Immunosuppression with ATG and cyclosporine is often administered as first therapy in severe aplastic anemia, since most patients lack a histocompatible sibling donor or are not suitable candidates for stem cell transplantation due to age, comorbidities, or lack of access to this treatment modality. Most published experience is with the horse formulation of the polyclonal antibody. In the past decade, rabbit ATG plus cyclosporine has gained in popularity due to its activity in relapsed and refractory severe aplastic anemia. In some centers in the US, rabbit ATG has been used as first therapy, and in Europe, Japan and Latin America, rabbit ATG is the only formulation currently available.
The reported experience with rabbit ATG (Thymoglobulin®) plus cyclosporine as initial therapy in severe aplastic anemia is limited to retrospective studies with conflicting results. In a small phase II study in the US of 13 patients with severe aplastic anemia, response to rabbit ATG was observed in 12 (92%) at about 3 months after therapy.21
In contrast, a retrospective analysis conducted in Brazil of 71 patients found a higher response rate at 6 months in those who had received horse ATG (60%) compared to rabbit ATG (35%), with a survival benefit noted for the former. In addition, rabbit ATG was an independent predictor of mortality in multivariate analysis in this study.22
In another recent and also retrospective report from Europe, there was no difference in overall response between horse (49%) and rabbit ATG (45%) when administered as first line therapy; 23
but the response rate to horse ATG of 49% was markedly lower than reported response rates of 60–70% with this regimen in large prospective studies in the US, Europe, and Japan.1
In the current work, in which a randomized prospective protocol with good matching of patients in randomization arms was executed to completion, rabbit ATG plus cyclosporine was inferior to horse ATG plus cyclosporine when administered as first line treatment. The hematologic response rate for rabbit ATG was about half that of horse ATG, which translated into about a 25% worse survival at 3 years. Despite a relative short period of follow-up, the rate of relapse and clonal evolution do not appear to differ between the 2 arms.
These results were unanticipated, given the success of rabbit ATG in treating relapsed and refractory severe aplastic anemia and the superiority of this regimen in protecting kidney allografts.13–16
The introduction of this regimen as first line in severe aplastic anemia was logical due to its greater immunosuppressive properties17
, and a higher response rate and improved survival anticipated. Our study was originally designed to test this hypothesis and powered to detect a 25% difference between 2 groups. Due to the large clinical difference between the 2 arms, the response rate and survival crossed statistical boundaries of significance at the conclusion of the study with confidence intervals between groups that did not overlap for hematologic response and survival.
Our data raise questions as to the mechanism by which hematopoiesis is restored following ATG administration in severe aplastic anemia. Despite undergoing apparently similar manufacturing processes, there are marked differences in vitro
and in vivo
between the horse and rabbit preparations of ATG. In human peripheral blood mononuclear cells co-cultured with different ATGs, expansion of regulatory T cells was observed with rabbit ATG but not with horse ATG.18
Furthermore, there was a marked difference in gene expression profile in human cells cultured with either horse or rabbit ATG.18
In humans, more prolonged lymphopenia follows rabbit ATG administration, and patterns of viral reactivations have been shown to differ between these two agents.17
Lot-to-lot variability among ATGs is unlikely to explain the observed large differences in outcomes. First, laboratory testing has not disclosed significant or consistent dissimilarity in cytotoxicity or antigen binding specificities among multiple horse and rabbit ATG lots24, 25
nor among different commercially available ATGs.26
Second, in our clinical experience over several decades, the response rate to horse ATG in sequential protocols for treatment-naïve aplastic anemia has been nearly identical, at an average of 62% (including the current study).10–12
Responses to rabbit ATG in refractory severe aplastic anemia have appeared stable in separate studies conducted over 10 years at our institution, at about 33%.16, 27
Third, as many more animals contribute to the preparation of rabbit ATG, less variability would be expected with this formulation. Fourth, the kinetics of lymphocyte depletion with either agent was consistent in patients. Finally, secular trends in the response observed in the current study remained steady, nor were there significant differences in response rates among patients treated with different lots of ATG in this study (data not shown).
Other, more plausible explanations likely account for our results. Both ATGs led to similar depletion of CD8+
cytotoxic T cells, but there was more profound CD4+
T cell depletion after rabbit ATG (). One possible inference is that CD8+
T cell depletion is linked to success of horse and rabbit ATG, as expected from the pathophysiology of aplastic anemia, but that loss of CD4+
T cells after rabbit ATG may be detrimental. The CD4 cell compartment is phenotypically and functionally heterogeneous. Contained within the large CD4 cell population are regulatory T cells, which regulate immune response. In the current study, the frequency of regulatory T cells was higher after rabbit ATG (as predicted from tissue culture experiments)18, 19
, but this effect was negated by the more potent deletion of CD4+
T cells compared to horse ATG (). CD4 cells have other positive effects on hematopoiesis and they may be important for hematologic recovery as well as in promoting tolerance in this setting (as after stem cell transplantation).28
In addition, horse serum might contribute to recovery of hematopoiesis by stimulatory effects in the bone marrow.29, 30
More prolonged lymphopenia after rabbit ATG might impair marrow recovery as stimulatory cytokines derived from T-cells are depleted.31
It is unclear if further intensification of immunosuppression will yield superior outcomes in severe aplastic anemia.32
The addition of mycophenolate mofetil10
added to horse ATG plus cyclosporine has not achieved this goal, and now the addition of more potent lymphocytotoxic agents (rabbit ATG and alemtuzumab) in place of horse ATG has yielded inferior results. Nevertheless, horse ATG plus cyclosporine should remain the immunosuppressive regimen of choice in severe aplastic anemia as first line therapy. As horse ATG is not available in Europe and Japan, these data have implications for the treatment of aplastic anemia worldwide, as well as to the mechanism of action of polyclonal antisera in general and in particular in this disease.