Effects of antithymocyte globulin on bone marrow CD34+ cells in aplastic anaemia and myelodysplasia

The mechanism of action of antithymocyte globulin (ATG) in the treatment of aplastic anaemia (AA) and myelodysplastic syndromes (MDS) is poorly understood and may involve many different mechanisms. The aim of this in vitro study was to investigate further the effect of ATG on haemopoietic progenitor cells. A total of 16 patients (10 AA and 6 MDS) and 12 normal control subjects were studied. Purified bone marrow (BM) CD34+ cells were cultured in committed progenitor assay in the presence of ATG and autologous serum, then scored on day 14 for granulocyte-monocyte colony-forming units (CFU-GM) and erythroid colonies. ATG was found to be inhibitory to haemopoietic progenitor cells at high concentrations (1000 microg/ml and 100 microg/ml). This was confirmed by CD34-FITC and 7AAD staining of purified normal CD34+ cells after overnight incubation with ATG. In contrast, at lower doses (0.1-10 microg/ml), ATG produced an increase in colony growth in most normal, MDS and AA BM CD34+ cells. The greatest effect was in patients with non-severe AA, in whom the greatest increase in CFU-GM was seen at 0.5 microg/ml (P < 0.02) and 0.1 microg/ml (P = 0.02) and erythroid colonies at 0.1 microg/ml (P < 0.05). Serum ATG levels peaked during infusion to levels that were found to be toxic to haemopoietic progenitor cells in vitro and fell thereafter to levels that were associated with the highest colony numbers (0.1 and 0.5 microg/ml) in vitro. These results suggest that an increase in haemopoietic progenitor cells by ATG may be one of several important mechanisms for haematological recovery in AA and MDS.     

Effect of androgens on the response to antithymocyte globulin in patients with aplastic anaemia

30 patients with aplastic anaemia (18/30 with severe aplastic anaemia) were prospectively randomized to be treated with 100 mg/kg ATG with or without the oral androgen Methenolone (3 mg/kg). 15 of 30 patients responded. Among the 15 patients receiving ATG plus androgen, 11 patients (73%) responded, including 8 complete and 3 partial responses. 4 of the 15 patients (31%) receiving ATG only responded, including 2 complete and 2 partial responses. The difference in response rate was statistically significant (p = 0.01). The survival rate in the total population of 30 patients was 64%. The survival rate in the group receiving ATG plus androgen was 87%; in the group receiving ATG only it was 43%. The difference in survival rates between both groups did not reach statistical significance (p = 0.15). Toxicity of ATG and androgens was considerable but manageable. These data support the result of the recent European reevaluation of a large pool of patients by the EBMT (39), that androgens in addition to ATG increase survival in patients with aplastic anaemia. They are, however, in contradiction to a controlled American study showing no benefit of a combined treatment with androgens as compared to ATG only. Further controlled studies on a larger number of patients are indicated to determine the therapeutic efficacy of androgens in addition to immunosuppression in aplastic anaemia.     

The incidence and significance of fevers during treatment with antithymocyte globulin for aplastic anaemia

Antithymocyte globulin (ATG) is a foreign protein used widely to treat aplastic anaemia (AA). Febrile reactions occurring during its administration may be impossible to distinguish clinically from fever due to sepsis, and are therefore routinely treated with intravenous antibiotics after collection of blood cultures. A statistically highly significant difference was found in positive blood cultures between 39 AA patients who developed fever during ATG therapy, and 38 febrile neutropenic patients with acute leukaemia, suggesting that most fevers developing during ATG treatment are not due to infection. It may therefore be reasonable to consider early discontinuation of intravenous antibiotics in patients who are clinically stable and have no proven sepsis.     

Favourable response to antithymocyte or antilymphocyte globulin in low-risk myelodysplastic syndrome patients with a 'non-clonal' pattern of X-chromosome inactivation in bone marrow cells

OBJECTIVE: Antithymocyte and antilymphocyte globulin (ATG/ALG) have a therapeutic effect in about 30% of patients with myelodysplastic syndromes (MDS). We were interested to know whether responding patients achieve clonal or polyclonal remissions. PATIENTS: Ten women with low-risk MDS received either ALG or ATG. Before treatment and 3, 6, and 12 months later, X-chromosome inactivation patterns of peripheral blood T lymphocytes were compared with those of peripheral blood granulocytes or bone marrow cells, using the human androgen receptor gene assay and the phosphoglycerate kinase-1 assay. RESULTS: Six women did not respond to therapy. Prior to treatment, four of them had a monoclonal, one had an oligoclonal, and one had a skewed X-chromosome inactivation pattern (XCIP). Four patients responded to ATG/ALG. Three of them were informative in our X-inactivation assays, and showed a non-clonal XCIP which did not change significantly after treatment with ATG/ALG. CONCLUSION: A non-clonal XCIP in the bone marrow was associated with a response to ATG/ALG. Non-clonal XCIPs do not necessarily imply that there is no pathological clone. By definition, they just indicate that there is no evidence of a clone contributing more than 50% of cells in a sample. Non-clonal XCIPs may therefore be attributable to incomplete clonal expansion. This, in turn, might be explained by a vigorous immune attack against the MDS clone, which simultaneously causes collateral damage in the remaining normal haemopoiesis. In such patients, ATG/ALG may improve normal haemopoiesis by relieving the immunological pressure on the innocent bystanders.     

A case of refractory anaemia with p53 point mutation at codon 249 (AGG to ATG)

Although mutations of the p53 tumour-suppressor gene have been observed occasionally in the advanced stages of myelodysplastic syndromes (MDS), they have not been detected in early refractory anaemia (RA) or RA with ring sideroblasts phases. Using single-strand conformation polymorphism analysis and direct sequencing, we searched for p53 mutations in a patient who progressed from RA to overt leukaemia. A p53 mutation at codon 249 (AGG to ATG) was observed in RA, RA with excess of blasts in transformation and overt leukaemia. We describe a case of MDS with p53 mutation at codon 249 detected in the RA phase.     

A case of refractory anaemia with p53 point mutation at codon 249 (AGG to ATG)

Although mutations of the p53 tumour-suppressor gene have been observed occasionally in the advanced stages of myelodysplastic syndromes (MDS), they have not been detected in early refractory anaemia (RA) or RA with ring sideroblasts phases.
Using single-strand conformation polymorphism analysis and direct sequencing, we searched for p53 mutations in a patient who progressed from RA to overt leukaemia. A p53 mutation at codon 249 (AGG to ATG) was observed in RA, RA with excess of blasts in transformation and overt leukaemia.
We describe a case of MDS with p53 mutation at codon 249 detected in the RA phase.     

Comparable TNF-alpha,IFN-gamma and GM–CSF production by purified normal marrow CD3 cells in response to horse anti-lymphocyte and rabbit antithymocyte globulin

Abstract: In vitro priming of T cell with horse antilymphocyte globulin (HALG) results in cytokine release, and this has been associated with its clinical efficacy in patients with severe aplastic anaemia (SAA). Rabbit antithymocyte globulin (RATG) has been studied less extensively. In this study we compare the in vitro priming effect of HALG and RATG on purified normal marrow T cells: end-points of the study were 1) levels of TNF-alpha (TNF-α), IFN-gamma (IFN-γ) GM–CSF in T cell supernatants, and 2) effect of T cell supernatants on colony formation with or without exogenous GM–CSF. TNF-α, IFN-γ and GM–CSF levels were comparable for HALG, RATG and phytohaemagglutinin (PHA). T cell supernatants showed comparable enhancement of colony formation in the presence of recombinant human GM–CSF (rhGM–CSF) and supported colony forming unit granulomacrophage (CFU–GM) growth in the absence of growth factor. This study shows that horse and rabbit derived ALG/ATG and PHA have a comparable in vitro priming effect on T cells: both agents should probably be tested for their clinical efficacy in SAA patients.     

Clinical characteristics predict response to antithymocyte globulin in paroxysmal nocturnal haemoglobinuria

Seven patients with paroxysmal nocturnal haemoglobinuria (PNH) were treated with antithymocyte globulin (ATG). Each patient received ATG (20 mg/kg/d) for 8 d and prednisone to prevent or control serum sickness. Three patients experienced a sustained improvement in at least one peripheral blood cytopenia, including one patient who had a complete trilineage response. Several pre-treatment clinical features appeared to be associated with response to ATG. All responding patients had hypoproliferative features including depressed platelet counts (<30×10⁹/l), and a minor degree of chronic haemolysis as indicated by relatively low reticulocyte counts (<100×10⁹/l), lactate dehydrogenase (<1000 U/l) and total bilirubin (<17μmol/l) levels. Responding patients continued to have chronic low-grade haemolysis after their response to immunosuppression that was similar to that observed prior to treatment. The non-responding patients had a classic haemolytic form of PNH characterized by elevated reticulocyte counts (>100×10⁹/l), lactate dehydrogenase (>2000 U/l) and total bilirubin (>17μmol/l) levels.
The impaired haemopoiesis that occurs in hypoproliferative PNH may respond to ATG treatment, but the haemolytic component of the disease, and hence the PNH clone, is not altered by immunosuppressive therapy.     

Rabbit antithymocyte globulin (r-ATG) plus cyclosporine and granulocyte colony stimulating factor is an effective treatment for aplastic anaemia patients unresponsive to a first course of intensive immunosuppressive therapy. Gruppo Italiano Trapianto di Midollo Osseo (GITMO)

About 30% of patients with severe aplastic anaemia (SAA) unresponsive to one course of immunosuppressive (IS) therapy with antithymocyte or antilymphocyte globulin can achieve complete or partial remission after a second IS treatment. Among various second-line treatments, rabbit ATG (r-ATG) could represent a safe and effective alternative to horse ALG (h-ALG). In a multicentre study, 30 patients with SAA (17 males and 13 females, median age 21 years, range 2-67) not responding to a first course with h-ALG plus cyclosporin (CyA) and granulocyte colony stimulating factor (G-CSF), were given a second course using r-ATG (3.5 mg/kg/d for 5 d), CyA (5 mg/kg orally from day 1 to 180) and G-CSF (5 microg/kg subcutaneously from day 1 to 90). The median interval between first and second treatment was 151 d (range 58-361 d). No relevant side-effects were observed, but one patient died early during treatment because of sepsis. Overall response, defined as transfusion independence, was achieved in 23/30 (77%) patients after a median time of 95 d (range 14-377). Nine patients (30%) achieved complete remission (neutrophils >/=2.0 x 109/l, haemoglobin >/=11 g/dl and platelets >/=100 x 109/l). The overall survival rate was 93% with a median follow-up of 914 d (range 121-2278). So far, no patient has relapsed. Female gender was significantly associated with a poorer likelihood to respond (P = 0.0006). These data suggest that r-ATG is a safe and effective alternative to h-ALG for SAA patients unresponsive to first-line IS treatment.     

Cyclophosphamide and antithymocyte globulin as a conditioning regimen for allogeneic marrow transplantation in patients with aplastic anaemia: a long-term follow-up

A total of 81 severe aplastic anaemia patients, aged 2-63 years, received human leucocyte antigen-matched related marrow grafts after cyclophosphamide + antithymocyte globulin followed by postgrafting methotrexate + cyclosporin. Median follow-up was 9.2 years. Ninety-six per cent of patients had sustained engraftment, 24% developed acute graft-versus-host disease (GVHD), grade II in all but two patients, and 26% developed chronic GVHD; all surviving patients eventually responded to immunosuppressive therapy. Six patients developed cancer: one fatal lymphoma and five carcinomas (all five patients are now free of cancer). Survival was 88%. The regimen appeared well tolerated and effective in heavily pretreated patients with aplastic anaemia.     

Treatment of red cell aplasia with antithymocyte globulin: repeated inductions of complete remissions in two patients

Two patients with red cell aplasia unresponsive to prednisone and cyclophosphamide were treated with antithymocyte globulin (ATG). Both patients developed reticulocytosis within 2-4 days after ATG treatment and had complete remissions. Within 4-6 months, they relapsed, and after retreatment with ATG both again developed reticulocytosis and remission. ATG should be considered for the treatment of patients with red cell aplasia who fail to respond to glucocorticoid/alkylator treatment.     

Antithymocyte globulin for patients with myelodysplastic syndrome

Twenty-five transfusion-dependent myelodysplastic syndrome (MDS) patients (with < 20% blasts) were treated in a phase II study with antithymocyte globulin (ATG) at 40 mg/kg/d for four doses and then followed with blood counts every 2 weeks and clinic visits every 3 months, for a median of 14 months (range 1–38 months). 11 (44%) patients responded and became transfusion-independent after ATG, including three complete responses, six partial responses, and two minimal responses. Responses were observed in 9/14 patients (64%) with refractory anaemia (RA) and 2/6 patients (33%) with refractory anaemia with excess blasts (RAEB). Median response duration was 10 months (range 3–38 months). The Kaplan-Meier estimate of overall survival was 84% at 38 months, with one early death due to pneumonia and two deaths from disease progression to leukaemia. Side-effects consisted mainly of mild serum sickness in all patients. A single course of ATG restored haemopoiesis in some patients with MDS and was well tolerated.     

Aplastic anemia associated with bone marrow suppressor T-cell hyperactivity: successful treatment with antithymocyte globulin.

In five patients with idiopathic aplastic anemia, the colony-forming unit (CFU-c) assay was used to quantitate stem cells and test the possible presence of suppressor cells inhibiting bone marrow differentiation. All five marrows failed to form CFU-c. In one out of the five cases tested, coculture of the patient's marrow with normal marrow suppressed the latter to form CFU-c. Removal of T-cells from the patient's bone marrow freed the aplastic marrow to produce more colonies and, in the coculture study, abrogated its inhibitory activity on the normal bone marrow. Treatment of the patient with horse antihuman thymocyte globulin (ATG), improved the peripheral blood count and myelopoiesis. Furthermore, the aplastic marrow increased its colony forming capacity from less than 1% pre-ATG to 46% post-ATG of the normal control, and its inhibitory activity on the normal marrow was ablated. In selected cases of aplastic anemia, with evidence of T-suppressor cell hyperactivity, treatment with ATG may offer an alternative approach to bone-marrow transplantation.     

Response to immunosuppressive therapy and an HLA-DRB1 allele in patients with aplastic anaemia: HLA-DRB1*1501 does not predict response to antithymocyte globulin

HLA-DRB1*1501, a subtype of HLA-DR2, has been shown to be closely associated with a good response to cyclosporine (CyA) therapy in patients with aplastic anaemia (AA). To determine whether this DRB1 allele can also predict a response to antithymocyte globulin (ATG) therapy in AA patients, we analysed the results of HLA-DRB1 typing in 59 Japanese patients who received ATG within 2 years after diagnosis of AA and also in 52 patients treated with CyA. All patients were divided into three groups: those with DRB1*1501, those with DRB1*1502, and those without either of these two alleles (DR2^–). The response rate to ATG in DRB1*1501⁺ patients (56%) was not significantly higher than that in DRB1*1502⁺ patients (47%) and in the other DR2^– patients (54%). In contrast, the response rate to CyA therapy in DRB1*1501⁺ patients (92%) was significantly higher than that in the DRB1*1502⁺ (41%) and in DR2^– patients (57%). Multivariate analysis revealed that possessing DRB1*1501 was an independent factor significantly predictive of a good response to CyA. These results indicate that although identifying the DRB1*1501 allele in AA patients prior to therapy is predictive of a good response to CyA therapy, it does not have a predictive value for ATG therapy.     

Successful treatment of chronic refractory pure red cell aplasia with antithymocyte globulin: correlation with in vitro erythroid culture studies

Two contrasting cases of chronic refractory pure red cell aplasia (PRCA) responsive to a commercial preparation of horse antihuman thymocyte globulin (ATG) are reported. Both cases were refractory to trials of cyclophosphamide, corticosteroids, and plasmapheresis. One patient developed a reticulocytosis after a single intravenous infusion of ATG; the other patient responded after administration of 14.7 g of ATG over a 28-day course. At presentation, erythroid progenitors (CFU-E and BFU-E) in one patient were normal; in the second patient, the number of erythroid progenitors was severely reduced. Neither patient had a serum IgG inhibitor to progenitor cells as judged by in vitro erythroid colony studies. Both patients had increased numbers of marrow T-cells and co-culture studies in one case were consistent with T-cell-mediated suppression of erythropoiesis. These studies confirm that ATG is a useful agent in the treatment of refractory PRCA. However, ATG may not act by removal of T suppressor cells in all cases.     

Immune hemolytic anemia following administration of antithymocyte globulin

Antithymocyte globulin is commonly used as a therapy for rejection of transplanted organs. Its use can be associated with many side effects. We report a significant hemolytic anemia following therapy with horse-derived antithymocyte globulin. Several commercial Coombs' sera failed to yield a positive Coombs' test with the patient's erythrocytes; however, we were able to detect horse immunoglobulin on the patient's cells as well as on control red cells incubated with this particular preparation of antithymocyte globulin.     

猪抗人淋巴细胞球蛋白治疗重型再生障碍性贫血的临床研究

目的：探讨一线应用猪抗人淋巴细胞球蛋白（P-ALG）联合环孢素（CsA）治疗重型再生障碍性贫血（SAA）的疗效及不良反应。方法回顾性分析我科应用 P-ALG 联合 CsA 治疗36例 SAA 患者的疗效、并发症及转归,并对患者的年龄、疾病严重程度、发病距应用 P-ALG 的时间等因素与疗效进行相关分析。结果36例患者中死亡4例,白细胞恢复中位时间为30天,脱离红细胞输注的中位时间为55天,脱离血小板输注的中位时间为60天。1年总有效率为86．2％,2年总有效率达91．7％,1年总有效率与性别、年龄、疾病分型、CD8比例无明显相关（P ＞0．05）,病程＜3个月及外周血中性粒细胞（ANC）≥0．2×109／L 患者的有效率高;与国内及欧美国家报道的利用马或兔抗胸腺细胞球蛋白（ATG）治疗 SAA 的有效率相当,而不良反应小,花费仅为马或兔 ATG 治疗的1／3～1／2。结论应用 P-ALG 联合 CsA 治疗重型再生障碍性贫血疗效显著,不良反应轻微。     

Pre-emptive therapy of acute graft-versus-host disease: a pilot study with antithymocyte globulin (ATG).

We have previously shown that patients at high risk of graft-versus-host disease (GVHD) and transplant-related mortality (TRM) can be identified on day +7 following an allogeneic bone marrow transplant (BMT), based on serum bilirubin and blood urea nitrogen levels. One possible approach to reduce the risk of GVHD and TRM, is pre-emptive treatment with T cell antibodies. We report a pilot study testing the feasibility of this approach in 18 high risk patients, with a median age of 41, 83% of whom had advanced disease, undergoing an alternative donor BMT (family mismatched in five and unrelated in 13). The patients received three doses of rabbit antithymocyte globulin (ATG) (Thymoglobuline; Sangstat) 1.25 mg/kg on alternate days, starting at a median interval of 11 days (range 7-13) after BMT. Controls were 20 historical unrelated donor transplants (median age 35, 63% with advanced disease), with a high score from our original publication in 1999. The actuarial 1 year TRM of the ATG-treated patients was 40% compared to 60% for untreated controls (P = 0.06). Severe grade III-IV aGVHD developed in 27% of the ATG-treated patients, and in 55% of the controls (P = 0.08). This study indicates that early pre-emptive treatment of aGVHD in day +7 high risk patients is feasible and may lead to a reduction of aGVHD and TRM. This approach is being tested in a prospective randomized trial.     

Retreatment with rabbit anti-thymocyte globulin and ciclosporin for patients with relapsed or refractory severe aplastic anaemia

The management of patients with severe aplastic anaemia (SAA) who do not have a matched sibling donor and fail a course of horse anti-thymocyte globulin (h-ATG)/ciclosporin (CsA) is uncertain. Repeated courses of ATG-based immunosuppression are often employed; in children and increasingly in adults, alternative donor haematopoietic stem cell transplantation is an option. We analysed the success rate of retreatment with rabbit ATG (r-ATG)/CsA in 43 patients treated at our institution in the last 5 years; 22 were refractory (20 adults; two children) to h-ATG/CsA-based regimens and 21 (17 adults; four children) had relapsed after h-ATG/CsA-based regimens. The overall response rate was 30% in patients who were refractory to h-ATG and 65% in patients who had relapsed following h-ATG. The 1000-d survival in patients who responded to r-ATG was 90% compared with 65% in non-responders. Six patients developed a clonal haematological disorder; two were responders, two were non-responders and in two the evolution occurred before the response could be assessed at 3 months following r-ATG. Thirteen patients died; three were responders, six were non-responders and four patients died prior to 3 months when response was assessed. In our study, the response rate in refractory patients was inferior to what has been previously reported.