The outcomes of transplanting bone marrow from genetically identical twins into patients with aplastic anemia may provide insight into the causes of bone marrow failure. The infusion of genetically identical bone marrow cells without pretransplantation conditioning should permanently correct bone marrow function in persons whose bone marrow failure has resulted from a lack of or defective hematopoietic stem cells. In contrast, persons whose bone marrow failure is caused by immune abnormalities may only improve when transplantation of genetically identical bone marrow is preceded by conditioning. Finally, persons with abnormal bone marrow microenvironments might never improve [8].

We addressed these issues using data on 40 patients with aplastic anemia who received transplants from their identical twins and were reported to the International Bone Marrow Transplant Registry.

Methods

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Patients

Forty patients with severe aplastic anemia who received one or more bone marrow transplants from their genetically identical twins between 1964 and 1992 were reported to the International Bone Marrow Transplant Registry by 31 centers worldwide. Severe aplastic anemia was defined according to published criteria [9]. Nineteen patients have been described in previous reports [4, 10-14]. Genetic identity was established by measuring concordance for HLA-A, -B, -C and -DR antigens (n = 39; HLA testing was not done in 1 patient); ABO blood group (n = 39); erythrocyte antigens (n = 1); and immunoglobulin allotypes (n = 1) and by a history of a single placenta (n = 40).

Outcomes

Hematologic response, graft-versus-host disease, and survival were the study outcomes. Complete response was defined as normalization of at least two of three hematologic variables (hemoglobin level > 12 g/dL, granulocyte count > 1.5 x 10⁹/L, and platelet count > 150 x 10⁹/L) and improvement in the third (hemoglobin level > 8 g/dL, granulocyte count > 0.5 x 10⁹/L, and platelet count > 20 x 10⁹/L without transfusion). Partial response was defined as improvement in two or more variables. Lack of response was defined as levels less than those indicated above and continuing dependence on transfusion. Responses were considered to be sustained if they persisted through the date of last contact. Diagnosis and grading of graft-versus-host disease were based on published criteria [15]. Persons at risk included those with complete or partial responses who survived 21 days or longer.

Statistical Analysis

We compared patient, disease, and transplantation variables using the Mann-Whitney and Fisher exact tests. Probabilities of survival were calculated using the Kaplan-Meier product limit estimator and were expressed as probabilities with 95% CIs. The latter were computed using the arcsine-square root transformation [16]. Survival distributions were compared using the log-rank test.

Results

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Patients

We studied 25 male patients and 15 female patients. The median patient age was 19 years (range, 4 to 69 years). Physicians at the transplantation centers ascribed aplastic anemia to drugs or toxins (7 patients), hepatitis (4 patients), or other causes (3 patients). We did not ascertain which criteria were used to make these designations. In 26 cases, no cause of aplastic anemia was identified. Treatments given between diagnosis and transplantation included corticosteroid therapy (9 patients), androgen therapy (1 patient), and both (7 patients). Twenty-three patients received no therapy other than transplantation. Thirty-seven patients received transfusions before transplantation (median number of transfusions, 14 [range, 1 to 263]). Median values for hematologic variables before transplantation were the following: hemoglobin level, 7.5 g/dL (range, 4.8 to 11.3 g/dL); leukocyte count, 1.7 x 10⁹/L (range, 0.5 to 5.2 x 10⁹/L); granulocyte count, 0.3 x 10⁹/L (range, 0 to 1.6 x 10⁹/L); and platelet count, 17 x 10⁹/L (range, 1.5 to 101 x 10⁹/L). Survivors were followed for a median of 7 years (range, 13 months to 29 years); all but one were followed for at least 2 years after transplantation.

Transplantation

First Transplantation Not Preceded by Conditioning

Twenty-three patients received a first transplant without pretransplantation conditioning. Fifteen patients who did not have a sustained complete response received one or more additional transplants after first receiving conditioning with the following regimens: Ten patients received cyclophosphamide alone (150 to 200 mg/kg of body weight); 2 received cyclophosphamide (200 mg/kg) and total-body radiation (3 to 4.5 Gy); 1 received cyclophosphamide (194 mg/kg) and cyclosporine; 1 received nitrogen mustard and antilymphocyte globulin; and 1 received azathioprine. A sixteenth patient who did not have a sustained complete response after the first transplantation received the second transplant without first receiving conditioning. The median dose of nucleated bone marrow cells for the first transplant was 3.6 x 10^8/kg (range, 0.4 to 9.8 x 10^8/kg). Four of 15 female donors had had transfusion or at least one pregnancy before donating bone marrow. No patients received immunosuppressive therapy after transplantation.

First Transplantation Preceded by Conditioning

Seventeen patients received conditioning before the first transplantation. Thirteen received cyclophosphamide alone (100 to 200 mg/kg), 1 received cyclophosphamide (152 mg/kg) and cyclosporine, 2 received cyclophosphamide (156 and 219 mg/kg, respectively) and busulfan (12 and 16 mg/kg, respectively), and 1 received cyclophosphamide (200 mg/kg) with total-body radiation (3.0 Gy). The median dose of nucleated bone marrow cells was 3.6 x 10^8/kg (range, 1.6 to 4.9 x 10^8/kg). After transplantation, 6 patients received immunosuppressive therapy: One received methotrexate, 1 received corticosteroids, 2 received cyclosporine, and 2 received cyclosporine and corticosteroids. We did not ascertain the reasons why immunosuppressive therapy was given after transplantation.

The likelihood that the first transplantation would be preceded by conditioning increased during the study years: Only 3 of the initial 20 (15%) patients receiving their first transplant received conditioning compared with 14 (70%) of the subsequent 20 patients receiving their first transplant (P = 0.05). Patients who had conditioning before receiving their first transplants had lower leukocyte, granulocyte, and platelet counts and had had more pretransplantation transfusions than the patients who did not have pretransplantation conditioning (Table 1).

Outcomes

The results of transplantation are summarized in Figure 1.

View larger version (27K): [in this window] [in a new window]   Figure 1. Outcome of transplanting bone marrow from genetically identical twins into 40 patients with aplastic anemia. *Patient died of fungal pneumonia 3.5 months after first transplantation; Patient died of septicemia 3.5 years after first transplantation; Patients died of fungal pneumonia (1 patient), the acute respiratory distress syndrome (1 patient), and diffuse alveolar hemorrhage (2 patients) 7, 12, 16, and 20 days after first transplantation, respectively. Patient died of interstitial pneumonia, pneumothorax, and graft-versus-host disease 5 months after first transplantation. Values in parentheses are numbers of patients. BMT = bone marrow transplantation.

Hematologic Response: First Transplantation Not Preceded by Pretransplantation Conditioning

Seven of the 23 patients (30%) who received a first transplant without first receiving conditioning had a sustained complete response; 16 did not. Five of the 16 had a complete response but had relapse a median of 35 weeks after transplantation (range, 11 to 103 weeks). Seven other patients had a partial response; relapse occurred a median of 10 weeks after transplantation (range, 8 to 16 weeks). Four patients had no response.

Granulocyte counts before transplantation were higher in the 12 complete responders than in the 11 patients who did not have a complete response (0.9 x 10⁹/L [range, 0 to 1.6 x 10⁹/L] compared with 0.3 x 10⁹/L [range, 0 to 1.2 x 10⁹/L]; P = 0.048). Granulocyte and platelet counts and number of transfusions before transplantation did not differ between the 7 patients who had sustained complete response and the 5 patients who had complete response and later had relapse. No correlation was seen between the likelihood of a sustained complete response and age, sex, cause of aplastic anemia, interval from diagnosis to transplantation, previous therapy, infections developing within 1 week before transplantation, previous transfusions, donor pregnancies, or number of cells transplanted.

The 16 patients who did not have a sustained complete response after receiving the first transplant without first receiving conditioning had one to four additional transplantations. One patient who did not have conditioning before receiving a second transplant had a sustained complete response. Eleven of 15 patients (73%) who received conditioning before the second transplantation had a sustained complete response. One of the 15 patients died of treatment-related toxicity 1 month after transplantation. Three of the 15 did not have a sustained complete response after receiving a second transplant: Of these, one patient with a partial response and one patient with no response received a third transplant after conditioning. Both had sustained complete responses after the third transplantation. After each of five transplantations, one patient had transient hematologic responses that lasted 11 to 38 months. He died after receiving the fifth transplant.

Hematologic Response: First Transplantation Preceded by Conditioning

Seventeen patients received their first transplant after first receiving conditioning. Thirteen (76%) had a complete response, and 11 (64%) had a sustained complete response. One patient had relapse 20 months after transplantation; a second transplantation preceded by conditioning resulted in a sustained complete response. Patients who received conditioning before receiving the first transplant had a higher incidence of sustained complete responses than did patients who did not receive conditioning before receiving the first transplant (P = 0.033; Fisher exact test).

Graft-versus-Host Disease

Four cases of graft-versus-host disease were reported among the 33 patients at risk for the disease. All 4 patients had grade 2 or higher acute graft-versus-host disease that involved the skin; 3 of the 4 also had gastrointestinal tract involvement. Acute graft-versus-host disease was confirmed by skin biopsy in 1 case and by skin and rectal biopsies in another. The latter patient subsequently developed chronic hepatic graft-versus-host disease that was confirmed by biopsy and autopsy. In 2 patients, diagnosis was made by clinical assessment only. All 4 patients with graft-versus-host disease had received conditioning before receiving their first transplant; after transplantation, 2 of the 4 also received immunosuppressive therapy with methotrexate (1 patient) or corticosteroids (1 patient). Four patients received immunosuppressive therapy after transplantation but did not develop graft-versus-host disease; they received cyclosporine, either alone (2 patients) or combined with corticosteroids (2 patients).

Survival

Seven patients died, including two whose first transplantation was not preceded by conditioning: One of the two died of fungal pneumonia 1 month after having a second transplantation that had been preceded by conditioning; the other died of septicemia after having four more transplantations, each preceded by conditioning, 3.5 years after the first transplantation.

Five patients whose first transplantation was preceded by conditioning died. Four died soon after transplantation, before hematologic recovery. None of these patients received immunosuppressive therapy after transplantation. Causes of death were fungal pneumonia (1 patient), the acute respiratory distress syndrome (1 patient), and diffuse alveolar hemorrhage (2 patients). One patient died of interstitial pneumonia, pneumothorax, and graft-versus-host disease 5 months after transplantation; this patient's bone marrow function was normal.

Thirty-three patients were still alive a median of 7 years (range, 1 to 29 years) after the first transplantation. The 10-year actuarial probability of survival is 78% (95% CI, 59% to 92%). According to the log-rank test of the equality of the survival functions, survival curves for patients whose first transplantation was not preceded by conditioning significantly differed from the curves for patients whose first transplantation was preceded by conditioning (P = 0.037). The 10-year probability of survival is 87% (CI, 66% to 99%) for patients whose first transplantation was not preceded by conditioning and 70% (CI, 47% to 89%) for patients whose first transplantation was preceded by conditioning. Although the log-rank test suggests that the two survival distributions are different, the 95% CIs for survival overlap at 10 years because of the relatively large estimated variability at this single point. All 7 patients who had sustained complete responses after a first transplantation that was not preceded by conditioning have survived a median of 7.5 years (range, 4.3 to 29 years) since transplantation. Fourteen of 16 patients who received no conditioning before their first transplantation and then received two to five more transplants had been alive at study end a median of 9 years (range, 1 to 14 years) after the first transplantation. At study end, 12 of 17 patients whose first transplantation was preceded by conditioning had been alive a median of 4.3 years (range, 2.1 to 14.3 years) since the first transplantation, including the 1 patient who received the second transplant because of recurrent bone marrow failure.

No hematologic or other malignant conditions were seen in the transplant recipients during a median observation period of 7 years (range, 1 to 29 years). No hematologic disorders were observed in donors during this interval.

Discussion

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Our study raises several interesting issues about aplastic anemia. First, although the numbers are small, the twins appear to have had discordance for this disease: None of the 40 normal twins developed any hematologic abnormalities during a median of 7 years of follow-up. Interpretation of this finding is influenced by the fact that the International Bone Marrow Transplant Registry records only those twin pairs in which one twin is normal at the time of transplantation.

Second, only 7 of 23 patients whose first transplantation was not preceded by conditioning had sustained complete recovery even though they received large numbers of normal bone marrow cells from their twin. These data suggest that many cases of aplastic anemia are caused by factors other than lack of or defective bone marrow cells. Although a few sets of patients and donors may have been incorrectly identified as twins, incorrect assignment is unlikely to account for failure to recover in most patients. Furthermore, most nonresponders recovered after a second or subsequent transplantation that was preceded by conditioning. This recovery may have occurred because some donors were HLA-identical siblings rather than genetically identical twins; however, this explanation is also unlikely because of the high recovery rate and absence of graft-versus-host disease despite the lack of administration of immunosuppressive therapy after transplantation in most patients. In previous studies of allografts from HLA-identical siblings that were transplanted without prophylaxis for graft-versus-host disease, a high incidence of early, severe acute graft-versus-host disease was found [17].

A more likely reason for the recovery of nonresponders after the second or third transplantation is that some or most cases of bone marrow failure were immune mediated. This interpretation is supported by the third interesting finding: a high rate of sustained complete responses (71%) in patients whose first transplantation was preceded by conditioning. Similar estimates of the frequency of immune-mediated bone marrow failure have been provided by studies of such immunosuppressive therapies as antithymocyte globulin and cyclosporine [1, 18, 19]. Additional support comes from in vitro studies of immune reactions to hematopoietic cells, involving antibodies, cytotoxic and regulatory T cells, natural killer cells, and cytokines [2, 7, 20-25].

The fourth point is that no clonal hematologic disorder or leukemia was seen in any patient despite prolonged follow-up. This finding contrasts with several reports of a high incidence of clonal hematologic disorders in persons with aplastic anemia who received immunosuppressive therapy but did not have transplantation [26-28]. Several recent studies reported an increased risk for cancer in patients who received allografts for aplastic anemia [29-35]. There are too few twins with which to analyze the effect of allogeneic compared with twin donors or the effect of conditioning on risk for cancer after transplantation.

One surprising finding was the four cases of graft-versus-host disease. Three of these cases were grade 2 or higher; only two were confirmed by histologic testing. Graft-versus-host disease occurred only in patients whose first transplantation was preceded by conditioning. No association was seen between development of graft-versus-host disease and immunosuppressive therapy after transplantation. None of the four patients who received cyclosporine developed graft-versus-host disease. Although it is difficult to comment critically on these cases, the fact that disease correlated with conditioning but not with cyclosporine therapy suggests that some diagnoses of graft-versus-host disease were incorrect. Two cases, however, were confirmed by biopsy, including the one in which chronic liver graft-versus-host disease developed. We cannot exclude the possibility that some pairs of patients and donors were not twins or that conditioning increases the likelihood of "syngeneic" graft-versus-host disease [36].

Our results suggest that because more early deaths occurred among the patients who received conditioning before transplantation, patients whose first transplantation was not preceded by conditioning had better survival. Whether this difference in survival is the result of the conditioning or of other differences between these groups cannot be definitively determined. Conditioning was more often given to patients who had more severe aplastic anemia, as reflected by lower leukocyte, granulocyte, and platelet counts and by greater numbers of transfusions before transplantation. In addition, we cannot exclude the possibility that conditioning was more likely to be given when physicians questioned the accuracy of the twin assignment. Finally, the number of patients in each group was small. Although times to death differed significantly, the 10-year probabilities of survival have overlapping 95% CIs; this suggests that caution should be used in drawing conclusions.

Several other aspects of our study, including its retrospective, uncontrolled design; variable data collection; and imperfect characterization of genetic identity, limit the certainty of our analyses. Most of these considerations are inherent to observational databases that evolve over several decades. Unfortunately, the infrequency with which transplants from genetically identical twins are used for aplastic anemia makes our approach the only possible study option.

In summary, these data on more than 40% of the twins throughout the world who received transplants for aplastic anemia help answer several important questions about the biology and treatment of aplastic anemia. They also raise new issues that need to be addressed, particularly the recent trend toward the use of pretransplantation conditioning for most patients receiving transplants from an identical twin.

From the International Bone Marrow Transplant Registry, Medical College of Wisconsin, Milwaukee, Wisconsin; Donauspital, Vienna, Austria; Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia; Rigshospitalet, Copenhagen, Denmark; University of Munich, Munich, Germany; University of Louisville, Louisville, Kentucky; and Salick Health Care, Inc., Los Angeles, California.

Dr. Gibson: Haematology Department, Royal Prince Albert Hospital, Missenden Road, Camperdown, New South Wales 2050, Australia.

Dr. Jacobsen: Bone Marrow Transplant Unit, Department of Haematology L4042, Rigshospitalet-9, Blegdamsvej, 2100 Copenhagen, Denmark.

Dr. Klein: Division of Biostatistics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226.

Dr. Kolb: Medizinische Klinik III, Klinikum Grosshadern, Universitat Munchen, Marchioninistrasse 15, 8000 Munchen 70, Germany.

Dr. Stevens: Division of Medical Oncology/Hematology, James Graham Brown Cancer Center, University of Louisville, 529 South Jackson Street, Louisville, KY 40292.

Dr. Gale: Bone Marrow and Stem Cell Transplantation, Salick Health Care, Inc., 8201 Beverly Boulevard, Los Angeles, CA 90048-4520.