Cyclosporin combined with levamisole for refractory or relapsed severe aplastic anaemia
About one-third of patients with severe aplastic anaemia (SAA) are refractory or relapse after treatment with immunosuppressive therapy (IST) by anti-thymocyte globulin (ATG) plus cyclosporin (CSA; Olnes et al, 2012). Allogeneic haematopoietic stem-cell transplantation (HSCT) could be effective as salvage therapy, but it is difficult to find a suitable donor, limited by the family planning policy in China. Intensification of current IST regimen seems to hit the ceiling (Passweg & Tichelli, 2009). However, patients in developing countries cannot afford the repeated courses of ATG due to the high-costs burden.
A novel IST regimen with a practicable and economical solution would be ideal. Levamisole (LMS), which had been originally designed for anthelminthic applications, has a broad range of immunomodulatory effects (Stevenson et al, 1991). We considered that its immunomodulatory potential had synergetic effects when combined with CSA in refractory or relapsed SAA. We herein conducted a small sample research of 12 refractory and four relapsed SAA patients between July 2008 and August 2012, who were salvaged by CSA alternately combined with LMS (CSA–LMS regimen). All of the patients or legal guardians provided signed informed consent. The research was approved by the Institutional Committee for Medical Care and Safety.
Cyclosporin and LMS were alternately administered every other day. The dose of CSA was 3 mg/kg per day in adults or 5 mg/kg per day in children aged 6–12 years. Oral LMS was alternately administered at the same time with a dose of 150 mg per day in adults or 2·5 mg/kg per day in children (<40 kg) in three divided doses. Both CSA and LMS were continued for 12 months, followed by a slow tapering rate of 25% reduction in dose for every 3–4 months according to response. Short courses of recombinant human granulocyte colony-stimulating factor (rhuG-CSF), red cell and platelet transfusions were administered as clinically indicated. Criteria for haematological response and clonal evolution were in accordance with our previous reports (Li et al, 2013).
Eleven of the 12 refractory patients were previously treated with one cycle of ATG plus CSA and Patient4 was refractory to two cycles of ATG plus CSA. Of the four relapsed patients, Patient 3 was a CSA-resistant non-SAA who progressed to SAA 36 months after the diagnosis, followed by a matched sibling donor haematopoietic stem cell transplantation (HSCT); she achieved complete response (CR) 3 months after HSCT, but her blood counts declined while tapering CSA and finally met the criteria of SAA after an Epstein-Barr virus infection. The median interval between relapse and CSA–LMS regimen was 6 (range 3–35) months. The initial clinical characteristics and previous treatments of the cohort of patients are summarized in Table 1. All the patients were transfusion-dependent except Patient 2. Median follow-up for this study was 25 (range 3–53·5) months.
| UPN | Sex | Aetiology | Age (years) | Severity of AA at diagnosis | Severity of AA at CSA–LMS | Previous treatment | Response to initial therapy | Time from previous treatment to CSA–LMS (months) | Disease duration (months) |
|---|---|---|---|---|---|---|---|---|---|
| 1 | M | Idiopathic | 42 | VSAA | SAA | ATG + CSA | PR | 69 | 72 |
| 2 | M | Idiopathic | 19 | VSAA | SAA | ATG + CSA | PR | 35 | 36 |
| 3 | F | Idiopathic | 25 | NSAA | SAA | Allo-PBSCT + CSA | CR | 58 | 66·5 |
| 4 | F | Idiopathic | 10 | SAA | SAA | dATG + CSA | NR | 15·5 | 16 |
| 5 | M | Idiopathic | 15 | VSAA | SAA | ATG + CSA | NR | 18 | 21 |
| 6 | F | Idiopathic | 25 | SAA | SAA | ATG + CSA | PR | 9·5 | 36 |
| 7 | M | Idiopathic | 27 | SAA | SAA | ATG + CSA | NR | 8 | 13 |
| 8 | F | Hepatitis | 21 | VSAA | VSAA | ATG + CSA | NR | 6 | 6 |
| 9 | F | Idiopathic | 40 | SAA | SAA | ATG + CSA | NR | 6 | 6 |
| 10 | F | Idiopathic | 14 | NSAA | VSAA | ATG + CSA | NR | 9 | 32 |
| 11 | M | Idiopathic | 17 | VSAA | SAA | ATG + CSA | NR | 6 | 6 |
| 12 | M | Idiopathic | 14 | SAA | SAA | ATG + CSA | NR | 6 | 8 |
| 13 | M | Hepatitis | 11 | VSAA | SAA | ATG + CSA | NR | 9 | 10·5 |
| 14 | F | Idiopathic | 15 | NSAA | VSAA | ATG + CSA | NR | 46 | 60 |
| 15 | M | Idiopathic | 48 | SAA | SAA | ATG + CSA | NR | 6 | 6 |
| 16 | M | Idiopathic | 16 | SAA | SAA | ATG + CSA | NR | 12 | 13 |
- UPN, unique patient number; M, male; F, female; AA, aplastic anaemia; SAA, severe aplastic anaemia; VSAA, very severe aplastic anaemia; N-SAA, nonsevere aplastic anaemia; ATG, antithymocyte globulin; dATG, double course of ATG; CSA, ciclosporin; LMS, levamisole; Allo-PBSCT, allogeneic peripheral blood stem cell transplantation; CR, complete response; PR, partial response; NR, non-responder.
Of the cohort of 16 patients, 14 were alive at a median follow-up of 28·0 (range 4–53·5) months. Five patients achieved CR and three met the criteria for partial response (PR) at last follow-up, including two relapsed (both achieved CR) and six refractory patients. The median haemoglobin concentration, absolute neutrophil count (ANC) and platelet count of the eight responders at last follow up were 122·5 (range 90–156) g/l, 2·55 × 109/l (range 0·9–3·4 × 109/l) and 99 × 109/l (range 33–142 × 109/l), respectively (Table 2).
| UPN | Pretreatment blood counts | Interval to last transfusion (d) | Current status | Recent blood counts | Follow-up (months) | Evolution | PNH assay (%) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Hb (g/l) | ANC (109/l) | PLT (109/l) | Red cell | PLT | Hb (g/l) | ANC (109/l) | PLT (109/l) | Pre | Post | ||||
| 1 | 68 | 0·40 | 6 | – | – | NR | 38 | MDS (−7) | GPI-AP (<5) | FLAER (<1) | |||
| 2 | 77 | 0·50 | 20 | a | a | CR | 120 | 3·40 | 98 | 49·5 | GPI-AP (<5) | FLAER (<1) | |
| 3 | 81 | 1·00 | 6 | a | 48 | CR | 145 | 3·16 | 100 | 19 | FLAER (<1) | FLAER (<1) | |
| 4 | 64 | 2·04b | 4 | 243 | 427 | PR | 90 | 2·58 | 33 | 53·5 | MDS (+21) | GPI-AP (<5) | FLAER (<1) |
| 5 | 54 | 1·87 | 14 | 65 | a | CR | 156 | 3·36 | 114 | 43·5 | +6 | GPI-AP (<5) | FLAER (3·72) |
| 6 | 62 | 1·23 | 6 | – | – | NR | 44 | GPI-AP (<5) | FLAER (<1) | ||||
| 7 | 63 | 0·31 | 12 | – | – | PBSCT | 27 | GPI-AP (<5) | GPI-AP (<5) | ||||
| 8 | 57 | 4·64b | 11 | – | – | Died | 23 | GPI-AP (<5) | GPI-AP (<5) | ||||
| 9 | 40 | 0·36 | 14 | 237 | 95 | CR | 125 | 1·65 | 142 | 38 | GPI-AP (<5) | FLAER (<1) | |
| 10 | 45 | 0·18 | 3 | – | – | NR | 6 | FLAER (2·47) | FLAER (2·73) | ||||
| 11 | 48 | 0·28 | 7 | 274 | 103 | CR | 145 | 2·52 | 132 | 29 | GPI-AP (<5) | FLAER (<1) | |
| 12 | 45 | 0·80 | 15 | 184 | 61 | PR | 90 | 0·90 | 60 | 15 | del 13 | FLAER (6·10) | FLAER (<1) |
| 13 | 57 | 0·28 | 10 | – | – | NR | 4 | FLAER (<1) | FLAER (<1) | ||||
| 14 | 64 | 0·00 | 4 | – | – | Died | 3 | FLAER (16·89) | – | ||||
| 15 | 60 | 0·80 | 13 | 71 | 45 | PR | 117 | 1·20 | 72 | 9 | FLAER (<1) | FLAER (<1) | |
| 16 | 55 | 0·77 | 5 | – | – | NR | 8 | FLAER (<1) | FLAER (<1) | ||||
- UPN, unique patient number; Hb, haemoglobin concentration; ANC, absolute neutrophil count: PLT, platelet count; PNH, paroxysmal nocturnal haemoglobinuria; PBSCT, allogeneic peripheral blood stem cell transplantation; CR, complete response; PR, partial response; NR, non-responder; GPI-AP, glycosylphosphatidyl-inositol anchored protein; FLAER, fluorescent aerolysin.
- a Transfusion-independent.
- b Granulocyte colony-stimulating factor.
Of the three very severe aplastic anaemia (VSAA) patients, Patient 8 never met criteria for response and died of sepsis 23 months after receiving the CSA–LMS regimen. Patient 14 developed pneumonia before CSA–LMS, her ANC never reached more than 0·2 × 109/l even following the administration of rhuG-CSF, and she died within 3 months after CSA–LMS treatment. Patient 10 was still red cell and platelet transfusion-dependent at last follow-up. Patient 7 acheived transient transfusion independence but relapsed later, and underwent sibling donor HSCT 27 months after CSA–LMS regimen. The other four non-responders were still transfusion-dependent.
Cyclosporin–Levamisole-associated toxicity was mild and required neither intervention or discontinuation of these drugs. Patients 1 and 4 progressed to myelodysplastic syndrome (MDS) 29 and 40 months after CSA–LMS regimen. Patients 5 and 12 developed cytogenetic clonal changes without the distinctive morphological features of MDS; but the abnormal clone (trisomy 6) disappeared in Patient 5 after he achieved CR. To date, no patient developed overt paroxysmal nocturnal haemoglobinuria (PNH). Three patients had a PNH clone at time of study entry (Table 2). The PNH clone of Patient 12 disappeared after he achieved PR. A new PNH clone was observed in a CR patient (Patient 5) 10 months after CSA–LMS regimen.
A second course of ATG lead to responses of 22–77% in refractory SAA patients and 47·1–65% in relapsed SAA patients (Tichelli et al, 1998; Di Bona et al, 1999; Scheinberg et al, 2006; Kosaka et al, 2008). High dose cyclophosphamide (HD-CTX) provided a response rate of 48% in refractory patients (Brodsky et al, 2010). Most refractory and relapsed SAA patients from poor regions in China have little access to further courses of ATG, salvage HD-CTX, or HSCT because they lack human leucocyte antigen (HLA)-matched siblings, or fully HLA-matched unrelated donors or health insurance.
Herein, these concerns have motivated a search for alternative therapy for SAA. Our study underscored the importance of excellent outcomes of refractory and relapsed SAA patients (50·0%) achieved by this novel immunosuppressive strategy. More importantly, in comparison to standard ATG + CSA, our strategy was less expensive, easier to administer and well tolerated. Clonal evolution occurred in 10–15% of AA patients overall, and the clonal progression was mostly seen in non-responders (Young et al, 2006). In this series, two patients developed MDS; both of them were long-term survivors with abnormal haematopoiesis. Interestingly, both the abnormal cytogenetic clone in a patient with CR (Patient 5) and the PNH clone of Patient 12 disappeared during follow-up.
In conclusion, the CSA–LMS regimen represents a promising strategy for refractory or relapsed SAA patients. Importantly, this oral regimen was well tolerated and inexpensive, suggesting that it could be reserved as a third-line choice for patients, especially those in developing countries not eligible for HSCT, repeated ATG or HD-CTX.
Acknowledgements
The authors would like to thank Prof. Robert A. Brodsky (The Johns Hopkins School of Medicine) for critical revision of the manuscript, important intellectual content and interesting scientific discussions. This study was supported by the Fundamental Research Funds for the Central Universities (2012N05). We thank the nursing staff of Severe Aplastic Anaemia Studying Programme and our physician colleagues for the excellent care of patients.
Author contributions
YZ was the principal investigator and takes primary responsibility for this study. YS, XL and YZ designed the research study. YS, XL and JS performed the research and analysed the data. MG, JH, ZH, JZ and NN coordinated the research. YS, XL, JS and YZ wrote the paper.
Conflict of interest
The authors declared that they have no commercial, proprietary, or financial interest in the products or companies described in this manuscript.
