Prospective Randomized Multicenter Study Comparing Cyclosporin Alone Versus the Combination of Antithymocyte Globulin and Cyclosporin for Treatment of Patients With Nonsevere Aplastic Anemia: A Report From the European Blood and Marrow Transplant (EBMT) Severe Aplastic Anaemia Working Party

THE OUTCOME of patients with severe aplastic anemia (AA) treated with immunosuppressive therapy or allogeneic bone marrow transplantation (BMT) has improved with time. In the early 1980s, antithymocyte globulin (ATG) was shown to significantly improve the survival of patients with AA in comparison to supportive care alone.1 It was initially uncertain whether there was any benefit to be gained by combining androgens with ATG, but a prospective randomized study later demonstrated that androgens improved the response rate to ATG therapy, although they had no impact on survival.2 Furthermore, the virilizing side effects of androgens were often unacceptable to female patients, in addition to the known hepatotoxicity.

The addition of cyclosporin (CSA) to ATG treatment has also improved the response rate, as demonstrated by Frickhofen et al3 in a multicenter randomized study. Although there was a trend for improved survival using the combination of ATG and CSA compared with ATG alone, with longer follow-up evaluation of patients, the survival advantage disappeared, since patients who did not respond to ATG alone could achieve rescue by a second course of immunosuppression with ATG and CSA.4 A direct comparison of ATG and CSA was reported by Gluckman et al5 in a French multicenter randomized study. At 3 months, if patients in either treatment arm showed no response to ATG or CSA, they were switched (crossover) to the other therapy (ATG or CSA). This study suggested that CSA alone may be as effective as ATG, but the number of responses in either study arm was low and the early crossover precluded further analysis of the response. A potential practical advantage of CSA alone would be that it can be administered to outpatients, reducing the cost of treatment. In contrast, hospital admission is required for ATG treatment.

The survival of patients with severe AA after immunosuppressive therapy is determined largely by the absolute neutrophil count. Patients with a neutrophil count >0.5 × 10⁹/L have a significantly better survival rate than patients with a neutrophil count <0.5 × 10⁹/L.6,7 In a recent analysis by the European Blood and Marrow Transplant (EBMT) Severe Aplastic Anaemia (SAA) Working Party in 1,765 patients treated from 1974 to 1996,8an even more striking difference in survival was found when comparing neutrophil counts of greater or less than 0.2 × 10⁹/L, although single centers have failed to show any difference in the survival of such patients.9,10 

There have been no prospective randomized studies of immunosuppressive treatment in patients with nonsevere AA (neutrophils ≥0.5 × 10⁹/L and transfusion dependence). The objective of this prospective randomized multicenter European study was to determine whether CSA alone is as effective as the current best treatment for severe AA, that is, the combination of ATG and CSA. The main study outcome parameter is the response at 6 months, as it was predicted that there would be no significant difference in the short-term outcome in the absence of severe neutropenia and with provision of intensive supportive care for both groups.

This multicenter study was organized by the EBMT SAA Working Party and involved 54 centers from eight countries. Eligibility criteria were as follows: no specific prior treatment for the disease, a neutrophil count of at least 0.5 × 10⁹/L, hypocellular bone marrow, and red blood cell and/or platelet transfusion dependence. Patients were excluded if they had congenital AA, paroxysmal nocturnal hemoglobinuria with evidence of significant hemolysis, a clonal cytogenetic abnormality, and severe uncontrolled infection or unexplained fever higher than 38°C. Consecutive patients meeting the above criteria for nonsevere AA were randomized and treated, and no patients were excluded from the analysis.

Patients were randomized to receive either CSA alone or the combination of ATG and CSA. CSA was administered orally from day +1 to day +180 at a dose of 5 mg/kg/d in two divided doses, with subsequent adjustment according to weekly whole-blood CSA and serum urea, creatinine, and bilirubin levels. The aim was to maintain trough whole-blood CSA levels between 75 and 200 ng/mL. CSA was continued in all patients, but if the blood cell count continued to increase at 6 months, CSA was continued at the therapeutic dose until the blood cell count plateaued, and then the dose was reduced gradually to help prevent a relapse of the aplasia. The rate of CSA dose reduction was decided by the individual physician in charge. Horse ATG (Lymphoglobuline; Merieux, Lyon, France) was administered at a dose of 1.5 vials/10 kg/d for 5 days (equivalent to 15 mg/kg/d) as an intravenous infusion. For prevention of serum sickness, prednisolone 1 mg/kg/d was administered orally from day +5, continued for 9 days, and then reduced to zero over 1 week.

The endpoint of the study was the hematologic response at 6 months. A complete response was defined as a neutrophil count >2.0 × 10⁹/L, a platelet count >100 × 10⁹/L, and transfusion independence; a partial response was defined as a neutrophil count >1.0 × 10⁹/L, a platelet count >30 × 10⁹/L, and transfusion independence; and patients who remained transfusion-dependent were classified as nonresponders regardless of the neutrophil and platelet count. Failure-free survival was defined as survival with response. Death, nonresponse by 6 months, disease progression requiring a second course of immunosuppressive treatment or a stem-cell transplant, and relapse were considered treatment failures. Follow-up evaluation was continued on these patients; however, they were excluded from the analysis of hematologic response and failure-free survival.

The study hypothesis was that there is no difference in the response rates between the two treatment groups. It was estimated that the required number of patients is 225 for each treatment arm to detect a difference in the response rate of about 10% with an expected response of 70% to 80%, with 95% confidence. Interim analyses were planned for every 100 patients randomized in case one group showed an unexpected significant difference in response, survival, and failure-free survival compared with the other group. The outcome parameters were the hematologic response and failure-free survival at 6 months after treatment, overall response, survival, and failure-free survival. The chi-square test was used to compare categoric variables, and the Mann-Whitney U test (nonparametric) or Studentt-test (parametric) were used to compare continuous variables. The probability of response and survival was analyzed using the method of Kaplan and Meier.11 

Informed written consent was obtained from all patients according to established procedures at each center, and the study was approved by the local hospital ethics committees.

The study commenced in April 1993, and this interim analysis was performed in March 1997, when 115 assessable patients were randomized. Sixty-one patients were randomized to CSA therapy alone and 54 to ATG and CSA. Patient characteristics are summarized in Table1. Patients in the ATG and CSA group were significantly younger (median age, 29 v 35 years,P = .04) and had a lower median platelet count (15 v20 × 10⁹/L) compared with the CSA group. The median follow-up period was similar, 343 days for the CSA group and 365 for the ATG and CSA group, respectively.

A complete response to CSA alone was observed in 14 patients (23%) and a partial response in 14 (23%), for an overall response rate of 46% (28 of 61). In contrast, in the ATG and CSA group, 31 patients (57%) had a complete response and 9 (17%) had a partial response for an overall response rate of 74% (40 of 54) (Table2). The difference in response between the two groups was statistically significant (P = .02). At 6 months, patients in the ATG and CSA group had a significantly higher median hemoglobin concentration of 11.8 g/dL as compared with 9.7 for the CSA group (P = .03), and a strikingly higher median platelet count of 84 × 10⁹/L as compared with 29 × 10⁹/L for the CSA group (P = .005). There was no difference in the median neutrophil count between the two groups at 6 months. Figure 1 illustrates not only that the probability of response at 6 months was higher in the ATG and CSA group but also that these patients responded earlier than those receiving CSA alone. Transfusion independence before 6 weeks was observed in some patients in both treatment groups.

Significantly more patients in the CSA group had evidence of disease progression before 6 months necessitating a second course of treatment during this period. In the CSA group, 15 of 61 (25%) patients required a second course of immunosuppression with ATG before 6 months, compared with only three of 54 (6%) in the ATG and CSA group (P = .005). A further 8 patients received an allogeneic stem-cell transplant before 6 months because of disease progression (5 in the ATG and CSA group and 3 in the CSA group). After censoring patients who received a second course of immunosuppression or a stem-cell transplant before 6 months, the overall response rate between the two groups remained significantly different, 87% for the ATG and CSA group and 65% for the CSA group (P = .001) (Table3).

The median interval from diagnosis to treatment was 28 days. There was no significant difference between an interval of less than 28 days or more than 28 days and the response (data not shown).

The overall probability of survival is 93% for the CSA group and 91% for the ATG and CSA group (P = .5) with a median follow-up period of 343 days (range, 19 to 1,454) and 365 days (21 to 1,227), respectively (Fig 2). The probability of failure-free survival was calculated by excluding not only deaths but also cases requiring a second treatment for nonresponse to the first course or for disease progression before 6 months, thus identifying patients who are alive, transfusion-independent, completing 6 months without crossover to a second course and/or responding to the initial therapy, and not transplanted. There were 4 early deaths in the CSA group (2 infections, 1 hemorrhage, and 1 infection with hemorrhage) and 2 early deaths in the ATG and CSA group (both from infection). There were 2 late deaths in the ATG and CSA group (1 hemorrhage and 1 post-BMT) and 1 death in the CSA group (post-BMT). At 6 months, 49 of 54 patients (90%) in the ATG and CSA group were alive and failure-free, compared with only 41 of 61 (67%) in the CSA group (P = .001) (Table 2). The probability of failure-free survival at 1,454 days was 80% for the ATG and CSA group, compared with 51% for the CSA group (P = .0005) (Fig3).

A total of 39 patients who failed to respond to either CSA alone or ATG and CSA received a second course of immunosuppression with ATG, of whom 27 were in the CSA group and 12 in the ATG and CSA group. Nine of 27 (33%) patients in the CSA group responded to a second course of ATG, and 6 of 12 (50%) in the ATG and CSA group responded to a second course of ATG (either horse or rabbit ATG; Merieux). There was no statistical difference in the response rate to the second course of treatment between the two groups (P = .32).

To date, there have been no relapses, 2 cases of myelodysplasia (both in the CSA group), no cases of acute myeloid leukemia, and 3 cases of paroxysmal nocturnal hemoglobinuria (2 in the ATG and CSA group and 1 in the CSA group), but the follow-up period is too short to evaluate the true risk of clonal evolution in this cohort of patients.

The results of this multicenter European study of nonsevere AA show that compared with CSA alone, the combination of ATG and CSA results in a significantly higher probability of hematologic response, an earlier response, a significantly better quality of response, and fewer cases of nonresponse and disease progression. The actuarial survival was similar between the two groups (91% to 93%), but failure-free survival was significantly higher in the ATG and CSA group (90%v 67% in the CSA group). This reflects a larger proportion of patients who failed to respond to CSA alone and a larger proportion who showed evidence of disease progression before 6 months after CSA therapy as compared with patients who received initial treatment with ATG and CSA.

Since the actuarial survival of patients in the two groups is similar, this study raises the question of whether an early response to immunosuppression is important, because it was shown that patients who fail to respond to CSA may respond later to ATG. Cost is a major factor when considering the use of CSA alone as initial therapy. CSA can be used for outpatients, thus avoiding inpatient hospital costs. Secondly, if successful, it avoids the immediate and late side effects (serum sickness) of ATG, in addition to the risk of early infective deaths reported by several studies.2,5 However, the investigators from one of these studies5 emphasize that if ATG is to be administered to patients with AA, it should only be used by physicians in centers who are familiar with the medication and its side effects. Furthermore, in this study, there were four early infective deaths in the CSA group, compared with only two in the ATG and CSA group, suggesting no reduction in early mortality with CSA. A lower risk of infective deaths is expected in patients with nonsevere AA versus severe AA. The concern about delaying the use of ATG until after an initial treatment with CSA is that patients would require a longer period of transfusion support, which would increase the risk of HLA alloimmunization antigens resulting in platelet transfusion refractoriness.12,13 Sensitization to minor histocompatibility antigens increases the risk of graft rejection after HLA-identical sibling transplantation. Other potential risks include transfusion-associated viral infection and, in the longer term, transfusional hemosiderosis. The response rate to ATG as a secondary therapy in this study appears to be lower than for ATG as an initial therapy in previously reported studies,14,15 although a larger number of patients would be required to confirm this trend. Evidence of disease progression while the patient is on CSA therapy would suggest a decreased chance of response to ATG subsequently.8 Furthermore, we have demonstrated that delaying ATG therapy after CSA does not compensate for the reduced response, so we encourage the use of ATG combined with CSA as the initial therapy for nonsevere AA.

It is possible that some of the early responses seen in both treatment groups may have represented spontaneous recovery of the aplastic anemia. In both groups, some patients became transfusion independent before 6 weeks. Because it is unlikely that all these cases were caused by spontaneous recovery, we suggest that this early transfusion independence after immunosuppression may be a feature of AA patients with nonsevere disease.

For patients with severe AA and who are ineligible for allogeneic BMT, intensive immunosuppression is now recommended.9,10,16-18In contrast, patients with nonsevere AA may not require such intensive immunosuppression and treatment with ATG alone, for example, may be sufficient. This study did not examine the use of ATG alone, but this could be evaluated in future studies. It has been suggested that two or more courses of ATG may increase the risk of later clonal disorders.19,20 

Because of the significant difference in response rate at 6 months between the two groups in this study, the study was closed at this initial interim analysis stage according to the original study plan. From this study we recommend that patients with nonsevere AA who are transfusion dependent are treated with ATG and CSA to achieve an earlier and higher chance of response. We believe it is important to follow up this cohort of patients over a long period of time, not only to assess later clonal disease and relapse but also to compare the incidence of HLA alloimmunization and transfusional hemosiderosis between the two groups.

At present, we conclude that there is not a good reason to use CSA alone as the initial therapy for nonsevere AA, given the lower response, poorer blood cell counts, and lack of reduction in early mortality as compared with the combination of ATG and CSA.

We are indebted to physicians from the following centers who entered patients into this study. Italy; P. Leoni, Ancona; D. de Mattia, Bari; R. Bassan, Bergamo; C. Finelli, P. Rosito, Bologna; P. Coser, Bolzano; T. Izzi, F. Porta, Brescia; G. Broccia, Cagliari; A. Gallamini, Cuneo; A. Lippi, Firenze; A. Bacigalupo, P. Mori, M. Gobbi, Genova; P. Foa, Milano; A. Locasciulli, E. Pogliania, Monza; L. Pinto, B. Rotoli, Napoli; A. Gabbas, Nuoro; I. Majolino, Palermo; F. Locatelli, P. Allesandro, Pavia; P. di Bartolomeo, Pescara; F. Caracciolo, Pisa; P. Iacopino, Calabria; W. Arcese, G. de Rossi, Roma; M. Carotenuto, S.G. Rotondo, P. Saracco, Torino; M. Baccarani, Udine; G. Todeschini, Verona; D. Bona, Vicenza; United Kingdom: D. Milligan, Birmingham; J. Hows, Bristol; S. McCann, I. Temperley, Dublin, Ireland; F. Matthey, East Surrey; A. Parker, Edinburgh; M. Lewis, Kidderminster; J. Marsh, E.C. Gordon-Smith, D. Samsom, E. Kanfer, A. Newland, London; J. Keidan, Norfolk; T. Littlewood, Oxford; F. Booth, Reading; S. Rassam, Sidcup; S. Roath, Southampton; Germany: V. Schillin, A. Neubauer, Berlin; M. Burk, Dusseldorf; W. Heit, Essen-Werden; Sonnen, Hamburg; Mengfelder, Homburg; Fischer, Karlsruhe; Hoffken, Jena; Wolf, Magdeburg; Lengfelder, Mannheim; H. Schrezenmeier, A. Raghavachar, Ulm; Turkey: O. Ilhan, K. Halluk, Ankara; Sweden: P. Ljungman, Huddinge; F. Celsing, Stockholm; E. Forestier, Umea; I. Nilsson, Karlstad; Finland: L. Volin, Helsinki; Spain: P. Marin, Barcelona; and The Netherlands: G. den Ottolander, Leiden.