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A Europe-wide Strategy to enhance Transplantation of highly sensitized patients on basis of Acceptable HLA Mismatches

Final Report Summary - EUROSTAM (A Europe-wide Strategy to enhance Transplantation of highly sensitized patients on basis of Acceptable HLA Mismatches.)

Executive Summary:
The presence of antibodies against the transplantation antigens (a kind of blood groups) of a possible kidney donor is a contraindication for transplantation as in this case there is a very high chance that the graft will be rejected within a few days. Some patients have a lot of these antibodies, due to previous blood transfusions, pregnancies en transplants. A significant proportion of these so called highly sensitized patients cannot be transplanted and dies on the waiting list because there is no suitable kidney donor available in their own donor population. The present study shows that international collaboration in Europe, taking advantage of the fact that the transplantation antigens differ between European populations will be beneficial for this patient group. Simulation studies clearly showed that collaboration between Eurotransplant and transplant organizations in the UK, Spain, Greece and the Czech Republic would lead to the identification of a suitable donor for about 25-30% of the 700 highly sensitized patients, who, at the moment, do not have any chance to be transplanted within their own organization. Furthermore, it was shown that extension of such collaboration to more countries and transplant organizations will lead to transplantation of even more of these patients. Of course, the introduction of such a program will be associated with many logistic, legal and ethical issues. The exact requirements for the start of such a program have been described in the final report of this Eurostam project. The next step will be the actual implementation, which will be coordinated by the project leader and some of the collaborating organizations in this project i.e. Eurotransplant and NHSBT in the UK. Several countries and organizations, which were originally not involved in the project, have expressed their interest in participation in this future European program.
The current method to search for a suitable donor is complicated by the fact that there are more than 10,000 different transplantation antigens. However, these antigens are not completely different from each other but share some characteristics, which determine whether an antibody will be detrimental or not. The project group identified an alternative method, which, in the future, may simplify this search by taking into consideration only the 180-200 relevant characteristics of the transplantation antigens.

Project Context and Objectives:
The presence of donor specific HLA antibodies is considered a contra-indication for renal transplantation as their presence is associated with the occurrence of hyperacute graft rejection. Highly sensitized patients have antibodies against many HLA antigens and it is very difficult to identify donors towards which they do not have donor specific HLA antibodies. For this reason, highly sensitized patients accumulate on the transplant waiting lists. As HLA antibodies are not only induced by blood transfusions and transplantation but also by pregnancy, the proportion of females in this group of patients is significantly increased. Considering the high mortality rate of patients on dialysis, special strategies are required to enhance transplantation of long waiting highly sensitized patients. One option is desensitization, which is the use of immunosuppressive agents to remove the antibodies followed by a transplant from a donor towards which the patient originally had specific HLA antibodies. This approach is effective in patients with weak antibodies, although it is associated with a higher incidence of humoral rejection, but fails in patients with strong cytotoxic HLA antibodies. Furthermore, the additional immunosuppressive treatment is not specific and associated with severe side effects.
A very successful alternative strategy is the acceptable mismatch program of Eurotransplant. Basis of this program is the exact definition of those HLA antigens, toward which the patient never formed antibodies, by extensive antibody screening of the patient sera. Donor selection is based on compatibility of the donor with the combination of the patients own HLA antigens and the acceptable HLA mismatches as one can predict that in this case the patient will not have any donor specific HLA antibodies. When such a donor becomes available in one of the Eurotransplant affiliated countries (i.e. Austria, Belgium, Germany, Luxembourg, Slovenia, Croatia and the Netherlands), the donor kidney is mandatorily shipped to the transplantation center of the highly sensitized patient. This strategy has proven to be very effective as 60% of the highly sensitized patients included in the acceptable mismatch program are transplanted within 2 years. The long term results of these transplants are excellent and similar to those of non-sensitized patients. However, for a significant proportion (about 35%) of the highly sensitized patients in the acceptable mismatch program no compatible donor can be found within the Eurotransplant area. These are often patients with a rare HLA phenotype in relation to the donor population. A similar problem occurs in other transplant exchange organizations within Europe. Background of this problem is the enormous polymorphism of the HLA system. However, HLA phenotype frequencies are different amongst European populations and rare HLA phenotypes in one population are more frequent in other populations. We hypothesize that extension of the potential donor pool by a Europe-wide collaboration will significantly increase the chance to find a suitable donor for a long waiting highly sensitized patient.
The major and final objective of this project is to determine the feasibility and advantage of a Europe-wide acceptable mismatch (AM) program to facilitate transplantation of highly sensitized patients all over Europe and to define the requirements for the actual implementation of such a program. A second objective is to test whether the definition of acceptable HLA epitopes rather than HLA antigens facilitates the identification of compatible donors. The current approach to define acceptable HLA mismatches is very complicated considering the more than 10,000 HLA class I antigens identified so far. Recently, we and others have focused our research on the inventory of the actual antibody epitopes present on these HLA antigens. HLA antigens can be considered as strings of epitopes which are shared between different HLA antigens. As 180-200 polymorphic residues are responsible for the induction of all HLA antibodies to the HLA class I antigens, the definition of acceptable epitopes would simplify the definition of acceptable mismatches. In a later stage , the same approach can be used for HLA class II. For such an epitope based strategy, an alternative HLA typing assay based on a quick identification of the relevant HLA epitopes is certainly of benefit, which is the reason why we tried to develop a prototype of such an assay during this project.
The third objective is to define the relevance of endothelium specific non-HLA antibodies for the outcome of transplants in highly sensitized patients. So far, only HLA antigens have been considered relevant targets for donor specific antibodies but it is clear that the endothelium in the graft, which is the main target for donor specific antibodies, also expresses other antigens.

Project Results:
1. Major goal: need, feasibility and guidelines for a Europe-wide acceptable mismatch program.
The simulation studies clearly showed the need and beneficial impact of a Europe-wide acceptable mismatch program taking advantage of the different HLA phenotype frequencies amongst the European populations. Currently, every transplant organization has a group of long waiting highly sensitized patients, who do not have a chance to be transplanted because they have formed many HLA antibodies due to previous pregnancies, blood transfusions or transplants. The presence of donor specific antibodies is a contra-indication for transplantation. Furthermore, this subgroup of patients has HLA phenotypes, which do not or hardly occur in the own donor population. The 5 transplant organizations participating in this project collected a group of 700 of such patients. The HLA types of the patients and their acceptable HLA antigens (antigens toward which they did not form antibodies) were included in a database and a search for compatible donors was done according to the principle of the acceptable mismatch program of Eurotransplant. For about 25-30% of these highly sensitized patients, who did not have any chance to be transplanted within their own organization, a compatible donor was readily available in one of the other donor populations. This was shown to be the case when only the populations of actual organ donors from the 5 Eurostam partners, i.e. NHSBT in the UK, Barcelona, Greece, the Czech Republic and Eurotransplant were included in the simulations.
Additional simulations, taking into consideration potential donors from additional countries in Europe, showed that the chance to find a compatible donor increases when more countries will participate and as a consequence more of these highly sensitized patients can be transplanted. For these additional simulations the HLA phenotypes of more than 500,000 individuals from 40 populations were collected. This database and a search tool are included in the HLA-net bioinformatics platform (hla-net.eu).
A next step will be the introduction of the actual Europe-wide acceptable mismatch program. On purpose, the Eurostam consortium partners did not spend all the grant money, originally allocated to the project. The reason for that was the fact that, during the 3 year period of the project, the main goals have been reached and the project leader and participants did not feel it appropriate just to spend all the money without any chance to reach additional milestone during the course of the project. The very successful simulation studies clearly show the benefit of a Europe-wide acceptable mismatch program to facilitate transplantation of highly sensitized patients. However, the time was too short for the actual implementation of such a program, taking into consideration all the logistic, legal and ethical aspects association with this implementation. Partners Eurotransplant and NHSBT have expressed their willingness to support the actual introduction of this program, according to the guidelines formulated in the end report of the Eurostam consortium. The future Europe-wide acceptable mismatch program will not be restricted to the partners of this consortium. The positive results of the simulation studies have been presented at several international meetings and as a consequence other transplant organizations, like Scandia Transplant, and other countries in Europe, not participating in Eurostam, have expressed their interest in participating in a future Europe-wide Acceptable Mismatch program. Furthermore, countries with an active paired donor exchange program for living donation experience similar problems as the ones described in this project for highly sensitized patients. For a number of patients with a potential living donor, who is not compatible with patient, paired donor exchange does not work in the own donor population. Also here it is opportune to take advantage of the different HLA phenotypes in other European donor populations. The guidelines formulated in the current project are equally relevant for this patient population. Therefore, the project leader will try to apply for a next grant in the context of Horizon 2020 in order to realize this cross-border exchange of donor kidneys taking advantage of the different HLA configurations in the European populations. For the realization of this goal, additional partners have already confirmed their willingness to participate in a future “Eurostam.2” project.
Acceptable HLA mismatches are defined by extensive antibody analyses. In order to guarantee an optimal quality of the data, the participants in the consortium have performed several proficiency testing rounds in order to establish the optimal laboratory conditions to define in a reliable way acceptable mismatches in different laboratories in Europe. Using uniform protocols and reagents, a high concordance rate was obtained but there were still some discrepancies. As the success of the program is fully dependent on compatibility of a potential donor with the acceptable mismatches, our recommendation is that all acceptable mismatches should be routinely checked in a reference laboratory before the patient is eligible for and can be introduced in an Europe-wide acceptable mismatch program, which is also the procedure of the current acceptable mismatch program of Eurotransplant.
2. Secondary goal: Future replacement of acceptable HLA antigens by acceptable HLA epitopes.
A second objective of Eurostam was to test whether the definition of acceptable HLA epitopes rather than HLA antigens facilitates the identification of compatible donors. The current approach to define acceptable HLA mismatches is very complicated considering the more than 10,000 HLA class I antigens identified so far. Recently, we and others have focused our research on the inventory of the actual antibody epitopes present on these HLA antigens. HLA antigens can be considered as strings of epitopes which are shared between different HLA antigens. As 180-200 polymorphic residues are responsible for the induction of HLA antibodies to all HLA class I antigens, the definition of acceptable epitopes would simplify the definition of acceptable mismatches
A computer algorithm to translate HLA antigens into epitopes, and the other way around was validated. Simulations showed the advantage of donor selection on basis of acceptable HLA epitopes as this will allow the definition of acceptable HLA antigens, which have never been tested before but which only express acceptable epitopes. At the moment quiet a number of HLA epitopes have been verified on basis of antibody reactivity and these are collected in a central data base: http://epregistry.ufpi.br/. However, the list of proven epitopes is not complete yet, which is essential before one can change the policy from acceptable HLA antigens to acceptable HLA epitopes. In order to speed up the definition of HLA epitopes, an HLA epitope workshop was organized by the project leader of Eurostam. A group of experts from the whole world came together during the last Eurostam meeting in order to define the current status of the inventory of HLA epitopes and their immunogenicity and to define specific projects, which will lead to a complete list of HLA antibody epitopes and their immunogenicity. As mentioned such a list is essential for the transition to a routine application of acceptable epitopes rather than acceptable HLA antigens. This successful workshop was attended by about 50 participants and led to the definition of two clear projects, described in the final report, which must lead to such a complete list in September 2017. These projects will be included in the International Histocompatibility and Immunogenetics Workshop, in which laboratories all over the world collaborate to solve problems, which can only be solved by international collaboration. The project leader of Eurostam is coordinator of these projects and member of the steering committee of this workshop.
For a proper implementation of a strategy based on acceptable HLA epitopes, the HLA typing strategy of organ donors should change. At the moment high resolution typing by (next generation) sequence-based typing is necessary to characterize the pivotal polymorphisms in the different HLA alleles. However, this type of high resolution typing is not routinely done in tissue typing laboratories involved in solid organ transplantation and, furthermore, in case of a deceased donor these assays take too much time. The SME Multiplicom provided its expertise and resources to development a multiplex PCR assay for the genotyping of HLA Class I epitopes. The final prototype showed a robust and efficient amplification of the target with an excellent sensitivity. This new typing technique has been validated by the laboratories in Leiden and Barcelona and showed more that more than 98% of tests were concordant. It is clear that this assay has the potential to define the crucial epitopes in a reliable way without the need of a full sequence of the HLA genes of a potential donor. In order to make this technique also useful for typing of deceased donors, where a typing should be available within 4-5 hours, partner Multiplicom is changing the read-out of the amplified products in collaboration with another company Mycartis. Furthermore, this assay will be further extended for a quick definition of the HLA class II epitopes.
3. The possible role of non-HLA antibodies in donor selection.
In order to define the possible role of non-HLA antigens on the selection of donors for highly sensitized patients, several prototypes of an assay to define complement dependent antibodies directed against endothelial cell were developed by SME Absorber. Validation studies by the other partners showed that these assays were not sufficiently robust and reliable. Therefore an alternative assay has been developed, which measures complement dependent cytotoxicity against monocytes, as several non-HLA targets are shared between endothelial cells and monocytes. However, the incidence of non-HLA antibodies was found to be very low. Based on these data and a recent study showing the excellent outcome of patients transplanted via the acceptable mismatch program of Eurotransplant (Heidt et al. Transplant Immunology 33: p51, 2015 ) , we conclude that antibodies against non-HLA targets are not very relevant for the selection of compatible donors for a population of highly sensitized patients.

Potential Impact:
1. More highly sensitized patients will be transplanted.
At the moment a significant proportion of the highly sensitized renal transplant candidates cannot be transplanted and dies on the waiting list because there is no compatible kidney donor available. This problem occurs in virtually all transplant organizations all over Europe. By introduction of a Europe-wide acceptable mismatch program, many patients with a rare HLA phenotype compared to their local donor population will be offered a chance to receive a suitable transplant. Considering the better quality of life and the lower mortality rate in transplant recipients compared to patients on dialysis, this is an important achievement. Furthermore, the costs of a transplanted patient are significantly lower than that of a patient on dialysis, which is an additional benefit for the community.
2. An easier definition of acceptable HLA mismatches.
The number of HLA alleles is still increasing and for the HLA class I antigens, which are the most important targets for the antibodies in highly sensitized patients, more than 10,000 alleles have been identified. Using classical strategies it is impossible to define all acceptable HLA mismatches for a highly sensitized patient. The epitope approach included in this project, in combination with an alternative HLA typing strategy focusing on the definition of the relevant HLA antibody epitopes, should lead to an easier and more exact definition of the acceptable mismatches.

List of Websites:
www.eurostam.eu
info@eurostam.eu