Rejection Prophylaxis in Corneal Transplant
A randomized study of HLA matching (the FANCY study)
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Background: Graft survival after corneal transplant is threatened by immunological graft rejection. Twenty to thirty percent of patients with corneal transplants experience at least one rejection episode in the first 5 years after transplantation. Prophylaxis through matching for human leukocyte antigens (HLA) is controversial. We herein report the results of the Functional ANtigen matChing in keratoplastY (FANCY) trial.
Methods: FANCY was a randomized, double-blind, multicenter clinical trial. The primary objective was to evaluate superiority of HLA matching versus random graft assignment. The primary endpoint was rejection-free graft survival. We included both normal-risk and high-risk indications. The study is registered with ClinicalTrials.gov (NCT00810472).
Results: 721 patients were included, 639 patients were randomized. 474 patients underwent keratoplasty within the study; 165 patients received grafts outside the trial. One patient died and one patient was lost to follow up. We observed 33 graft rejections in the HLA matching arm (n = 224). The corresponding estimated cumulative incidence rate of immune reactions after two years was 15.7%. We observed 40 rejections in the control arm (n = 249). After two years this yields an estimated cumulative incidence rate of 17%.
Conclusion: In our heterogenous study group, HLA matching did not show a significant advantage compared to random graft assignment. The rejection rate in our sample was lower than expected. Therefore no definite conclusions can be drawn as to whether HLA matching is beneficial in corneal transplantation.
All diseases affecting the shape or transparency of the cornea are potentially vision threatening. Corneal disease is among the most common causes of blindness (1). In Germany, Fuchs endothelial dystrophy, bullous keratopathy, keratoconus, and corneal scars are the most prominent indications for corneal transplants. For more than a hundred years now, keratoplasty has been the most commonly performed transplantation procedure worldwide (2).
The ocular immune privilege
The success of keratoplasty is owed to a phenomenon known as the ocular immune privilege: Allogeneic material is tolerated in the anterior eye chamber whereas it is vigorously rejected in almost all other body locations (3). The immune privilege of the eye is attributed to sequestration behind an efficient blood–retina barrier, local humoral immunosuppression, and the anterior chamber-associated immune deviation (ACAID). ACAID is maintained by antigen-specific antigen-presenting cells that migrate from the eye and induce specific regulatory T cells that systemically suppress graft rejection (4).
However, despite regular prophylaxis with topical steroids, 20 to 30% of patients with corneal transplants experience at least one rejection episode in the first 5 years after transplantation (2, 5, 6). Whenever the non-regenerative graft endothelium is immunologically destroyed, the graft fails, either directly or later on. Graft rejection thus threatens the long-term success of the keratoplasty procedure (7). Repeat keratoplasty carries an elevated risk of graft rejection, irrespective of the primary indication (8). This is relevant because every single keratoplasty comes with a certain risk of blindness and the number of keratoplasty procedures that can be performed per eye is limited.
The situation is worse in eyes with compromised immune privilege. This occurs as a result of trauma, chronic surface inflammation, or due to the patient requiring an oversized graft. In these “high-risk” situations, almost all grafts are rejected in the first 3 years after transplantion, despite topical and systemic immunosuppression (9). One reason may be that long-term adherence to systemic immunosuppressive medications such as cyclosporine A or mycophenolate mofetil is substantially limited (10).
This is, at least partially, due to controversial evidence. Table 1 summarizes our search of Pubmed using the keywords “keratoplasty AND HLA AND matching”. Only two of the retrieved publications reported on prospective trials, with the collaborative corneal transplant study (CCTS) being the only randomized controlled trial (13). The CCTS did not detect a beneficial effect of HLA matching in keratoplasty. This result was later questioned, however, after errors in HLA typing were discovered (14). The other prospective trial, the corneal transplant study II (CTFS II), did not assess the efficacy of HLA matching per se, but the influence of HLA class II matching on allograft rejection in patients well matched at HLA class I (15).
A search of two clinical trials registries (https://clinicaltrials.gov/ and http://apps.who.int/trialsearch/) using the same above-mentioned keywords yielded no further trials.
HLA matching has been questioned after animal experiments using mouse models have shown that the “minor” (H) transplantation antigens may be more important in graft rejection than the major histocompatibility complex (16, 17, Table 1).
It is currently unclear whether HLA matching is effective in rejection prophylaxis in human keratoplasty. It is also unexplained whether H transplantation antigens play a comparably significant role in human corneal transplantation as they do in animal models. We designed the Functional ANtigen matChing in keratoplastY trial (FANCY) to answer both questions.
The study protocol of the FANCY trial has been published elsewhere (29). FANCY was a randomized, controlled, double-blind, multicenter clinical trial with two parallel arms. We included all patients scheduled for penetrating or lamellar endothelial keratoplasty. Patients with deep anterior lamellar keratoplasty were excluded. Another major exclusion criterion was an anticipated waiting time for a matching donor of more than 6 months. This was predicted based on the rareness of patients’ HLA phenotypes (30).
More methodological data including statistical analyses and sample size calculation can be found in the eMethods section.
We included a total of 721 patients. 68 patients were excluded due to rare HLA phenotypes and 14 for other reasons. 639 patients were randomized. 165 patients withdrew early from the trial and were not followed (5 were found in retrospect not to have met the inclusion criteria, one patient died, 97 withdrew their informed consent while on the waiting list, and 62 patients dropped out for other reasons). 474 patients underwent keratoplasty (337 received penetrating keratoplasty, 13 limbo-keratoplasty, and 123 lamellar endothelial keratoplasty). Only one patient was lost to follow-up. A CONSORT flow diagram showing the flow of participants through the trial is provided in Figure 1 (CONSORT, Consolidated Standards of Reporting Trials).
473 patients received keratoplasty as part of the FANCY study and were followed for at least one day. The baseline characteristics of the intention-to-treat (ITT) population (study arms as randomized, irrespective of actual treatment) are summarized in Table 2. For 39 patients in the HLA matching arm no suitable donor was found within the study period. These patients received transplants from the next suitable donors. The actual degrees of HLA matching are given in eTable 1.
We observed 33 graft rejection episodes and 19 graft failures in the matching arm (n = 224). The corresponding cumulative incidence rate of immune reactions after two years is 15.7% (95% confidence interval: [11.4; 21.5]). In the control arm, we observed 40 rejection episodes and 14 graft failures (n = 249). This yields a cumulative incidence rate of 17% [12.7; 22.8] after two years. The Fine and Gray model that compares the cumulative incidence rates of graft rejection in the ITT population shows an estimated subdistribution hazard ratio of 0.97 [0.61; 1.54], p = 0.90. The incidence rates of immune reactions in the ITT population are shown in Figure 2.
The as-treated (AT) population comprises 386 patients with complete HLA-typing from the reference laboratory. We subdivided the AT population on the basis of actual HLA mismatches into a well-matched group (up to 2 out of 6 possible HLA mismatches at the HLA loci A, B and DR) and an unmatched control group (between 3 and the maximum of 6 HLA mismatches at the loci A, B and DR). To foster comparability with the earlier studies, HLA typing was performed at low resolution (”broad alleles”).
As a third group, we also included in the AT population patients without complete HLA-typing. No clinically relevant differences were observed in the AT population (eTable 2, eTable 3).
For high-resolution matching we also conducted an HLAMatchmaker analysis. We dichotomized the patients on the basis of 10 mismatched HLAMatchmaker eplets both for HLA class I (A and B) and class II (DR) separately. This threshold was chosen to achieve group sizes comparable to the conventional HLA matching. We assessed the degree of HLA class I matching against a background of HLA class II matching as this had been specifically targeted in the CTFS II (15). To this end, we formed 4 subgroups:
- Subjects well-matched both at HLA-class I and HLA-class II,
- Subjects well-matched only at HLA-class I,
- Subjects well-matched only at HLA-class II
- Subjects neither matched at HLA-class I nor at HLA-class II.
On this basis we observed a weak beneficial tendency for matching at HLA-DR in HLA-class I matched grafts. However, this tendency was not statistically significant: testing with a Fine and Gray regression model yielded a p-value of 0.58. A comparison of the subgroups AB <10, DR <10 versus AB ≥ 10, DR ≥ 10 yielded a p-value of 0.40.
Minor histocompatibility antigens
We typed donors and recipients for the minor H antigens HA-3, HA-8, ACC-1, and ACC-2 as these have been identified to be of importance in bone marrow transplantation and graft-versus-host disease (GvHD) (31). Imunological constellations were rare. Therefore, these antigenes are of limited clinical use in keratoplasty patients (Table 3). In the FANCY trial, no gender difference in graft rejection rates was observed for women reveiving a man’s cornea. The H-Y effect was not stronger when the HLA-A1 and A1 restrictions were taken into account (data not shown).
HLA matching as standard treatment
FANCY was designed in 2007. At that time, penetrating keratoplasty was the undisputed gold standard. Graft rejection was the major barrier towards graft survival. FANCY was designed to address the question whether HLA matching should be introduced as a standard treatment. Therefore, we deliberately included the low-risk situations (e.g. keratoconus and Fuchs endothelial dystrophy). It is important in patients with low-risk indications to limit their waiting time for a matching donor, because the risk for and the impact of graft rejection is low compared to high-risk indications. For ethical reasons, we limited the waiting time to 6 months because the delay of surgery had to be balanced against the putative benefit from HLA matching. To achieve this, we assigned the next available graft whenever a patient waited longer than 6 months. However, we maintained the randomized group assignment irrespective of the actual graft assignment. This intention-to-treat approach therefore enabled assessment of the usefulness of HLA matching within a maximum waiting time of 6 months. We found that HLA matching was not clearly beneficial under these circumstances. Interestingly, a total of 97 patients withdrew informed consent while on the waiting list. Unfortunately, we did not collect data on the individual motivations for these decisions. We hypothesize, however, that a prominent reason may have been the desire to schedule the procedure instead of having to wait for an indefinite time in the trial.
The efficacy of HLA matching
The negative result with respect to the primary endpoint is unexpected because several retrospective publications report a benefit from HLA matching (Table 1). This may be attributable to our intention-to-treat approach by which patients randomized into the HLA matching arm who had been waiting in vain for a matching donor for 6 months were allocated the next available random graft. We therefore conducted an “as-treated” analysis. This analysis too showed no advantage of HLA matching in preventing immune reactions (see eMethods). However, analysis using HLAMatchmaker revealed that transplant recipients’ immunological risk is increased in situations where donor materials are well-matched at HLA class I but mismatched at HLA class II. This is in accordance with a large retrospective study on the influence of class II matching against a background of histocomatibility at HLA class I in penetrating keratoplasty (28).
One reason for the negative outcomes may be that FANCY is underpowered, considering that substantially fewer cases of graft rejection were observed than had to be expected. No evidence of underreporting was documented in the on-site monitoring reports. The degree of immunosuppression was not higher than expected (data not shown). The most likely explanation for this is the remarkable success of the posterior lamellar techniques, particularly of Descemet membrane endothelial keratoplasty (DMEK). This method was introduced during the recruitment phase of FANCY. However, the reduced risk of graft rejection in DMEK grafts was not recognized until mid-2012 (32), when patient enrollment in FANCY was completed and 33 patients with DMEK had been included. However, we did not see a stronger matching effect in a subgroup analysis of only penetrating keratoplasties, with the caveat of a lowered statistical power (data not shown).
While most retrospective studies with positive results were larger than FANCY, some smaller studies also reported a beneficial effect of HLA matching (Table 1). These smaller studies, however, might be confounded by factors such as the surgeon’s learning curve or by bias from HLA matching being offered to a selected group of patients only.
Taking into consideration the above-mentioned statistical power considerations, it is still possible that HLAMatchmaker-based HLA matching might be beneficial in keratoplasty. An HLAMatchmaker-based approach in particular might help avoid unfavorable constellations with respect to graft tolerance induction.
Minor (H) antigens in human keratoplasty
Alternatively, the lack of effect from HLA matching could be due to a lack of biological relevance of HLA antigens in keratoplasty. Accordingly, minor (H) transplantation antigens have been associated with graft rejection in mouse models of keratoplasty, whereas this is not the case for the major histocompatibility complex (33, 34). A recent genome-wide analysis has linked corneal graft rejection to several minor loci in a swine keratoplasty model (16). Minor antigens have also been studied in human keratoplasty (35). In particular, H-Y antigens have been assessed in more detail. A recent meta-analysis has shown that grafts from male donors represent an immunological risk factor in female patients (36).
In the FANCY trial, we opted to analyze ACC-1, ACC-2, HA-3, and HA-8 in addition to the H-Y antigens. This choice was based on their importance in bone marrow transplantation. Our data suggest that the immunological role of the minor antigens ACC-1, ACC-2, HA-3, and HA-8 in keratoplasty is subordinate. Their apparently small effect on graft rejection and the sparsity of the mismatched constellations limit their clinical relevance (Table 3). Interestingly, FANCY did not confirm the positive findings of the H-Y meta-analysis (36). One explanation might again be that the statistical power is lower than anticipated. By comparison, the incidence rate of graft failure due to rejection in the CCTS, for example, was as high as 26% after 3 years (13).
FANCY is the largest clinical trial on HLA matching in keratoplasty with high resolution HLA and minor-antigen typings. Our major finding is that HLA matching is not beneficial as a standard treatment in a mixed group of patients undergoing normal-risk or high-risk keratoplasty.
However, the study is underpowered due to an event rate that was lower than anticipated. This renders definite conclusions impossible. It is still possible that HLA matching is of benefit in selected patients, provided they are willing to wait for a matching donor.
FANCY was funded by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG). The Bio Implant Services Foundation (NL) provided financial support for study planning.
Conflict of interest statement
The authors declare that no conflict of interest exists.
Manuscript received on 19 July 2017; revised version accepted on 11 January 2018
Prof. Dr. med. Daniel Böhringer
Klinik für Augenheilkunde,
Killianstraße 5, 79106 Freiburg
Eye Center, Medical Center—University of Freiburg, Faculty of Medicine, University of Freiburg, Germany: Prof. Dr. med. Daniel Böhringer, Prof. Dr. med. Thomas Reinhard
Clinical Trials Unit, Medical Center—University of Freiburg, Faculty of Medicine, University of Freiburg, Germany: Dr. phil. Birgit Grotejohann, Dr. rer. nat. Gabriele Ihorst
ADMEDICO Augenzentrum Olten, Switzerland: Dr. med. Helga Reinshagen
Laboratory for Translational Immunology, University Medical Center Utrecht, The Netherlands:
Eric Spierings, PhD
Members of the FANCY study group (in order of recruitment numbers):
Eye Center, Medical Center—University of Freiburg, Faculty of Medicine, University of Freiburg, Germany: Prof. Dr. med. Reinhard
Saarland University Eye Hospital, Homburg/Saar: Prof. Dr. med. Seitz
Department of Ophthalmology, University Medical Center of the Johannes Gutenberg University Mainz, Germany: Prof. Dr. med. Pfeiffer, PD Dr. med. univ. Dr. med. Lorenz
Clinic of Ophthalmology, University Medical Center Hamburg-Eppendorf (UKE): PD Dr. med. Linke
Department of Ophthalmology, Universitätsklinikum Erlangen: Prof. Dr. med. Kruse
University Eye Hospital, Ludwig Maximilians University (LMU), Munich: Prof. Dr. med. Messmer
Department of Ophthalmology, University of Muenster Medical Center:
Prof. Dr. med. Uhlig
The Department of Ophthalmology, Goethe-University Frankfurt am Main : Prof. Dr. med. Kohnen
Department of Ophtalmology , University Hospital Schleswig-Holstein,
Campus Kiel: Dr. med. Noelle
Department of Ophthalmology, Universitätsklinikum Würzburg:
Prof. Dr. med. Hillenkamp
Department of Ophthalmology, Essen University Hospital:
Prof. Dr. med. Bornfeld
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