Clinical significance of acquired somatic mutations in aplastic anaemia.

Aplastic anaemia (AA) is frequently associated with other disorders of clonal haemopoiesis such as paroxysmal nocturnal haemoglobinuria (PNH), myelodysplastic syndrome (MDS) and T-large granular lymphocytosis. Certain clones may escape the immune attack within the bone marrow environment and proliferate and attain a survival advantage over normal haemopoietic stem cells, such as trisomy 8, loss of heterozygosity of short arm of chromosome 6 and del13q clones. Recently acquired somatic mutations (SM), excluding PNH clones, have been reported in around 20-25 % of patients with AA, which predispose to a higher risk of later malignant transformation to MDS/acute myeloid leukaemia. Furthermore, certain SM, such as ASXL1 and DNMT3A are associated with poor survival following immunosuppressive therapy, whereas PIGA, BCOR/BCORL1 predict for good response and survival. Further detailed and serial analysis of the immune signature in AA is needed to understand the pathogenetic basis for the presence of clones with SM in a significant proportion of patients.

Allogeneic stem cell transplantation using alemtuzumab-containing regimens in severe aplastic anemia.

Alemtuzumab, a humanized anti-CD52, IgG1 monoclonal antibody, is used to reduce graft-versus- host disease (GVHD) and aid engraftment after allogeneic haemopoietic stem cell transplant (HSCT). Its associated low incidence of GVHD makes it an attractive alternative to anti-thymocyte globulin (ATG) in transplant conditioning regimen for severe aplastic anaemia (SAA). We have reviewed the use of alemtuzumab-based conditioning regimen for HSCT in SAA and show that it results in sustained haematological engraftment, a very low incidence of chronic GVHD without an increase in viral infections. Intriguingly, alemtuzumab appears to induce tolerance post-HSCT with the findings of stable mixed T cell chimerism with full donor myeloid chimerism and the absence of chronic GVHD, and which persist on withdrawal of post-graft immunosuppression. Finally, its low toxicity profile may permit future application of HSCT to older patients with SAA who fail to respond to immunosuppressive therapy.

Treatment of aplastic anaemia in elderly patients aged >60 years.

The treatment of aplastic anaemia (AA) in the elderly patient population is not always straightforward, as other factors apart from age per se and disease severity need to be considered in clinical management decisions. The presence of comorbidities needs to be evaluated carefully. In general, elderly patients have an inferior outcome following immunosuppressive therapy and haematopoietic SCT; hence patients need to be evaluated on an individual patient basis and their specific wishes respected, as quality of life is an extremely important outcome measure to consider. In this review we aim to provide some guidance on how to approach the options for treatment in the elderly patient.

Unrelated donor search and unrelated donor transplantation in the adult aplastic anaemia patient aged 18-40 years without an HLA-identical sibling and failing immunosuppression.

Currently at least 75% of patients with severe aplastic anaemia can be successfully transplanted using a matched unrelated donor (UD) haematopoietic SCT (HSCT). For children, outcomes are similar to matched sibling donor (MSD) HSCT. This improvement in outcome over time is likely due to improved HLA tissue typing to identify better matched donors, improvements in the conditioning regimen, particularly fludarabine-based regimens, and improved supportive care. Graft rejection occurs in ∼15% of adults, but is less frequent in children. Chronic GVHD remains a concern but may be reduced by using Alemtuzumab instead of ATG. UD HSCT should be considered early after failure to respond to one course of immunosuppressive therapy, but for children who lack a MSD up front matched UD HSCT may be considered.

Implications of CD34+ cell dose on clinical and haematological outcome of allo-SCT for acquired aplastic anaemia.

The precise effects of CD34+ cell dose on the outcome of allogeneic transplantation for aplastic anaemia (AA) are not known. Previous studies have used the total mononuclear cell count to quantify stem cell dose. We evaluated the effects of CD34+ cell dose on the clinical and haematological end points of transplantation. The transplant variables and outcome parameters on 46 patients with acquired AA were assessed by comparing low vs high CD34+ cell doses. Infusion of less than 2 x 10(6)/kg of CD34+ cells was associated with an increased incidence of graft failures (P=0.03), higher incidence of bacterial infections (P=0.006) and a delay in the engraftment of neutrophils (P=0.046). The latter was found to be an effect of stem cell source (non-PBSC) rather than the CD34+ count. Other parameters, such as plt engraftment (P=0.63), red cell (P=0.94) and plt (P=0.31) transfusion independence, chimerism, acute and chronic GVHD (P=1.0) and OS (P=0.57), were not significantly influenced by the CD34+ cell dose. These findings are different to the published studies on the relevance of CD34+ cell dose in allogeneic transplantation for haematological cancers.

Aplastic anaemia in association with coeliac disease: a series of three cases.

We report a series of three patients in whom the diagnoses of aplastic anaemia (AA) and coeliac disease were made concurrently. Haematological manifestations of coeliac disease are well described but this is the first report to suggest an association with aplastic anaemia. 'Silent/atypical coeliac disease', in the absence of gastrointestinal symptoms, is increasingly recognised and patients may present with generalised symptoms, such as malaise and fatigue, which are easily attributable to AA. Immunosuppressive therapy for AA could modulate the course of celiac disease. We recommend clinicians should be vigilant for signs of coeliac disease in patients with AA.

Lack of clinical efficacy of rituximab in the treatment of autoimmune neutropenia and pure red cell aplasia: implications for their pathophysiology.

We describe 11 patients with severe refractory autoimmune cytopenias treated with the anti-CD20 monoclonal antibody rituximab. Six patients had autoimmune neutropenia (AIN), two had pure red cell aplasia (PRCA), one had AIN and autoimmune haemolytic anaemia, one had AIN and immune thrombocytopaenia purpura (ITP) and one had PRCA and ITP. Rituximab was administered at a dose of 375 mg/m(2) as an intravenous infusion weekly for 4 weeks. Six of eight patients with AIN and all three patients with PRCA did not respond. Two patients died: one with resistant AIN and autoimmune haemolytic anaemia died of pneumocytis pneumonia infection, and one with PRCA and ITP died of an acute exacerbation of bronchiectasis. Rituximab in AIN and PRCA appears to be less effective than Campath-1H when compared to historical data from our group. This supports the hypothesis that T cells may be important in the pathophysiology of AIN and PRCA.

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

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 0.5vials/10kg/day for 5 days (5mg/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.

Bone marrow transplants from mismatched related and unrelated donors for severe aplastic anemia.

For patients with acquired severe aplastic anemia without a matched sibling donor and not responding to immunosuppressive treatment, bone marrow transplantation from a suitable alternative donor is often attempted. We examined risks of graft failure, graft-versus-host disease and overall survival after 318 alternative donor transplants between 1988 and 1998. Sixty-six patients received allografts from 1-antigen and 20 from >1-antigen mismatched related donors; 181 from matched and 51 from mismatched unrelated donors. Most patients were young, had had multiple red blood cell transfusions and poor performance score at transplantation. We did not observe differences in risks of graft failure and overall mortality by donor type. The probabilities of graft failure at 100 days after 1-antigen mismatched related donor, >1-antigen mismatched related donor, matched unrelated donor and mismatched unrelated donor transplants were 21, 25, 15 and 18%, respectively. Corresponding probabilities of overall survival at 5 years were 49, 30, 39 and 36%, respectively. Although alternative donor transplantation results in long-term survival, mortality rates are high. Poor performance score and older age adversely affect outcomes after transplantation. Therefore, early referral for transplantation should be encouraged for patients who fail immunosuppressive therapy and have a suitable alternative donor.

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

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.

Remission induced by Campath-1H for thymoma-associated agranulocytosis.

Thymoma-associated agranulocytosis is a rare but almost universally fatal condition. Reports to date have described several immunosuppressive therapies including steroids, cyclophosphamide and vincristine as adjuvants to thymectomy, in an effort to improve neutropenia. We report the response to the monoclonal antibody Campath-1H of a patient with a thymoma and associated agranulocytosis with complete absence of bone marrow granulocyte precursors, which had failed to respond to thymectomy. Treatment with Campath-1H resulted in complete responses of promising durability sustained with the addition of cyclosporin and mycophenolate mofetil as maintenance therapy.

Stem cells in paroxysmal nocturnal haemoglobinuria and aplastic anaemia: increasing evidence for overlap of haemopoietic defect.

The clinical association between paroxysmal nocturnal haemoglobinuria (PNH) and aplastic anaemia (AA) has long been recognized. Haemolytic PNH, as confirmed by a positive Ham's test, can occur in the setting of AA, and conversely AA can be a late complication of PNH. With the development of sensitive flow cytometry to quantify the expression of phosphatidylinositolglycan (PIG)-anchored proteins on blood cells, a small PNH clone can now be detected in a large number of patients with AA at diagnosis. PIG-A gene mutations can also be demonstrated in some AA patients. In haemolytic PNH, there is always marrow suppression despite a morphologically cellular marrow. In vitro cultures show markedly diminished proliferative capacity in both short-term and long-term marrow cultures, similar to that seen in AA. A similar autoimmune process, through the T-cell cytotoxic repertoire, is probably responsible for the pathogenesis of both AA and PNH. PIG-deficient cells may be resistant to immunological attack by autoreactive cytotoxic T cells, because they lack PIG. They are also more resistant to apoptosis than the PIG-normal cell population. This results in the selection of the PIG-deficient clone, in contrast to the PIG-normal stem cells which possess the PIG anchor and hence are targeted and depleted by the autoreactive T cells.

Autologous recovery following non-myeloablative unrelated donor bone marrow transplantation for severe aplastic anaemia.

We report the outcome of nine unrelated bone marrow transplants performed for acquired severe aplastic anaemia at a single centre. Six patients received transplants from fully matched donors. Three donor/recipient pairs were mismatched, two at a single allele on high resolution typing. Pre-transplant conditioning consisted of cyclophosphamide and in vivo Campath-1 monoclonal antibody. One patient also received total body irradiation (TBI), and another patient with a coexisting paroxysmal nocturnal haemoglobinuria (PNH) clone received additional busulphan. Cyclosporin A was given for 12 months as prophylaxis against graft-versus-host disease (GVHD). Six of nine patients are alive and transfusion independent with a mean follow-up of 24 months (range: 1.5-94). All six patients who received fully matched transplants are alive; the three who received mismatched grafts died. Four long-term survivors developed autologous haematological recovery following rejection of their grafts. Acute GVHD grade II+ occurred in two patients. We highlight the importance of high-resolution HLA typing, including Cw matching in reducing the incidence of graft rejection and GVHD, resulting in improved survival in our patient group. This study also shows that autologous recovery with long-term survival can occur following non-irradiation conditioning regimens.