Low dose antithymocyte globulin for the treatment of older patients with aplastic anaemia

Leukemia Research 30 (2006) 1517–1520

Low dose antithymocyte globulin for the treatment of older patients with aplastic anaemia S.B. Killick a,∗ , J.D. Cavenagh b , J.K. Davies b , J.C.W. Marsh c a

Department of Haematology, Royal Bournemouth Hospital, Castle Lane East, Bournemouth BH7 7DW, UK b Department of Haematology, St. Bartholomew’s and The Royal London Hospitals, UK c Department of Haematology, St. George’s Hospital and St. George’s University of London, UK Received 1 February 2006; received in revised form 1 February 2006; accepted 2 February 2006 Available online 10 March 2006

Abstract We report 14 older patients with aplastic anaemia (AA) who were treated with ‘low dose’ antithymocyte globulin (ATG). The aims of the study were to assess the efficacy and safety of reduced dose ATG in patients over the age of 60 years. Median age was 71 years (range 62–74 years). At the study endpoint (response to treatment at 6 months) 12 patients were evaluable. All patients received lymphoglobuline (horse ATG; Genzyme) at a dose of 0.5 vials/10 kg/day for 5 days (5 mg/kg/day, equivalent to one-third of the standard dose). There were no deaths attributed to ATG. Two patients died during follow-up, from sepsis and anaphylaxis following platelet transfusion, respectively. Only one of the 12 evaluable patients responded to treatment and remains transfusion independent at 14 months after ATG. These results suggest that this lower dose of ATG, though well tolerated, had low efficacy in the treatment of AA. © 2006 Elsevier Ltd. All rights reserved. Keywords: Aplastic anaemia; Antithymocyte globulin; ATG

1. Introduction Antithymocyte globulin (ATG) is widely used in the treatment of aplastic anaemia (AA), and more recently its use has been explored in a subgroup of patients with myelodysplastic syndromes (MDS). Response rates are approximately 60–80% in AA [1–3] and 30–50% in low risk MDS [4–6]. Previous data suggests that older patients with AA have similar response rates to younger patients [7], however, the treatment tends to be less well tolerated with a higher mortality from infections and bleeding compared with younger patients. For this reason, older patients often do not receive ATG as part of their treatment, with supportive therapy alone favoured. Within the class of ATGs, there are several different products marketed; although broadly similar in mode of action, they differ in potency and thus dose. The product

∗

Corresponding author. Tel.: +44 1202 704783; fax: +44 1202 300248. E-mail address: [email protected] (S.B. Killick).

0145-2126/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2006.02.003

most widely used and studied in Europe is lymphoglobuline, which was used in this study. Previous in vitro work has shown that high concentrations of lymphoglobuline (similar to in vivo peak infusional drug levels) are toxic to haemopoietic progenitor cell growth in cell culture and also result in increased cell death of CD34+ cells by apoptosis. In contrast, lower concentrations of ATG stimulate colony growth from bone marrow CD34+ cells of patients with AA and MDS [8]. These data led us to investigate using lower doses of ATG in older patients. The aims of this study were to determine the efficacy of low dose ATG (lymphoglobuline, Genzyme) in older patients with AA, and to assess the safety of its use within this population.

2. Patients and methods A total of 14 AA (7 with non-severe AA, 4 severe AA and 3 very severe AA) patients ≥60 years of age from three hospitals in the UK were treated with horse ATG

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(lymphoglobuline, Genzyme). Of these, 12 patients had received no prior therapy and none had previously received ATG. A single treatment course was given at a dose of 0.5 vials/10 kg/day for 5 days, equivalent to 5 mg/kg/day. A test dose (one-tenth vial) was given prior to the full dose as an intravenous infusion. Before each daily dose of lymphoglobuline, platelets were infused if the platelet count was <50 × 109 /l. All patients received antibacterial, antifungal and antiviral prophylaxis. Oral prednisolone (0.5 mg/kg/day) was given for the prevention of serum sickness for 9 days commencing on day 5 of therapy. Diagnosis and severity of disease for AA patients were classified according to peripheral blood and bone marrow findings [9,10]. The study end point was response at 6 months. Response was defined as published by Camitta [11]. For this pilot study, a minimum of 14 patients (Gehan design) was required to address the aims [12]. Informed consent was obtained from all patients, and the Local Research Ethics Committees approved the study.

3. Results Table 1 shows the patient characteristics. The median age was 71 years (range 62–74 years). The median time from diagnosis to treatment was 2 months (range 1–11 months). HLA DR15 status was assessed in 12 patients and was positive in all those tested. PNH clones were assessed by flow cytometry and were detected in two patients pre-treatment; one had a small neutrophil PNH clone (CD16 87%) and one had a large clone deficient in GPI-anchored proteins affecting neutrophils and monocytes (CD14 56%, CD16 34%) [13]. All patients tolerated the full 5-day course; 13 patients had first dose fever and rigors and 5 patients experienced serum sickness requiring intravenous hydrocortisone. There were 12 AA patients evaluable at the study end point. During the study period there were two deaths; UPN 4 at 3 months following treatment with ATG due to anaphylaxis following a platelet transfusion and UPN 8 at 5 months from neutropenic sepsis.

Table 1 Patient characteristics UPN

Age (years)/gender

Disease

Cytogenetics pre-treatment

Time from diagnosis to treatment (months)

Previous treatment

Pre-treatment FBC Hb (g/dl), neut (×109 /l), plts (×109 /l)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

67/F 71/F 65/M 72/M 67/M 74/M 72/F 74/M 62/M 72/M 72/F 71/M 69/M 64/M

VSAA NSAA NSAA SAA SAA SAA NSAA NSAA NSAA VSAA SAA VSAA NSAA NSAA

Failed Normal Normal Normal Normal 45,X,-Y 45,X,-X Normal Normal Normal Normal Normal Normal – FISH onlyb Normal

3 4 1 1 2 1 11 1 2 2 7 1 6 1

None None None None None None CSA None None None CSA None None None

7.8a , 0.1, 9a 9.9, 1.2, 12 8.0a , 0.6, 4a 10.7a , 0.3, 28a 10.9a , 0.2, 11a 9.4a , 0.3, 18a 11.1a , 3.4, 11a 9.4a , 0.7, 32a 9.6a , 1.3, 4a 9.6a , 0.1, 37a 7.9a , 0.2, 66a 7.6a , 0, 53a 9.0a , 1.0, 63a 10.9a , 0.8, 15a

HLA DR15

PNH clone

Response at 6 months/outcome

Positive Positive Positive Positive ND Positive Positive Positive Positive Positive Positive Positive ND Positive

No No No No No Small Large No No No No No No No

NR NR NR NR died at 3 months NR NR PR NR died at 5 months NR NR NR NR NR, transformed to AML NR

Legend: M, male; F, female; VSAA, very severe aplastic anaemia; SAA, severe AA; NSAA, non-severe AA; CSA, ciclosporin; ND, not done. Response: PR, partial response; NR, no response [11]. Hb, haemoglobin level; neut, neutrophil count; plts, platelet count. a Denotes counts supported with either red cell or platelet transfusions, respectively. b UPN 13 did not have a standard karyotype performed. FISH analysis showed no abnormality for deletion 20, trisomy 8, deletion 5q, and monosomy 5; deletion 7/monosomy 7 failed.

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Only one patient with AA (UPN 7) had a response to ATG at 6 months follow-up. This patient continues to have a partial response to treatment at the time of publication (14 months) and remains transfusion independent.

4. Conclusion This study recruited the required number of older AA patients and only one of 14 patients responded to low dose ATG. The rationale for this study using low dose ATG is summarised as follows: (i) patients >60 years of age have an increased risk of dying from infection and bleeding after ATG compared with younger patients with AA [7]; (ii) ATG is now being used in MDS patients who are often elderly raising additional concerns about toxicity [14] and (iii) high doses of ATG in vitro are toxic to bone marrow colony forming cells [8]. ATG is a polyclonal IgG preparation. The most commonly used preparations are lymphoglobuline (horse ATG, Genzyme) and Thymoglobuline (rabbit ATG, Genzyme). These two ATGs are prepared by immunising horses or rabbits, respectively, with human thymocytes. Other ATGs are also manufactured by immunising horses or rabbits, but use different immunogens which result in slightly different antibody specificities. ATG is a mixture of antibodies, many of which are directed against non-T-cell specific antigens. ATG reacts with many cell types and molecules, namely T-cells, B-cells, NK-cells, monocytes/macrophages, neutrophils and platelets, and endothelial cells. ATG also reacts with molecules involved in the immune response, integrins and their ligands, and chemokine receptors [15,16]. Studies comparing the antibody specificities between thymoglobuline and lymphoglobuline are in broad agreement, but there are fewer studies with lymphoglobuline and those reported are older because the product is older and has been less extensively developed. Retrospective clinical experience reflects the laboratory data and suggests that there is no difference between these two products, although there have been no randomised studies comparing these two preparations of ATG in AA. Additionally, care must be taken when treating patients as the potency and thus dose requirements are very different. The mechanisms of its immunosuppressive action include T-cell depletion, apoptosis of activated B-cells [17], effects on dendritic cells resulting in interference with T-cell activation [18] and modulation of cell surface molecules [15]. In AA, ATG may act by depletion of auto-reactive cytotoxic T-cell clones. Risitano and colleagues have recently demonstrated disappearance of expanded CD8+ T-cell clones looking at the T-cell repertoire with TCR ␤-CDR3 sequencing [19]. ATG is also mitogenic in the absence of complement resulting in release of haemopoietic growth factors [20]. ATG reduces apoptosis and Fas antigen expression on AA bone marrow CD34+ cells [21]. ATG also stimulates normal haemopoietic progenitor cells and induces differentiation of HL60 cells [22,23]. More recently, it has been shown that ATG also

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directly stimulates normal and AA CD34+ cells at serum levels between 0.1 and 10 ␮g/ml but is toxic to colony growth between 100 and 1000 ␮g/ml. It is notable that peak serum levels measured after horse ATG given to patients with AA were between 90 and 650 ␮g/ml [8]. ATG binds to normal and AA CD34+ cells, as assessed by FACS. Higher levels of binding are found in patients who respond to ATG compared with non-responders, untreated patients and those who relapse after ATG treatment [24]. Several mechanisms may account for the T-cell depletion induced by ATG, some depending on the concentration of ATG, as well as the animal source used: (1) complementmediated lysis occurs only at high ATG concentrations (100–1000 ␮g/ml) with both horse and rabbit ATG [25]. (2) Activated T-cells are susceptible to antibody dependent cellmediated cytotoxicity (ADCC) induced by ATG and this occurs at low rabbit ATG concentrations (0.1–1 ␮g/ml). It does not occur with horse ATG [25]. Activation-induced cell death (AICD) occurs after several days and is mediated by Fas expressed on activated lymphocytes and is dose dependent [25]. Recently, another mechanism of apoptosis by ATG has been documented using high ATG concentrations. Early release of cathepsin-B from lysosomes into the cytosol triggers apoptosis independently of cytochrome C release and caspase activation [26]. Using low dose horse ATG one might anticipate loss of some action of ATG through complement mediated lysis of Tcells and cathepsin-B dependent apoptosis of T-cells. ADCC is preserved at low rabbit ATG concentrations but not with horse ATG. AICD activity is preserved at low ATG concentrations. The low response rate seen in this study suggests that AICD may not be an important mechanism of horse ATG (lymphoglobuline) in inducing response in AA. Unfortunately serum ATG levels were not measured in our study so we do not know if enough ATG was present in the serum to act via the other mechanisms when using ATG at one-third the standard dose (5 mg/kg/day for 5 days instead of the standard dose of 15 mg/kg/day for 5 days). We conclude that low dose ATG (lymphoglobuline), when given at one-third of the standard dose, was well tolerated but had low efficacy in inducing a sustained haematological response in older patients with AA. For such patients we suggest using the standard dose of 15 mg/kg/day for 5 days, but each patient should be assessed clinically very carefully to determine whether they are fit enough or not for ATG treatment. 5. Conflict of interest This study was supported in part by Genzyme. Acknowledgement We would like to thank Peter Thomas for his statistical advice.

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Contributions. Dr. S. Killick was the co-ordinator of the trial and was the key author of the manuscript. Dr. Cavenagh and Prof. Marsh were main collaborators and recruited patients. They both had a major input into the trial design and the final manuscript. Dr. J. Davies collected a significant amount of the data and was involved in the writing of the manuscript.

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