Allografting

Marrow transplants from matched unrelated donors for aplastic anaemia using alemtuzumab, fludarabine and cyclophosphamide based conditioning

Summary:

Graft failure, regimen-related toxicity and graft-versus-host disease (GVHD) are the critical barriers to unrelated donor transplants for aplastic anaemia (AA). We investigated the use of a novel conditioning regimen consisting of alemtuzumab (humanized CD52 antibody), fludarabine and cyclophosphamide in seven patients with AA, who underwent bone marrow transplant procedure using matched unrelated donors. The aetiology of AA was acquired (n=3), Fanconi's (n=3) and congenital (n=1). Median age was 13 years (range 8–35). All the donors were fully matched for HLA class I and II antigens using high-resolution typing. All the patients engrafted at a median of 18 days (range 13–35). Two patients died of transplant-related complications: one of adenovirus disease and the other developed extensive chronic GVHD of skin followed by cytomegalovirus (CMV) disease. Three patients developed Grade II acute GVHD disease (GVHD); none had Grade III–IV acute GVHD. Of the six evaluable patients, only one developed chronic GVHD. We conclude that this conditioning regimen for unrelated donor transplants for AA is sufficiently immunosuppressive to allow stable engraftment and appears to have a favourable impact on the incidence and severity of GVHD, warranting further investigation.

Main

The long-term survival of aplastic anaemia (AA) with bone marrow transplantation (BMT) using HLA-identical family donors has improved over the years and is in the range of 75–90%.1,2,3 However, in comparison, the survival of patients transplanted from unrelated donors has only been in the range of 20–50%.2,4,5,6 Main causes of transplantation failure in unrelated donor transplants in patients with AA (acquired as well as Fanconi's anaemia) are a high-incidence of graft failure, toxicities related to conditioning regimen and graft-versus-host disease (GVHD).5,6 To improve the outcome of unrelated donor transplants, a multidimensional approach using strategies addressing all the above issues is needed.

The optimum conditioning regimen for unrelated donor transplants in AA is not known. Excellent long-term outcomes have been reported with cyclophosphamide (CY) and antithymocyte globulin (ATG) in the conditioning of acquired AA patients using HLA-identical family donors.3 However, a similar approach with unrelated donors resulted in a high-incidence of graft failure.7 Irradiation containing regimens result in better engraftment, but add substantial regimen-related toxicity and more GVHD resulting in no improvement in survival.8,9,10,11 The role of low-dose total body irradiation (TBI, 200–300 cGy) in acquired AA has also been investigated and recommended as a measure to reduce graft rejection.12,13 Even with low-dose of TBI, fatal pulmonary toxicity such as diffuse alveolar damage has been observed.12

Alemtuzumab (Campath-1H) is a humanized IgG1 anti-CD52 monoclonal antibody (MoAb) and effectively depletes lymphocytes. In a pilot study, we previously reported a low incidence of GVHD in marrow transplants from HLA-identical sibling donors using in vivo anti-CD52 MoAb [Campath-1G (rat IgG2b)] plus CY in patients with AA.14 However, the incidence of graft failure was high both for related (24%, unpublished observation) as well as for unrelated (55%) donor transplants.15 As intensive immunosuppression is a key factor for the successful outcome of these transplants, we hypothesized that the addition of fludarabine to the conditioning regimen based on CY and CD52 MoAb may result in sustained haematopoietic reconstitution in patients with AA undergoing transplantation from unrelated donors. In this report, we present our preliminary observations on seven patients using this approach. The primary outcomes of interest were engraftment, regimen-related toxicity and GVHD.

Patients and methods

Patients

Between November 2000 and January 2004, seven patients with AA underwent marrow transplants from matched unrelated donors at St George's Hospital in London. The baseline characteristics are shown in Table 1. The aetiology of AA was: acquired (n=3), Fanconi's anaemia (n=3) and congenital (non-Fanconi) (n=1). Median time from diagnosis to BMT was 16 months (range 4–228). Two patients with acquired severe AA (UPN 3 and 5) had failed two courses of immunosuppressive therapy with ATG in combination with cyclosporine (CSA). One patient with very severe AA (UPN 7) developed invasive fungal infection of lung soon after diagnosis, which was responding poorly to antifungal therapy. Marrow transplantation from an unrelated donor was considered in preference to immunosuppression to facilitate quicker neutrophil recovery. All three patients with acquired severe AA were heavily transfused (>50 transfusions) prior to BMT. Patient (UPN 6) with congenital AA was dependent on oxymetholone for more than 15 years and had developed liver function abnormalities. One patient with Fanconi's anaemia (FA) (UPN 2) had progressive cytopenias, whereas the other two (UPN 1 and 4) had stable cytopenias. All the three patients with FA/AA had >3 somatic abnormalities. All patients or their guardians consented for the BMT. The study was approved by the local research and ethics committee.

Table 1 Donor and recipient characteristics and outcome of aplastic anaemia patients undergoing MUD transplants

Donors

The donors were identified from national and international registries. HLA compatibility was carried out by molecular methods. All donors were fully matched for HLA class I and II antigens using high-resolution typing. Donor–recipient characteristics relevant to BMT are shown in Table 1.

Transplantation regimen and GVHD prophylaxis

The conditioning regimen consisted of alemtuzumab 0.2 mg/kg/day i.v. for 5 days for children and 20 mg/day i.v. for 5 days for adults. Fludarabine was given in a dose of 30 mg/m2/day i.v. for 4–5 consecutive days. CY was given in a dose of 10–20 mg/kg × 4 days for patients with FA and 300 mg/m2/day × 4 days for other patients. One patient (UPN 3) with acquired AA had CY in a dose of 50 mg/kg × 4 days. The dosage schedule of pre-BMT conditioning is shown in Table 1. No planned G-CSF was given to any patients to facilitate neutrophil recovery.

Unmanipulated bone marrow stem cells were used in all patients. GVHD prophylaxis was with CSA alone in all except one (UPN 2). UPN 2 received CSA, short course of methotrexate and alemtuzumab in a dose of 0.1 mg/kg i.v. on alternate days from days +2 to +14. Supportive care was given as described in our previous report.15

Definitions and evaluation of response

Engraftment was defined as the first of 3 consecutive days with an absolute neutrophil count >0.5 × 109/l. Patients were evaluable for engraftment if they survived at least 21 days post transplant. Patients who did not have a neutrophil count >0.5 × 109/l for 3 consecutive days were defined as primary graft failure and those who achieved initial engraftment but subsequently had a severely hypocellular marrow or ANC <0.5 × 109/l were defined as secondary graft failure.

Patients with sustained donor engraftment who survived more than 14 days and more than 3 months after transplantation were evaluable for occurrence of acute and chronic GVHD, respectively. The severity of acute and chronic GVHD was scored according to the previously defined criteria.16,17

Chimerism testing was carried out sequentially on PB and BM using Y-chromosome-specific complementary DNA probes in patients with a donor of opposite sex or probes specific for polymorphic DNA sequences (short tandem repeats) at approximately 1, 3, 6 and 12 months after the transplant and where clinically indicated.

Results

The outcome of the study cohort is summarized in Table 1. All patients engrafted at a median of 18 days (range 13–35). Chimerism studies were carried out in four patients and showed complete donor or predominantly stable donor (>90% donor cells) chimerism (Table 1). Two patients (UPN 1 and 3) died from transplant-related complications on days +54 and +425, respectively. UPN 1 developed severe haemorrhagic cystitis and pneumonia. Adenovirus was isolated from the blood and urine, and there was cytomegalovirus (CMV) reactivation in the terminal phase. UPN 3 developed extensive cGVHD of skin and CMV retinitis. Lymphocyte subset studies in this patient at 1-year showed markedly low CD4 count 0.28 × 109/l and CD 4/8 ratio of 0.33. His clinical course was further complicated by renal failure and pulmonary aspergillosis.

Three patients developed aGVHD (grade II involving skin), which resolved rapidly with oral steroids in 5–7 days. None developed Grade III–IV aGVHD. Of the six evaluable patients for cGVHD, only one developed cGVHD as described above. Infectious complications before day 100 in all the patients are shown in Table 1. Of the six patients surviving 100 days, one patient (UPN 3) developed CMV disease as described above. None of the other patients developed any significant infectious complication beyond day 100.

No evidence of severe mucositis or veno-occlusive disease was seen in any of the patients and none required parenteral nutrition. No unusual regimen-related toxicity was observed in first 30 days. With a median follow-up of 289 days (range 211–995), all survivors are transfusion independent and have performance status of 100%.

Discussion

All patients engrafted in this study and none developed late graft failure. Whereas three of seven patients developed Grade II aGVHD, none developed Grade III–IV aGVHD and only one of six evaluable patients had cGVHD. Given the nature of the transplants and pre-BMT characteristics, all patients were at high risk of graft failure and GVHD. The favourable impact on engraftment and incidence as well as severity of GVHD in the present study is probably multifactorial. Better donor selection using high-resolution molecular HLA typing is probably one of the most important contributory factors as previously demonstrated by the studies from the National Marrow Donor Program and Japanese Marrow Donor Program (JMDP).18,19 Another study from JMDP evaluating the outcome of unrelated transplants for SAA showed that survival was significantly inferior for HLA-A and B-locus mismatch.10 All donors in the present study were fully matched by high-resolution typing. While the contribution of better donor selection to favourable engraftment and GVHD cannot be ignored, this seems unlikely to be the only factor. Previous observations by the Seattle team support this notion.7 Whether this conditioning regimen can overcome the histocompatibility differences between donor and recipient remains to be seen.

We chose fludarabine in preference to TBI because of concerns of pulmonary toxicity and risk of long-term complications, such as endocrine, infertility and secondary malignancies.20 In addition, the patients with FA are at high risk of head and neck cancers using CY plus thoraco-abdominal irradiation (15 year incidence: 53%).21 Although the impact of fludarabine on fertility is unclear at present, preliminary data point towards less gonadal failure.22 The selection of alemtuzumab, in preference to ATG, was based on our previous experience.14 No unusual regimen-related toxicity was observed in any of the patients in first 30 days. Although the aetiology of AA in our study cohort is heterogenous, the magnitude of graft failure, regimen-related toxicity and GVHD was the same in these patients. A noteworthy point in this study is that this regimen was well tolerated by all patients irrespective of the aetiology of AA.

A multicentre EBMT study reported preliminary results on 46 patients using fludarabine, CY and ATG conditioning for AA (acquired, 33; Fanconi's anaemia, 13) using alternate donors (unrelated, 41; mismatched family, 5).23 In this study, the incidence of graft failure, grade II–IV aGVHD and cGVHD for acquired AA were 24, 12 and 31%, respectively. The patients with FA had a higher incidence of acute (36%) and chronic GVHD (36%).

With alemtuzumab containing regimens, increased risk of viral complications such as CMV and adenovirus is of concern.24,25 Delayed immune reconstitution, persistent lymphocytopenia and potent immunosuppression contribute to the higher incidence of these infections. Out of four recipients positive for CMV, two developed CMV reactivation. One patient developed adenovirus disease. Though it is difficult to compare the infectious complications in this study with others, the overall spectrum of various infective complications is not much different than expected in this high-risk population (Table 1). Nevertheless, it is important that better prophylactic, surveillance and pre-emptive strategies are developed in future to prevent the morbidity and mortality associated with viral complications.

We conclude that this conditioning regimen for unrelated donor transplants for AA is sufficiently immunosuppressive to allow sustained engraftment. No unexpected acute toxicity is observed with this regimen. Furthermore, the impact on incidence as well severity of GVHD is favourable. These observations, albeit based on a small number of patients, merit further investigative attention and need to be confirmed on a larger cohort of patients with longer follow-up.

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Correspondence to J C W Marsh.

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Gupta, V., Ball, S., Sage, D. et al. Marrow transplants from matched unrelated donors for aplastic anaemia using alemtuzumab, fludarabine and cyclophosphamide based conditioning. Bone Marrow Transplant 35, 467–471 (2005). https://doi.org/10.1038/sj.bmt.1704799

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Keywords

  • aplastic anaemia
  • Fanconi's anaemia
  • alemtuzumab
  • CD52
  • fludarabine

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