, should not be transplanted into recipients with a negative HCV serology (HCVR–)16. In parallel, the question to be answered was whether these organs could be safely transplanted into HCVR+. There were arguments against this approach: (i) anti-HCV antibodies are not protective and not indicative of a viremic state, and (ii) several HCV genotypes have been described, so superinfection with another HCV genotype could potentially occur17. But there were also strong arguments in favor of this policy: (i) the prevalence of HCV antibodies among organ donors may be high in specific countries or geographical areas, so universally discarding these organs could exacerbate organ shortage; (ii) cardiovascular-related rather than liver-related morbidity and mortality is by far the most frequent after kidney transplantation; and (iii) there is still today a residual risk of discarding a donor with a false positivity for HCV antibodies.

Experiences with the use of kidneys from HCV-positive donors into HCV-positive recipients Based on the abovementioned arguments in favor of their use, in March 1990 two Spanish kidney transplant units initiated a

Beatriz Domínguez-Gil, et al.: Kidney Transplantation from Donors with a Positive Serology for Hepatitis C

pilot experience with the transplantation of kidneys from HCVD+ into HCVR+. First results revealed the short-term safety of this policy. Graft and patient survival of HCVR+ was similar regardless of HCV serology of their donors. A similar percentage of patients in both groups developed biochemical criteria consistent with CLD (ALT levels > 2.5 times the upper normal limit during more than six consecutive months)18,19. Nonetheless, the policy did not prevent the transmission of HCV infection. Retrospectively, HCV RNA was assessed in donors and recipients through the polymerase chain reaction (PCR) technique. Three different situations were described when using kidneys from HCVD+ into HCVR+19. First, when HCV RNA was detectable in both the donor and the recipient, no negative clinical consequences were apparent in the posttransplant period. As expected, also no negative consequences were observed if the donor was HCV RNA negative and the recipient exhibited a positive HCV RNA. The situation to be avoided was when the donor was HCV RNA positive and the recipient HCV RNA negative, a circumstance described in five patients within the series. Four of them became HCV RNA positive after transplantation and two developed CLD, as previously defined. As a result of these findings, in March 1993 both Spanish groups modified their policy of using kidneys from HCVD+ by limiting their use to those patients in the waiting list who exhibited a positive HCV RNA before transplantation. This approach was then nationally adopted with the support of the Spanish National Transplant Organization. Other single-center experiences with the same approach as the Spanish one have later been published (Table 1)20-25. Conclusions are rather similar among these groups: no outstanding differences are observed in HCVR+ who have received a kidney transplant from an HCVD+ compared to those transplanted from an HCVD-, at least in the short term. Moreover, some of these series have demonstrated that

time in the waiting list for HCVR+ is significantly shorter when these patients are transplanted from HCVD+21,22,24. Furthermore, according to these experiences in kidney transplantation, livers from HCVD+ have been transplanted into HCVR+ with good results26. The information derived from these previously described experiences have been the basis for international guidelines and recommendations on the use of kidneys from HCVD+ for transplantation in a safe way, avoiding their loss at a moment of organ shortage27-29. In contrast to the positive results obtained in center-based experiences, registry studies have offered contradictory results. By using the U.S. Renal Data System registry, Abbot, et al. evaluated the outcome of recipients transplanted from HCVD+ versus HCVD30,31. No apparent differences were noticed in terms of graft survival. However, patient survival was significantly worse in recipients transplanted from HCVD+, irrespective of HCV serology of the recipient. The increased risk of death among recipients of HCVD+ kidneys was delayed for two years, which suggested the development of an intermediate complication that resulted in a later increased risk of death32. The observed higher incidence of posttransplant diabetes mellitus (PTDM) among recipients of kidneys from HCVD+ could be the reason behind this32. These data made the authors conclude that caution should be paid to the use of organs from HCVD+ and that careful and complete information should be provided to the potential recipient of these organs before transplantation33. However, when taking a careful look at these papers it is important to note that kidneys from HCVD+ had been used into patients with a worse baseline clinical and immunological situation compared to recipients of kidneys from HCVD-. Factors associated with the use of kidneys from HCVD+ were advanced donor and recipient age, African American race, and a high rate of dialysis 131

Trends in Transplantation 2010;4

Table 1. Main results of the center-based experiences with the use of kidneys from HCV-positive versus HCV-negative donors in HCV-positive recipients Ali20

Kasprzyk25

D+/R+ D-/R+

D+/R+ D-/R+

Number

28

Follow-up (months)

36 (12-60)

Acute rejection

16

50%

60

199

12-156

68%

Mandal21

Morales35

D+/R+

D-/R+

D+/R+

D-/R+

19

10

162

306

15.4 (SD = 2)

–

Veroux24 D+/R+ D-/R+ 28

74.5

16 23

Woodside22 D+/R+

D-/R+

20

20

26.3

34.9

42%

50%

42.1%

37.2%

10%

14.2%

20%

25%

Graft survival

86%

78%

70%

89%

70%

47% 58.5% (10 yr) (10 yr)

90%

88%

89% (1 yr)

79% (1 yr)

Patient survival

86%

95%

85%

89%

90%

72.7%

100%

94%

89% (1 yr)

94% (1 yr)

Acute liver dysfunction

16%*

–

–

16%‡

10%‡

16.1%* 11.6%*

–

–

–

–

Chronic liver dysfunction

9%†

11%§

10%§

9.8%¶

6.2%¶

–

–

–

–

–

–

9

24

Time in the waiting list (months)

–

–

–

–

9 29 (SD = 3)** (SD = 3)**

76.5%

9.9 17.8 (SD = 1.8)** (SD = 3.3)**

D: donor; R: recipient; SD: standard deviation. *ALT > 2.5 times the upper normal limit for more than 2 weeks, but less than 6 months. †ALT > 2.5 times the upper normal limit for more than 6 consecutive months. ‡ALT > 2 times the upper normal limit. §ALT > 2 times the upper normal limit for more than 3 months. ¶Decompensated liver disease: At least one episode of ascites, hepatic encephalopathy and/or gastrointestinal bleeding due to ruptured gastrointestinal varices. **p < 0.05.

access complications31,32. It is also important to note that the previously described studies reflected a lack of a specific policy on the use of organs from HCVD+, since they were also used into HCVR-, and there was no information available on the HCV RNA status of the recipients at the time of transplantation. Finally, also by using the U.S. Renal Data System registry, it has been shown that receiving a kidney from an HCVD+ is independently associated with improved patient survival compared with remaining in the waiting list (adjusted HR: 0.76; 95% CI: 0.60-0.96)34. Latest evidence on the safety of transplanting kidneys from HCVD+ into HCVR+ has been offered by the Spanish groups piloting the first experiences (Table 1)35. For the very first time, information has been offered 132

on the long-term outcome (mean follow-up 74.5 months) of 162 HCVR+ transplanted from HCVD+ (group 1) versus 306 HCVR+ transplanted from HCVD- (group 2). No differences were observed in patient survival. Only three deaths in group 1 and two deaths in group 2 were liver disease related. On the contrary, there was a trend towards a lower death-censored graft survival and a significantly lower non censored for death graft survival in patients transplanted from HCVD+. This could be due to differences in baseline demographic and clinical variables: group 1 exhibited a higher donor and recipient age and, as expected, a more frequent recipient pretransplant viremic state (HCV RNA positive), resulting from the allocation policy applied since 1993. This theory is supported by Mahmoud, et al. who have described a higher frequency

Beatriz Domínguez-Gil, et al.: Kidney Transplantation from Donors with a Positive Serology for Hepatitis C

Table 2. Factors independently associated to patient death, graft loss, and decompensated chronic liver disease in the multivariate analysis performed in the Spanish experience35 Patient death*

Decompensated CLD†

Graft loss*

P

OR

95% CI

P

OR

95% CI

P

OR

95% CI

0.22

0.709

0.412-1.223

0.18

1.248

0.902-1.726

0.92

1.048

0.429-2.560

–

–

–

< 0.001 1.022

1.012-1.032

–

–

–

< 0.001

1.075

1.049-1.102

–

–

–

–

–

–

–

1.367-2.674

–

–

–

Pretransplant cardiovascular disease

0.05

1.850

0.997-3.432

–

–

–

–

–

–

Delayed graft function

–

–

–

0.03

1.417

1.031-1.949

–

–

–

Acute rejection

–

–

–

< 0.001 1.778

1.304-2.425

–

–

–

0.003

2.883

1.447-5.746

–

–

–

–

–

–

–

–

–

–

–

–

< 0.001

9.462

3.887-23.030

0.03

2.883

1.447-5.746

–

–

–

–

–

–

HCVD+ Donor age Recipient age PRA ≥ 50%

NODAT Moderate CLD‡ Decompensated CLD§

–

–

< 0.001 1.912

CLD: chronic liver disease; HCVD+: positive serology for HCV; PRA: panel-reactive antibody; NODAT: new onset diabetes after transplantation. *Cox regression analysis. †Logistic regression analysis. ‡ALT > 2.5 times the upper normal limit for more than 6 consecutive months. §At least one episode of ascites, hepatic encephalopathy and/or gastrointestinal bleeding due to ruptured gastrointestinal varices.

of chronic allograft nephropathy among HCV RNA-positive recipients36. Nevertheless, the Cox-regression analysis performed in the Spanish experience (Table 2) could not identify the donor HCV-positive serology as a significant risk factor for death or graft loss. Moreover, decompensated CLD (at least one episode of ascites, hepatic encephalopathy and/or gastrointestinal bleeding due to ruptured gastrointestinal varices) occurred in 10.3 vs. 6.2% of the patients (p = ns), respectively in both groups. Donor HCV-positive serology was not an independent risk factor for the evolution towards a situation of advanced liver disease, as previously defined (Table 2). Although de novo PTDM occurred more frequently in group 1, HCVD+ was not identified as an independent risk factor in the multivariate analysis. No differences were observed in the incidence of posttransplant glomerular disease between the two groups. Limitations of this latest experience are challenges for research in the near future:

–– Information on HCV RNA among HCVD+ was lacking, but the practice of testing donors with nucleic acid testing has only been recently suggested27. Knowledge about the HCV RNA of donors, however, should not substantially modify the allocation strategy applied to the use of kidneys from HCVD+. –– No information has been provided on the HCV genotype of both donors and recipients, something important to evaluate the incidence of superinfection and its consequences. –– Information on the evaluation of HCV liver disease has been assessed clinically but not histologically. Because liver biopsies were not routinely performed in the series, whether the histological outcome of HCVrelated liver disease is different (stable or progressive liver fibrosis)37 in HCVR+ transplanted from HCVD+ versus HCVDstill remains to be answered. 133

Trends in Transplantation 2010;4

Decreasing the risk of HCV transmission when using kidneys from HCV-positive donors into HCV-positive recipients As demonstrated by the Spanish groups, the policy of transplanting kidneys from HCVD+ into HCVR+ does not completely prevent the transmission of HCV infection. Hence, this option should be limited to those candidates for kidney transplantation with a positive HCV RNA in the waiting list. This means that patients with a positive serology for HCV and a positive HCV RNA are the ones to be offered the possibility of receiving a kidney from an HCVD+, always with appropriate information on the special characteristics of these potential donors. In a very elegant exercise, Natov and Pereira analyzed the consequences of four different approaches to the use of kidneys from HCVD+38. The following assumptions were made: 2.4% prevalence of HCV antibodies among deceased donors, second generation ELISA test with 100% sensitivity and 98% specificity, 100% transmission of infection with the use of kidneys from HCV RNA-positive donors, 20% prevalence of HCV infection among patients under dialysis therapy, and absence of clinical consequences of HCV superinfection. No restriction on the use of organs from HCVD+ (all organs used irrespective of HCV serology of the recipients) would be related to 0% of graft losses, but 2.4% of transmission of the infection and 2% of new infections. With a universal restriction on the use of these organs (no organ used irrespective of HCV serology of the recipient), no transmission or new infection would occur but 4.2% of organs would be lost. By using organs from HCVD+ into HCVR+, 0% of graft losses would occur but a risk of transmission (2.4%) and new infection (0.5%) would persist. The best balance seemed to be achieved with the restriction of organs from these donors to recipients with a positive HCV RNA 134

before transplantation, with a theoretical occurrence of 2.4% of transmission of HCV infection but 0% of new infections and no graft losses. Therefore, the policy of using kidneys from HCVD+ into HCVR+ seems to be safer when the organs are exclusively placed into recipients with a positive HCV RNA before transplantation. But superinfection with a different HCV genotype may still occur. Studies in a posttransfusion hepatitis C infection model in chimpanzees have demonstrated that a preexisting infection with HCV did not protect from reinfection with a different genotype or even the same viral genotype39. Likewise, kidney transplant patients with a baseline HCV infection are not protected from a superinfection with a new HCV genotype17. Although mixed infection has not been associated with an increased mortality in a recent study40, at least one clinical report on a severe liver disease has been published when using a kidney from an HCVD+ into an HCVR+, when donor and recipient were infected by a different HCV genotype (genotype 1 to genotype 2)41. Therefore, matching donor and recipient according to the HCV genotypes involved should still improve results by reducing the risk of HCV transmission, although limited by obvious time constraints. Besides, depending on the HCV genomic heterogenicity within a specific geographical area, the possibilities of a mismatch between donor and recipient should be balanced.

Is there a place today for the use of HCV-positive donors into HCV-positive recipients: making this policy compatible with interferon therapy before transplantation It has been documented that survival of HCVR+ is significantly better than that of matched patients who remain in the waiting list34,42,43. Therefore, kidney transplantation is

Beatriz Domínguez-Gil, et al.: Kidney Transplantation from Donors with a Positive Serology for Hepatitis C

the best therapy for patients with HCV infection and end-stage renal disease. However, HCVR+ have proven to exhibit a worse long-term graft and patient survival than HCVR-44-50. Also, HCV infection has been related to the development of posttransplant complications, such as de novo PTDM51, posttransplant glomerulonephritis52-54, proteinuria and chronic allograft nephropathy55, after kidney transplantation. Notably, treatment with interferon (IFN) before kidney transplantation may be related to a decreased incidence of posttransplant HCV-related glomerulonephritis56. Interferon therapy in 50 HCV RNA-positive patients significantly decreased the incidence of chronic allograft nephropathy57. In spite of this, treatment with IFN before transplantation in HCVinfected patients has not been related yet to benefits in terms of graft or patient survival. The problem of anti-HCV therapy is that IFN increases the risk of allograft dysfunction and therefore its use in kidney transplant patients is contraindicated, with the exception of patients with fibrosing cholestatic hepatitis15,27,58,59. Therefore, the best strategy is to treat HCV infection in patients on dialysis before transplantation15,27,52,58-62. While in the past recommendations on end-stage renal disease patients with HCV infection were based on the liver clinical and histological situation15, the negative clinical consequences of HCV infection after kidney transplantation constitute the basis to indicate therapy with IFN, independently of the stage of the liver disease, in order to improve the outcomes after transplantation27. Treatment of HCV infection before transplantation with the aim of a sustained virologic response is obviously not compatible with the use of HCVD+ into HCVR+. However, the limitations of HCV antiviral therapy should be taken into consideration: a wide range of adverse events has been described with IFN therapy, the rate of nonresponding patients is not negligible63,64, the treatment is long and

during this time the patients should be excluded from the waiting list, and finally it is an expensive treatment not universally affordable. Therefore, a group of end-stage renal disease HCV RNA-positive patients would not be candidates for antiviral treatment, some will refuse to be treated, or will not respond or withdraw the therapy. These patients, despite presenting a positive HCV RNA before transplantation should be placed into the waiting list since their outcome will be better than remaining under dialysis34,65-67. It is in this context where the possibility of being transplanted with a kidney from an HCVD+ could be offered, with the potential advantage of reducing the time in the waiting list. Even the possibility of preemptive kidney transplantation with organs from HCVD+ for these recipients could be offered. The rationale behind this is simple. The prevalence and the incidence of HCV infection is decreasing among patients with endstage renal disease. The number of HCV RNApositive patients in the list is progressively less and most of them are immunologically highrisk patients. Hence, there are a number of kidneys from HCVD+ which are not transplanted because of the lack of an appropriate recipient. Organizational measures should hence be developed in order to allow preemptive transplantation in these exceptional cases. Finally, some countries may probably not consider the universal approach of treating HCV RNA-positive patients under dialysis because of economic reasons. Unfortunately, these countries are usually those with a higher prevalence of HCV infection among their donors and their recipients. The policy of using HCVD+ for HCVR+ could be a safe approach in these populations.

Conclusions The use of kidneys from HCVD+ into HCVR+ seems to be a safe approach in the long term and a way of using these kidneys 135

Trends in Transplantation 2010;4

that otherwise would be lost. Results provided by center-based experiences show a favorable outcome of HCVR+ in terms of graft survival, patient survival, and HCV-related liver disease when transplanted from HCVD+. Registry studies have raised doubts on safety but do not represent a standardized policy. The safety of this approach can be improved by limiting the transplantation of these kidneys to patients with a positive HCV RNA before transplantation and, ideally, by matching donors and recipients according to their HCV genotype. Donor HCV-positive kidneys are being lost today because of remaining doubts that seem to be reasonably overcome nowadays and by the lack of appropriate recipients. Organizational measures such as devising preemptive transplantation for HCV RNA-positive recipients accepting to be transplanted with HCVD+ kidneys and international cooperation seem essential to avoid the loss of these organs at a moment of dramatic organ shortage.

References

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Trends in Transplant. Transplantation 2010;4:138-44 2010;4

Living Donor Liver Transplantation Juan Carlos García-Valdecasas1, Itxarone Bilbao Aguirre2, Ramón Charco Torra3, Constantino Fondevila Campo4, Josep Fuster Obregón5, Juan Carlos García-Valdecasas1, Paloma Jara Vega6, Rafael López Andújar7, Pedro López Cillero8, Juan Carlos Meneu-Díaz9, Miguel Navasa Anadón5 and Fernando Pardo Sánchez10 1

Department of Surgery, Hospital Clínic i Provincial, Barcelona, Spain; 2Liver Transplant Unit, Hospital Vall d´Hebron, Barcelona, Spain; Department of HPB and Transplant Surgery, Hospital Vall d’Hebron, Barcelona, Spain; 4Division of Gastroenterology and General Surgery, Hospital Clínic i Provincial, Barcelona, Spain; 5Liver Surgery and Transplantation Unit, Institute of Digestive and Metabolic Diseases,Hospital Clínic i Provincial, Barcelona, Spain; 6Pediatric Liver Care and Transplant Center, Children’s University Hospital, La Paz, Madrid, Spain; 7 Liver Transplantation Unit, Hospital Universitario La Fe, Valencia, Spain; 8Division of General and Gastrointestinal Surgery, Hospital Reina Sofía, Córdoba, Spain; 9Division of Gastroenterology and General Surgery, Hospital 12 de Octubre, Madrid, Spain; 10Department of HPB and Liver Transplant Surgery, Clínica Universidad de Navarra, Pamplona, Spain 3

Abstract Living donor liver transplantation was introduced for the purposes of increasing the number of donors, reducing mortality and morbidity rates, and improving long-term survival of the recipients. The procedure for living donor liver transplantation is the same as for cadaveric liver transplantation. The suitability of potential donors is established following exhaustive evaluations of the donor’s liver and overall health. In adult transplantation cases, living donor liver transplantation outcomes are as good as in cadaveric transplants, but donor morbidity continues to be significant as are biliary complications, whereas outcomes in pediatric liver transplants from living donors are more successful than those from cadaveric liver grafts. Living donor liver transplantation is a valid alternative to cadaveric transplantation that can offer improvement of survival rates in the future if we manage to select suitable candidates and overcome a few technical difficulties. (Trends in Transplant. 2010;4:138-44) Corresponding author: Juan Carlos García-Valdecasas, [email protected]

Key words Liver transplantation. Living donor. Pediatric liver transplatation. Adult liver transplatation. Trends in liver transplantation.

Correspondence to: Juan Carlos García-Valdecasas Servicio de Cirugía General y Digestiva Hospital Clinic de Barcelona c/ Villarroel, 170 08036 Barcelona, España E-mail: [email protected]

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Juan Carlos García-Valdecasas, et al.: Living Donor Liver Transplantation

Introduction The main objective of living donor liver transplantation (LDLT) is to increase the number of organs available for transplantation. Although the potential risks to the donor’s life are low, they very much govern the performance of this type of transplantation. Nevertheless, the mortality rate for patients on the waiting list for a donor justifies the use of this procedure. This document details the outcomes of discussions held at the consensus meeting of the Spanish groups, whose objective was to detect problems and provide possible solutions.

Donation process Trends in living donor transplantation Over the past few years, advances in liver transplantation have allowed the survival rate after one year to rise to nearly 95%. Although Spain has one of the highest transplant rates in the world, availability of cadaveric organs for transplantation is not sufficient at this time to cover existing needs. Waiting list mortality has hovered around 7-8% for the last few years; the probability of undergoing transplantation was 51% in 2008. While in recent years a slight increase in living donor kidney transplantations has been observed1, in LDLT the trend has been in the opposite direction2. One of the reasons for this decline has been the fact that application of the model for end stage liver disease (MELD) causes the urgency for donations to decrease as patients with a higher risk of death are identified. Other reasons are donor mortality and morbidity rates, the risk of worse outcomes in recipients according to their etiology or the seriousness of their illness, the potential donor

evaluation process itself, which means only between 9 and 17% are accepted for donation, as well as issues related to the donors’ quality of life following transplantation. At this time, LDLT continues to be a complex procedure that involves morbidity and mortality risks for donors as well as risks for recipients due to the need for complex vascular and biliary reconstruction. Nevertheless, the general opinion is that this type of transplantation is justified due to the fact that the waiting list mortality rate still remains too high3. In addition, the prevalence of hepatocellular carcinoma (HCC) means it is impossible to cover all of the need for liver transplants so that HCC should be considered as an indication criterion for LDLT. Improvement of LDLT requires proper identification of appropriate candidates, such as MELD exceptions or those with HCC, reduction of donor morbidity and mortality and improvement in their quality of life following the donation, compensation for financial loss and, lastly, the introduction of more aggressive options such as programs for crossmatching donor and recipient or programs for use from donors with blood group incompatibility. The majority of LDLT that take place in Spain are being performed in pediatric recipients4. So far, more than 2,000 implantations of left lateral segment grafts, which is an option that parents frequently request, have been performed worldwide. Selection of potential recipients is based on pediatric endstage liver disease criteria that predict mortality within three months of being included on the waiting list. This modification of the adult “score” does not appear to identify all of the children in urgent need of transplantation. Those in exceptional situations or serious cases account for approximately 50% of those who receive a transplant. In addition, children over the age of 12 compete with adults, which 139

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makes giving them priority more difficult. The solution would be to systematically prioritize pediatric patients, giving status as a child whenever necessary and making it an obligation for this pediatric grouping to make division of the graft a priority so as to be able to transplant into both an adult and a child at the same time. The graft will thus have been assigned to two patients and there would be no predominance of one group over the other which, in the majority of cases, is a source of conflict. To maintain the offer of a living donation is also a duty for those parents or relatives who are willing to make a donation. In some cases, LDLT has been proven to have survival outcomes that are equivalent5 or superior to full grafts. However, and in spite of being associated with a somewhat greater survival rate, the risk of graft loss is somewhat higher for divided or split grafts than for full ones.

Incentives for live donations In order to encourage living donations, the objective must be to reduce to the minimum the negative impact that transplantation has on the donor, as much physically as psychologically or even financially. One of the main drawbacks is the scar, which can be resolved through the use of laparoscopic surgery6,7. Economic obstacles for the donor could be solved through the creation of protection mechanisms that would guarantee employment maintenance or provide access to long-term care insurance. The majority of hospitals are not candid with patients on the waiting list about the possibility of opting for a LDLT. To improve on this situation in the future, informing patients on the waiting list about this option should be mandatory, as should advising them about referral centers when need be. 140

Table 1. Characteristics of the ideal live donor –  Age: 18-55 years –  BMI: < 30 kg/m2 –  No cardiopulmonary, renal or metabolic disease –  Residual liver volume (LLL): > 40% –  Graft (RLL): > 0,8% recipient weight –  Steatosis < 20% –  Favorable anatomical suitability –  Donor/recipient must be ABO-compatible –  Significant relationship with the recipient –  Independence and competence of the donor

Donor evaluation The key to LDLT lies mainly in the consideration of the risk to the donor, which should be minimal, and the benefit for the recipient. The donor must weigh the risk of possible mortality, aftereffects, and social, economic, and work aspects. The importance of these factors may vary according to survival of the recipient. The risk of minor complications for the donor is about 27%, that for potentially serious complications that are successfully resolved is 26%, about 2% for lifethreatening conditions, and 0.8% for death8. Extensive evaluation of the donors is key to achieving good short- and long-term outcomes. The risk of complications in the donor is currently about 37%, about half of which are minor while the remainder are considered to be potentially serious, according to the Clavien classification system. Therefore, one of the most pressing objectives is to try and reduce this number by means of thorough prior testing and a meticulous surgical technique to ensure the highest standard of quality of life for the donor following the operation. Table 1 shows the factors that determine the selection of donors and table 2 shows the protocols for the selection of poten-

Juan Carlos García-Valdecasas, et al.: Living Donor Liver Transplantation

Table 2. Phases in the process of donor evaluation Preliminary general health evaluation

First informed consent form Detailed medical history Physical examination Blood tests, blood group, hepatitis serology

Psychological evaluation

Mental stability Voluntary nature and willingness Relationship between donor and recipient Informing the donor sufficiently about the surgical procedure

Anatomy

Cholangio-MRI CT angiography All-in-one MeVis®

Overall risks of the surgical procedure

Lab tests: biochemical, lipid profile, iron, ferritin, transferrin, α1-antitrypsin, ceruloplasmin, immunoglobulin levels, thyroid function tests, tumor markers, coagulation factors Hyper-coagulation profile* Chest X-Ray Lung function test Stress test-ECG Echocardiogram

Liver biopsy†

Presence of steatosis (contraindicated: if > 20% or if 10-20% and RLVBWR < 0,8) Discovery of other histologic findings: (portal and sinusoidal fibrosis; NASH; portal inflammation and necroinflammatory changes)

Preparation for surgery

Autologous blood donation Second psychological evaluation Evaluation by hepatologist Assessment by anesthetist Final consent Ethics committee Civil registry

RLVBWR: remnant liver volume body weight ratio; NASH: nonalcoholic steatohepatitis. *If donor has a history of deep vein thrombosis. †in patients with abnormal liver function test results, radiologic abnormalities (steatosis and others), BMI > 30 kg/m2, or relatives of recipients with primary biliary cirrhosis, primary sclerosing cholangitis or autoimmune disease.

tial donors. The donor must be informed of the risks and drawbacks associated with transplantation before giving consent. Despite all this, a comprehensive preoperative evaluation does not guarantee the absence of postoperative morbidity in the donor. The mortality risk for donors is five times greater in LDLT than in living kidney donation 9,10. Progressive experience and improvements in donor selection may reduce mortality and morbidity rates in the future, although they are not expected to ever be as low as in living donor kidney transplantation. The need for total transparency regarding outcomes for donors and awareness that

morbidity and mortality rates will never be zero justify the need for establishing a prospective donor morbidity registry and establishing a standard system for recording complications in donors. In the meantime, surgery complications should be recorded following the Clavien classification system or one of the recent adaptations based on it11. Follow-up of donors is essential and should be made, at the very least, for the first three years following surgery, at a rate of once every three months during the first year and at 12-month intervals after that. It is recommended that the tests to be required should include complete lab tests and volume calculations using magnetic resonance imaging. It is necessary to have a long time of follow-up 141

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of all donors because we don’t know what the long-term complications could be. The majority of programs consider that during the first year, follow-up should be performed every three months.

Evaluation of the liver The liver is an organ that has surgically important vascular variations, both venous and arterial, in addition to the biliary tract, which makes evaluating it very complex. Current imaging technology is extremely efficient and allows the anatomic distribution of all of its structures, including the biliary tract, to be described with great exactitude. Helical computed tomography with multiplanar reconstruction and magnetic resonance imaging allow the visualization of all of these structures in a single exploration. In addition, the possibility of including a reconstruction of the liver through use of a special program (MeVis®) allows three-dimensional images to be obtained that further increase safety in surgical planning. Even so, imaging during the surgical intervention itself must be used to guarantee the anatomical orientation suggested by the preoperative evaluation. In a major surgical procedure, such as that performed on the donor, where meticulous dissection of the hilum of the liver is required, there is no room for guesswork and each step of the process must be performed with the maximum possible safety and knowledge of the possible consequences.

Selection of candidates for liver transplantation The ideal candidate for LDLT is a person who would benefit from receiving a cadaveric liver, but who has a low probability of receiving one for transplantation because of the seriousness of their disease, and in addition, is someone who has not previously 142

suffered from significant deterioration in quality of life. At present, the MELD system is able to identify and prioritize those patients with the highest probability of pretransplant death. That is why LDLT currently targets all those patients who are not correctly identified and therefore not prioritized. From our point of view, HCC represents a leading indication for LDLT, both for patients who meet the Milan criteria and for those who exceed these criteria but are known to have a relatively good prognosis (Barcelona criteria, Kyoto criteria, etc.)12, or those who respond following chemoembolization or radiofrequency ablation and survive for at least three months within the Milan criteria.

Donor operation in adult-to-adult and adult-to-child living donor liver transplantation Donor surgery in adult-to-adult procedures In the majority of cases, the surgical technique for the adult donor consists of a right hepatectomy including segments V-VIII, and in which the middle hepatic vein remains with the donor.

Donor surgery in adult-to-pediatric recipient procedures The surgical technique for the adult donor normally includes resection of liver segments II and III. Anatomical variability is significantly lower, especially where the bile duct, which is unique in 90% of cases, is concerned. This makes the surgery easier to perform and minimizes the need for bankedblood.

Juan Carlos García-Valdecasas, et al.: Living Donor Liver Transplantation

Recipient operation in adult-toadult and adult-to-child living donor liver transplantation

Results

Recipient surgery in adult-to-adult procedures

As has already been mentioned, the objective of LDLT is to increase the number of donors, reduce mortality and morbidity rates among donors, and improve the longterm survival of recipients.

Vascular reconstruction depends on achieving the best possible venous drainage, which means not only performing anastomosis of the right hepatic vein, but also reconstructing all of the veins, whether they be accessory veins of the right lobe or tributaries of the middle hepatic vein, for which cryopreserved grafts are frequently necessary. Although the artery is small (only 3-5 mm in diameter) its reconstruction rarely causes problems. Continuous hemodynamic monitoring is needed to ensure adequate arterial flow. Last of all come the bile ducts, which have a diameter between 2 and 4 mm and are the “Achilles heel” of this type of transplantation. The ideal is to perform a systematic duct-to-duct biliary reconstruction, and when this is not possible, to perform a hepaticojejunostomy.

Recipient surgery in adult-topediatric recipient procedures Surgical techniques in pediatric living donor transplantation depend largely on the patient’s original disease. Vascular reconstruction is essentially the same as in adults, although in this case the size of the liver is always larger than required so that it is not necessary to maneuver to ensure venous drainage. On the contrary, because of its association with congenital anomalies, insufficient portal flow must be ruled out (due to hypoplasia of the portal vein). On the other hand, the size of the bile ducts, which is frequently insufficient, make it necessary to always perform a hepaticojejunostomy, something that is absolutely necessary in cases of biliary atresia.

Adults

Outcomes for LDLT have improved in the last few years. Although survival rates are now comparable to those from cadaveric donors, the incidence of biliary complications affects long-term outcomes. Nevertheless, according to follow-ups for periods of more than five years in the USA as well as in Europe, the presence of these complications does not appear to affect long-term outcomes. The current trend in Western countries towards progressive reduction of this type of transplantation is not due to poor outcomes, but rather to sporadic cases of donor death, which have led to the closing of LDLT programs at hospitals where these have occurred. There have been a variety causes, from those due to the absence of an appropriate level of care to those where the pressure on the medical staff has had an impact on care delivery. A total of 232 adult and 91 pediatric LDLT were performed in Europe during 2007. Both patient and graft survival rates are better in LDLT. Since MELD scores in LDLT patients are lower than in patients receiving cadaveric transplants, there is a need for caution when comparing figures. The experience in Spain is small, although at present the absence of donor mortality associated with good outcomes in both pediatric and adult recipients allow for the consideration of the need for joint action by those hospitals where LDLT is performed in 143

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order to increase activity. The identification of those patients in need of LDLT is of paramount importance as are systematic information and referral of patients to those centers with the experience, when the case arises. In the last 15 years, 188 LDLT have been performed in Spain13. As yet no deaths have been recorded, although there has been an 8.3% rate of reoperations to solve complications. Graft survival is similar in patients who are living donor recipients (80% at one year and 65% at five years) as in those who are cadaveric donor recipients (82 and 66%, respectively). The big problem in this type of donation is the high number of complications involved.

Living donor liver transplantation in the pediatric population Outcomes in the pediatric age group cause many fewer problems. The family relationship with the child is more reasonable and outcomes are better than those obtained with grafts that come from cadaveric donors. Only the systematic division of all liver grafts would reduce the need for this type of transplantation. Even so, at present living donation allows ensuring absence of mortality on the waiting list, something unthinkable in the 1990s when mortality on the waiting list was around 30%. Between 1993 and 2009, survival of pediatric recipients of living donor transplants in Spain was 84.8% at one year and 79.8% at five years.

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Final considerations The most important aspects to be resolved in LDLT are the establishment of standardized registries, the resolution of technical difficulties, shortening the learning curve, and improving quality of life for the donor and the efficiency of the procedure. It is also necessary to assess the possibility of expanding indications for LDLT, allowing expected survival in recipients of up to 30%.

References

1. Steinbrook R. Public solicitation of organ donors. N Engl J Med. 2005;353:441-4. 2. Clavien PA, Dutkowski P, Trotter JF. Requiem for a champion? Living donor liver transplantation. J Hepatol. 2009;51:635-7. 3. García-Valdecasas JC, Fuster J, Fondevila C, Calatayud D. Adult living-donor liver transplantation. Gastroenterol Hepatol. 2009;32:577-83 4. ONT. Registro Español de Trasplante Hepático. Memoria de resultados 2009. [Spanish Hepatic Transplant Registry. History of results 2009]. Available at: http://www.ont.es/infesp/ Registros/MEMORIA_RETH_2009.pdf 5. Bourdeaux C, Darwish A, Jamart J, et al. Living-related versus deceased donor pediatric liver transplantation: a multivariate analysis of technical and immunological complications in 235 recipients. Am J Transplant. 2007;7:440-7. 6. Baker TB, Jay CL, Ladner DP, et al. Laparoscopy-assisted and open living donor right hepatectomy: a comparative study of outcomes. Surgery. 2009;146:817-23. 7. Suh KS, Yi NJ, Kim T, et al. Laparoscopy-assisted donor right hepatectomy using a hand port system preserving the middle hepatic vein branches. World J Surg. 2009;33:526-33. 8. Ghobrial RM, Freise CE, Trotter JF, et al. Donor morbidity after living donation for liver transplantation. Gastroenterology. 2008;135:468-76. 9. Brown RS. Live donors in liver transplantation. Gastroenterology. 2008;134:1802-13 10. Cotler SJ, McNutt R, Patil R, et al. Adult living donor liver transplantation: Preferences about donation outside the medical community. Liver Transpl. 2001;7:335-40. 11. Hata T, Fujimoto Y, Suzuki K, et al. Two cases of central venous catheter-related thrombosis in living liver donors: how can the risk be minimized? Clin Transplant. 2009;23:289-93. 12. Mazzaferro V, Llovet JM, Miceli R et al. Predicting survival after liver transplantation in patients with hepatocellular carcinoma beyond the Milan criteria: a retrospective, exploratory analysis. Lancet Oncol. 2009;10:35-43. 13. SETH-ONT. Memoria de resultados 1984-2008. [History of results 1984-2008]. Available from: http://www.sethepatico.org.

VALCYTE 50 mg/ml polvo para solución oral. VALCYTE 450 mg comprimidos con cubierta pelicular. COMPOSICIÓN CUALITATIVA Y CUANTITATIVA: VALCYTE polvo para solución oral: cada frasco contiene 5,5 g de hidrocloruro de valganciclovir, por 12 g de polvo para solución oral. La solución reconstituida contiene 50 mg por ml de valganciclovir (como hidrocloruro). VALCYTE comprimidos con cubierta pelicular: cada comprimido contiene 496,3 mg de hidrocloruro de valganciclovir, equivalente a 450 mg de valganciclovir (base libre). Para consultar la lista completa de excipientes, ver apartado lista de excipientes. FORMA FARMACÉUTICA: VALCYTE polvo para solución oral: polvo para solución oral. El polvo es un granulado de color blanco a ligeramente amarillento. Cuando el polvo es disuelto, la solución es clara, de incolora a parda. VALCYTE comprimidos con cubierta pelicular: comprimidos con cubierta pelicular. Comprimidos con cubierta pelicular de color rosa, convexo y ovalado, con el grabado “VGC” en una cara y “450” en la otra. DATOS CLÍNICOS: Indicaciones terapéuticas VALCYTE está indicado para el tratamiento de inducción y mantenimiento de la retinitis por citomegalovirus (CMV) en pacientes con síndrome de inmunodeficiencia adquirida (SIDA). VALCYTE está indicado para la prevención de la enfermedad por CMV en pacientes seronegativos al CMV que han recibido un trasplante de órgano sólido de un donante seropositivo. Posología y forma de administración: Advertencia – se deben seguir estrictamente las recomendaciones sobre la posología para evitar sobredosificación (ver apartados Advertencias y precauciones especiales de empleo y Sobredosis). Después de su administración oral, el valganciclovir se metaboliza de forma rápida y extensa a ganciclovir. 900 mg de valganciclovir por vía oral, dos veces al día, es equivalente terapéuticamente a 5 mg/kg de ganciclovir administrado dos veces al día. La exposición sistémica a ganciclovir con 900 mg de valganciclovir solución oral es equivalente a 900 mg de valganciclovir en comprimidos. Posología habitual en adultos: Tratamiento de inducción de la retinitis por CMV: La dosis recomendada para los pacientes con retinitis activa por CMV es de 900 mg de valganciclovir dos veces al día durante 21 días (en el caso de Valcyte comprimidos con cubierta pelicular: dos comprimidos de 450 mg). Un tratamiento prolongado de inducción puede incrementar el riesgo de toxicidad para la médula ósea (ver apartado Advertencias y precauciones especiales de empleo). Tratamiento de mantenimiento de la retinitis por CMV: Después del tratamiento de inducción, o si se trata de pacientes con retinitis inactiva por CMV, se recomienda administrar una dosis de 900 mg de valganciclovir una vez al día (en el caso de Valcyte comprimidos con cubierta pelicular: dos comprimidos de 450 mg). Se puede repetir el tratamiento de inducción en aquellos pacientes en los que la retinitis empeore; sin embargo, se debe tener en cuenta la posibilidad de resistencia viral al fármaco. Prevención de la enfermedad por CMV en el trasplante de órgano sólido: La dosis recomendada en pacientes que han recibido un trasplante es de 900 mg una vez al día (en el caso de Valcyte comprimidos con cubierta pelicular: dos comprimidos de 450 mg), comenzando dentro de los 10 días del trasplante y continuando hasta los 100 días post-trasplante. Instrucciones posológicas especiales: Pacientes con insuficiencia renal: Los niveles séricos de creatinina o el aclaramiento de creatinina se deben vigilar cuidadosamente. El aclaramiento estimado de creatinina (ml/min) se puede calcular según la creatinina sérica mediante las siguientes fórmulas: para los varones = (140 – edad [años]) x (peso corporal [kg])/(72) x (0,011 x creatinina sérica [micromoles/l]). Para las mujeres = 0,85 x valor de los varones. VALCYTE polvo para solución oral: hay que ajustar la posología según el aclaramiento de creatinina, tal y como se indica en la siguiente tabla (ver apartado Advertencias y precauciones especiales de empleo).

CrCl (ml/min) ≥ 60 40 – 59 25 – 39 10 – 24