Aplastic anaemia: Management

Blood Reviews (2000) 14, 157–171 © 2000 Harcourt Publishers Ltd doi: 10.1054/ blre.2000.0133, available online at http://www.idealibrary.com on

State of the art

Aplastic anaemia: Management

S.B. Killick, J.C.W. Marsh Acquired, idiosyncratic aplastic anaemia (AA) is a rare but potentially fatal haematological disorder. Severe AA constitutes an acute medical emergency, and supportive therapy is needed to prevent overwhelming sepsis or a life threatening haemorrhage. Specific therapy for the disease includes the choice between allogeneic stem cell transplantation (SCT) from an HLA-identical sibling or immunosuppressive therapy with anti-thymocyte globulin (ATG) and cyclosporin A (CSA). Longterm cure rates of 75–90% are now achieved following HLA (human leukocyte antigen) identical sibling bone marrow transplant. The use of donors other than HLA-id siblings for transplantation in AA remains experimental. Transplantation offers the patient a chance of cure, whilst treatment with immunosuppressive therapy carries a long-term risk of relapse and clonal transformation. The haemopoietic growth factors, apart from granulocyte colony stimulating factor (G-CSF), have been shown to be potentially toxic when given to patients with AA. A short course of G-CSF may be useful to help treat severe infection, but its longer-term use with ATG and CSA remains controversial. Results from immunosuppressive treatment continue to improve with time, as a result of the additional use of CSA with ATG, the use of repeat courses of ATG for non-responders and improvements in the supportive care of patients. © 2000 Harcourt Publishers Ltd

fragility particularly when exposed to DNA crosslinking agents. Malignant AA presents particularly as acute lymphoblastic leukaemia in childhood, which may present as AA. This review will focus solely on the management of acquired idiosyncratic AA.

INTRODUCTION Classification of aplastic anaemia Aplastic anaemia (AA) may be classified as acquired, inevitable, inherited or malignant. Acquired AA is an idiosyncratic reaction with unpredictable recovery and severity that may follow exposure to a drug or virus infection. Inevitable AA follows exposure to cytotoxic drugs or radiation, and is predictable with dose dependent onset and duration. The commonest type of inherited AA is Fanconi anaemia, characterized by delayed onset, increased risk of leukaemia and solid tumours and chromosomal

Definition and pathogenesis of acquired AA Acquired AA results from the failure of normal haemopoiesis. It is defined as peripheral blood pancytopenia associated with a hypocellular bone marrow in the absence of an abnormal infiltrate or increased reticulin. There is an absence or marked reduction of haemopoietic cells in the bone marrow and an increase in fat cells. Specific blood criteria for defining AA are that the peripheral blood must show at least two of the following three criteria: (1) haemoglobin < 10 g/dl (2) platelets < 50 × 109/1 (3) neutrophils < 1.5 × 109/.1

Sally B. Killick, Judith Marsh, Department of Haematology, St George’s Hospital Medical School, Cranmer Terrace, London SW17 ORE, UK. Tel.: 0208 725 3545; Fax: 0208 725 0245 Correspondence to: J. Marsh

157

158

Blood Reviews

Grades of severity of AA are defined below. Many pathogenetic mechanisms have been suggested to explain the bone marrow failure seen in AA, and current views on the pathogenesis of AA have recently been summarized.2,3 They include a primary stem cell defect, a secondary stem cell defect due to abnormal regulation by cellular or humoral factors, a deficiency in the stromal microenvironment and immune suppression of haemopoiesis by activated T-suppressor cells. Because immune mechanisms appear to play an important part in the maintenance and/or progression of acquired AA, this is the rationale for the use of immunosuppressive therapy as one option in the treatment of the disease, and for the use of an intensively immunosuppressive conditioning regimen required for successful bone marrow transplantation in acquired AA.

Table 1 Aetiology of aplastic anaemia Idiosyncratic AA Idiopathic Hepatitis: 19 non-A, non-B, non-C, non-G in most cases; less common hepatitis A and B Drugs: 20–23 chloramphenicol (not eye drops), sulphonamides, gold, penicillamine, NSAID, remoxipride Chemicals: 24,25 benzene, pesticides Rarely:26–29pregnancy, anorexia nervosa, tuberculosis, systemic lupus erythematosus, transfusion associated graft versus host disease Predictable AA Radiation Chemotherapy Inherited AA20 Fanconi anaemia Dyskeratosis congenita Shwachman’s syndrome Malignant AA Childhood acute lymphoblastic leukaemia30

Definition of disease severity There are established criteria for the severity of AA. Severe AA has been defined by the International AA Study Group in 1976.4 This requires (1) bone marrow cellularity to be < 25% of normal, or 25–50% of normal with < 30% residual haemopoietic cells and (2) two out of three criteria: neutrophils < 0.5 × 109/1, platelets < 20 × 109/1, reticulocytes < 1%. Very severe AA, defined as for SAA but with a neutrophil count < 0.2 × 109/L, has since been added.5 Patients not fulfilling the criteria for SAA or VSAA have non-severe AA. These criteria are still widely used although the neutrophil count appears to be the most important prognostic indicator.5,6 Aetiology and incidence Most cases of acquired AA are idiopathic. In a third of cases, an underlying cause may be suggested although causality is often difficult to prove7,8 (Table 1). There is a close association between AA and paroxysmal nocturnal haemoglobinuria (PNH): AA can evolve from PNH, or progress to PNH later in the disease.9–11 Following immunosuppression, there is a 10–20% risk of developing myelodysplasia (MDS) or acute myeloid leukaemia (AML).12–14 In the West, the incidence of AA is approximately 2 million per year15,16 so that in the UK, 100–150 new cases are seen each year. New data on the incidence and aetiological factors for AA in the UK are awaited from the recent ly completed UKAAS epidemiological case control study. The incidence of AA seems to vary worldwide. This is thought to be due to environmental rather than genetic factors.15 The overall incidence in Bangkok is 3.6 million per year,17 where it has been shown to be related to socio-economic status.18 This is not a cause

for AA but may represent a surrogate marker for environmental agents such as infections and toxins associated with poor residential areas. AA can occur at any age, although two peaks have been observed: adolescents and young adults, and the elderly. Men and women are affected equally.

MANAGEMENT OF AA Initial assessment of the patient Prior to commencing treatment, it is important to exclude other conditions that may also present with pancytopenia and a hypoplastic BM, such as hypoplastic MDS, hypoplastic AML, acute lymphoid leukaemia in children, hairy cell leukaemia and myelofibrosis. Differentiation of AA from hypoplastic MDS can sometimes be difficult.31,32 In both conditions macrocytosis is very common: dyserythropoietic changes in the bone marrow occur in more than half the cases of AA and abnormalities of monocytes may also be seen in AA. In contrast, dysplastic neutrophils, abnormal megakaryocytes, increased blasts and the presence of fibrosis are not features of AA. The presence of an abnormal cytogenetic clone does not exclude a diagnosis of AA. Small clones can be detected in 4–11% of patients with otherwise typical AA,33,34 and they may disappear or remain stable without evidence of progression to MDS or AML.35,36 It is important not to treat such patients with chemotherapy; response to immunosuppressive therapy appears to be similar to AA patients lacking an abnormal cytogenetic clone.35 The patient should also be investigated for evidence of a co-existing PNH clone with the Ham test and the

Aplastic anaemia: Management

more sensitive technique of flow cytometry to detect small populations of cells (red cells, neutrophils, monocytes and sometimes platelets) deficient in the expression of phosphatidylinositol glycan (PIG)anchored proteins. The urine should also be examined for haemosiderin as evidence of chronic intravascular haemolysis. If the presence of a PNH done is confirmed, but in the absence of clinical or laboratory evidence of haemolysis, it is reasonable to treat the patient along similar lines to AA patients lacking a PNH clone, but with careful monitoring of the size of the PNH clone. The presence of haemolytic PNH would influence not only the type of treatment but also the conditioning regimen if allogeneic sibling BMT is indicated, when a myeloalative regimen may be considered necessary to eliminate the PNH clone as opposed to the usual immunosuppressive conditioning regimen used for standard acquired AA. Treatment offered to a patient with AA will depend on a number of variables including (1) the severity of disease, (2) the age and general medical condition of the patient and (3) the availability of an HLAmatched donor. Supportive care Much of the improved survival of patients with AA has been due to the increased quality of supportive care, including platelet transfusion, improved antibiotics, antifungal agents and possibly the use of recombinant human haemopoietic growth factors such as granulocyte colony stimulating factor (G-CSF). Transfusion of red cells and platelets should not be withheld when clinically indicated. Prophylactic platelets should be given to maintain the platelet count > 10 × 109/1 in the absence of fever, infection or haemorrhage. However, steps to avoid sensitization of the patient to major histocompatibility antigens should be taken,37–39 such as the use of leucocytedepleted transfusions from the time of first presentation. Sensitization of patients results in refractoriness to random donor platelets necessitating the use of HLA matched platelets.37 Sensitization to minor histocompatibility antigens results in an increased risk of graft rejection following HLA-identical haemopoietic stem cell transplantation.40 Family members should not be used as blood donors as the recipient may become sensitized to minor histocompatibility antigens from the potential donor. G-CSF alone has limited value in AA. Its use may be indicated for a short course for severe infections which are not responding to parenteral antiboiotics and antifungal agents. It can produce a temporary rise in the neutrophil count but only in those with residual granulocytic activity in the BM,41 that is, those with

159

less severe disease. Most patients with severe, and particularly with very severe AA, more often fail to show a significant rise in the neutrophil count with G-CSF. If no response occurs by one week, it is reasonable to discontinue the drug. If a response occurs, when the drug is stopped the neutrophil count invariably returns to its baseline level. Haemopoietic growth factors such as G-CSF should not be used on their own in newly diagnosed patients.42 AA is not due to a deficiency of any of the known growth factors, and endogenous serum levels of the majority of growth factors are markedly elevated. Treatment with a growth factor alone will result in delay in commencing specific therapy, during which time the patient may become sensitized to HLA and non-HLA antigens from multiple blood transfusions, or become infected. Finally, growth factors such as GM-CSF, IL-2, IL-3, II-6 and stem cell factor, have been associated with serious toxicity in AA patients. G-CSF appears to be the only growth factor which is well tolerated clinically in AA patients and without serious side-effects.42 Haemopoietic stem cell transplantation or immunosuppressive therapy? Once the diagnosis of AA has been confidently established and the patient has been stabilized clinically in terms of controlling bleeding and treating active infection, the decision whether to start immunosuppressive therapy or to proceed to BMT as definitive treatment for AA needs to be carefully considered (see Fig. 1). Bone marrow transplantation HLA identical sibling BMT 1. Indications. Patients should be offered an HLA identical sibling BMT as first line therapy if (1) the patient has severe AA; (2) is less than 45–50 years old (although it is reasonable to consider BMT in older patients up to perhaps the age of 60 years if they have failed immunosuppressive therapy and are in good medical condition); and (3) the patient has an HLA matched sibling. Unfortunately only 20–30% of patients with severe AA in N. America and Europe have an HLA genotypically identical sibling. The EBMT group has recently devised a model to predict for a particular patient the outcome of BMT versus immunosuppressive therapy based on the patient’s age and neutrophil count.6 2. Conditioning regimen and GVHD prophylaxis. Currently the best conditioning regiment for HLA identical sibling BMT is cyclophosphamide 50

160

Blood Reviews

Diagnosis of AA established

Non-severe AA (transfusion dependent)

Severe AA or very severe AA

ATG + CSA

< 45* yrs

> 45 yrs

HLA identical sibling ATG + CSA

Yes

No

Allogeneic sibling BMT

*See text for further discussion on age Fig. 1 First-line therapy for aplastic anaemia.

mg/kg × 4. The addition of ATG (1.5 vials/10 kg × 3, lymphoglobuline or thymoglobuline) may further improve the outcome.43,44 A prospective IBMTR study is currently in progress to try to determine whether the addition of ATG confers benefit over cyclophosphamide alone. GVHD prophylaxis is with cyclosporin alone or the combination of cyclosporin and short course methotrexate. Continuation of cyclosporin to 9 months followed by tailing of the drug over a further 3 months is important to help prevent late graft failure. Recent animal data suggest that the post graft immunosuppression may also reduce host versus graft reactions, thereby reducing the risk of graft failure; in a dog model the combination of cyclosporin and methotrexate was found to be more effective than cyclosporin in this regard.45

3. Dose and source of stem cells. The dose of bone marrow cells is very important in AA, as the incidence of graft failure correlates inversely with the number of marrow cells infused. It is recommended to harvest a minimum of 3 × 108/kg nucleated cells.46,47 The source of stem cells is also of particular importance in AA. Previous attempts to reduce the incidence of graft failure in AA using unirradiated donor buffy coats as an additional dose of stem cells were successful in this regard, but resulted in a high incidence of chronic GVHD.48 There has also been recent concern about a higher incidence of chronic GVHD in patients transplanted for leukaemia using G-CSF mobilized PBSC compared with bone marrow cells.49,50 Two recent analyses by the IBMTR and EBMTR have independently shown a significantly worse survival (60%) with PBSC compared with 75%

Aplastic anaemia: Management

survival with bone marrow cells among patients with AA transplanted from HLA identical sibling donors (personal communications, H. Schrezenmeier and M. Horowitz, 2000). 4. Outcome. Current long-term survival figures for HLA identical sibling BMT vary between 75% and 90%.44,47,51 Predictive factors for survival are (1) patient age (younger patients do better [see Fig. 2]; (2) year of transplant [see Fig. 3] (3) transfusion history (multi-transfused patients do worse46,47); and (4) pre-transplant Karnovsky score. The incidence of graft failure after 1990 is 5–20%. The incidence has fallen with time and the EMBT reports an incidence of 5% for patients transplanted after 1992.52 Other predictive factors for graft failure are (1) low marrow cell dose (see above); (2) multitransfused patients; (3) the use of irradiation which reduces graft failure but is associated with more toxicity, increased incidence of secondary malignancy and problems with growth, development and fertility;53–56 and (4) the possible addition of methotrexate to cyclosporin as GVHD prophylaxis (see above).45 Acute and chronic GVHD remain problems and have adverse effects on survival. Acute GVHD grade II-IV occurs in 20–40% of patients and risk factors are older age and the use of methotrexate alone.47,51 The incidence of chronic GVHD varies

161

from 0–25%. A history of acute GVHD, older recipient age and the use of unirradiated donor buffy coats are risk factors. In addition, irradiation and the use of PBSC are also probable risk factors for chronic GVHD.49,50,57 Because irradiation is not recommended for HLA identical sibling BMT, younger patients grow and develop normally, fertility is preserved, and the risk of second malignancies associated with radiation is avoided. A joint retrospective analysis from Seattle and Paris reported the long-term outcome of 700 AA patients, and risk factors for solid tumours were azathioprine used for treatment of chronic GVHD and irradiation.58 The follow up of patients transplanted at the Fred Hutchinson Research Centre in Seattle now approaches more than 20 years, and an important recent review on 212 patients transplanted between 1970 and 1993 revealed a 20 year survival of 89% for patients without and 69% for patients with chronic GVHD. At 2 years, 83% had returned to school or work and at least half the patients had become pregnant or fathered children (although it was not possible to determine accurately how many had attempted to have children). No new haematological disorders were observed and the majority of patients surviving beyond 2 years had returned to a fully functional life.54

Age Effect 100 90

% of patients surviving

80

A

70

B A

60

B

C

50 04 30

C

20 10

≥ 1990; BMT

< 1990; BMT

0 12

24

36

Months from BMT

48

60

12

24

36

48

60

Months from BMT

Fig. 2 The effect of age and year of transplant on outcome of HLA identical sibling BMT. Actuarial survival of patients transplanted before 1990 (left, n = 915) and after 1990 (right, n = 844). A = age <16 years; B = age < 40 years; C = age > 40 years (with kind permission of A. Bacigalupo, data from EBMTR).6

162

Blood Reviews

1.0

0.8

Probability of survival

1988–92 (n=471) 1981–87 (n=648)

0.6

1976–80 (n=186)

0.4

log – rank test for trend P =0.0001 0.2

pairwise comparisons: 1976– 80 vs 1981 – 87 P=0.0002 1981– 87 vs 1988 – 92 P=0.009 1976– 80 vs 1988 – 92 P=0.0001

0.0 0

12

24

36

48

60

72

Months

Fig. 3a Probability of survival following HLA identical sibling BMT for aplastic anaemia (IBMTR). Curves show improvement in survival with time for the periods 1976–1980, 1981–1987 and 1988–1992. Data from IBMTR, with kind permission of J. Passweg.51

Alternative donor BMT 1. HLA phenotypically matched BMT. The probability of finding an HLA phenotypically matched family donor among AA patients in N. America and Europe is 1%. When the patient’s parents are first cousins, or when the patient has at least one common HLA haplotype, it is worth performing an extended family donor search. Results of BMT using phenotypically matched family donors were previously reported to be as successful as HLA genotypically matched sibling BMT,58–60 although a more recent analysis of a larger series of patients has shown a significantly worse survival among HLA phenotypically matched family donors.61 Consequently, more data are required on phenotypically matched family transplants before useful recommendations can be made. 2. Partially matched family donor BMT. The chance of having a one antigen HLA mis-matched family donor is 5–7% in N. America and Europe.62 One antigen mis-matched family donor transplants have been performed in patients with AA, although most were done in patients heavily

sensitized to HLA and non-HLA antigens through multiple blood transfusion, and usually done as last ditch procedures in patients with poor performance scores. Results of these transplants were, and remain, very poor with high mortality from graft failure, GVHD and infection.59,63 3. Identical twin BMT. In the rare event of a patient with AA having an identical twin, syngeneic BMT would be indicated. Out of 12 patients transplanted in Seattle, 6 had sustained engraftment after simple bone marrow infusion from the twin donor without prior conditioning. The other 6 were successfully re-grafted using standard cyclophosphamide as for HLA identical sibling BMT.64 Hinterberger, on behalf of the International Bone Marrow Transplant Registry (IBMTR), analysed 40 patients transplanted from identical twins.65 Of 23 patients transplanted without conditioning, only 8 had a full haematological revovery. The other 15 received a second transplant with conditioning. There was only one graft failure among 17 patients who received conditioning with their first transplant. Because less than half the patients transplanted without conditioning will achieve sustained

Aplastic anaemia: Management

163

% 100

July 1988 – 1997

75

Survival

1976–June 1988

50 1970–1975

25

0 0

10

20

30

Years following Marrow Transplantation

Fig. 3b Changing survival for HLA identical sibling BMT for aplastic anaemia (Seattle). Single centre experience from Fred Hutchinson Cancer Research Centre demonstrating improved survival of patients with time, attributable to (a) the use of the combination of CSA and methotrexate since 1981 instead of methotrexate alone and (b) the addition of ATG to cyclophosphamide since 1988 without unirradiated donor buffy coat infusions. With kind permission of S. McCann, 2000.2000

engraftment, it may be reasonable to suggest that AA patients with an identical twin should receive cyclophosphamide with the initial marrow infusion. 4. HLA matched unrelated donor BMT. The probability of finding a well matched unrelated donor is highly dependent on (1) the patient’s HLA phenotype and (2) whether the patient has the same ethnic background as most registry donors. Since the majority of donors are Caucasian, Caucasian patients have the best chance (around 70%) of finding a donor but for other ethnic groups the chance is much lower.66 Kernan, on behalf of the National Marrow Donor Program (NMDP), reported a 2 year survival of only 29% for 31 severe AA patients transplanted from unrelated donors.67 Better survival of 58% at 2 years was reported from the Milwaukee group among 30 young patients, 13 using matched donors and 17 using one or more antigen mismatch donors. These patients received a very intensive regimen of cyclophosphamide, cytosine arabinoside, TBI, ATG and partial T-cell

depletion of the donor marrow.68 Encouraging results are also reported among children from the Japan Marrow Donor Program, where there would be a greater probability of finding a well-matched donor within a population with more similar HLA haplotypes.69 A recently published large retrospective study examined the outcome of 195 patients reported to the IBMTR, European BMT Registry (EBMTR), International Marrow Unrelated Search and Transplant (IMUST) Study Group and patients transplanted at the Fred Hutchinson Cancer research Centre (FHCRC), between 1986 and 1995 using closely HLA matched unrelated donors.61 The 3 year survival was only 33%, graft failure occurred in 19%, grade II-IV acute GVHD in 56% and chronic GVHD in 56%. There was no improved outcome among children from this study. Thus, BMT using unrelated donors remains an experimental procedure and is still associated with high morbidity and mortality. Recent guidelines from a group of international experts70 propose that two courses of immunosuppressive therapy should be

164

Blood Reviews

given before considering alternative donor BMT. Such a procedure should be restricted to children and young adults (<40 years old). The optimal conditioning protocol is not yet established. The inclusion of irradiation reduces the incidence of graft failure but is associated with significant toxicity. Deeg and colleagues have recently reported their preliminary results using deescalating doses of TBI with cyclophosphamide and ATG. Significant pulmonary toxicity was observed down to a dose of 4 Gy TBI, but the use of 2 Gy TBI with cyclophosphamide and ATG was associated with 67% survival at 1.5 years, in a small number of patients.71 The use of new agents such as fludarabine remains to be explored. The degree of HLA matching appears to be important in this setting and only donors matched at the DNA level using high resolution DNA typing techniques should be used. A retrospective analysis from the NMDP demonstrated a significant improvement in survival of 56% at 3 years for patients transplanted from donors matched at both serological and allele level DRBI compared with only 15% for DRBI mis-matched patients.72

B and NK cells, monocytes, activation and adhesion receptors and signalling molecules.75,76 It is not known how exactly ATG works in aplas tic anaemia. ATG stimulates production and release of haemopoietic growth factors by blood lymphocytes,77–79 but serum levels of growth factors are already very high in patients with aplastic anaemia. Direct antilymphocytic activity is more likely and ATG results in a rapid blood lymphopenia, probably due to complement mediated lysis,80 which may involve cytotoxic T-cells implicated in the disease. At low doses ATG selectively induces apotosis of activated T-cells.80 ATG was also known to induce proliferation and differentiation of HL60 cells81 and normal haemopoietic progenitor cells,82 and more recent work has demonstrated that it stimulates colony formation from AA bone marrow CD34+ cells.83 AA CD34+ cells are more apoptotic than normal CD34+ cells, and after ATG treatment they become less apoptotic. Despite the poor understanding of the mechanism(s) of action, clinical studies have shown that ATG can stimulate the residual haemopoietic stem cells in the bone marrow in a significant proportion of patients even with very severe disease. Definition of response to ATG

Antithymocyte globulin Preparations and possible mechanism(s) of action Mathe first reported the use of ATG in the setting of BMT as a conditioning agent.73 However, it was Speck and colleagues who investigated its use in the treatment of AA following work on a benzene-induced model of AA in rabbits.74 ATG is a polyclonal antibody (IgG) preparation. There are several different preparations which vary according to the animal source and immunogen used, for example, ATG from IMTIX-Sangstat (formerly Merieux) is prepared by immunizing horses or rabbits with human thymocytes collected from children at time of cardiac surgery, and this preparation is widely used throughout Europe and many other countries worldwide. The term antilymphocyte globulin (ALG) refers to the previous method of immunization of horses or rabbits with lymphocytes derived from the thoracic duct, instead of using human thymocytes, but the two terms are often used synonymously. ATG-Fresenius is sera from rabbits which have been immunized with the T-cell Jurkat cell line. Upjohn-ATGAM preparation is obtained by immunising horses with thymocytes, and has been the preparation predominantly used in the USA. ATG contains many antibodies, many of which are not T cell specific, for example, antibodies against

Response to therapy with ATG can be difficult to differentiate from spontaneous recovery,84 and the latter appears to depend on disease severity.85–87 There are some ‘late responders’ to ATG (but this may represent spontaneous recovery) that may occur due to the persistence of some antibodies long after the administration of ATG.75 There has been much confusion over the best definition of response to treatment following immunosuppression, and a new definition has recently been proposed88 (see Table 2). Changes in ATG protocols from its use as a single agent to combination therapy with other agents 1. ATG alone (with/without corticosteroids). During the 1970s, ATG was generally used as a single agent. The first prospective randomized study reported in 1983, comparing ATG with supportive care alone, demonstrated a significantly improved survival with ATG.89 Early reports of remission of AA with very high doses of methylprednisolone90,91 led to the common practice of giving ATG with high dose of corticosteroids in the mistaken belief that corticosteroids improved the response rate with ATG, but there is no evidence for such an effect. Furthermore, their use is associated with significant short-and long-term toxicities

Aplastic anaemia: Management

165

Table 2 Definition of response to immunosuppression in aplastic anaemia88 Severe AA

NR PR CR

Non-severe AA

NR PR

CR

still SAA transfusion independent no longer meets definition for SAA haemoglobin normal for age and gender neutrophils > 1.5 × 109/l platelets > 150 × 109/l worsening AA or not meeting criteria above transfusion independence (if prior requirement), or doubling or normalization of one or more cell line, or increase of one or more blood values by – haemoglobin 3 g/dl – neutrophils 0.5 × 109/l (if lower than 0.5 initially) – platelets 20 × 109/l (if lower than 20 initially) as for severe AA

Criteria must be fulfilled by 2 or more counts at least 4 weeks apart. NR = no response; PR = partial response; CR = complete response.

(especially avascular necrosis of bone).92 Consequently, there is no role for the use of corticosteroids as single agents in the treatment of AA and they are currently used with ATG only in a short course, low dose regimen to help prevent serum sickness. 2. ATG with androgens. It is well recognized that androgens alone are active in some patients with AA. Their effect is most marked in patients with non-severe disease, and their greatest effect is to stimulate erythropoiesis although occasionally there may be some response in the neutrophil and platelet count. During the 1980s, several groups examined the impact of androgens in combination with ATG, and results suggested an improved response with androgen therapy [reviewed in reference 93]. One small prospective study from UCLA suggested no benefit in using androgens with ATG,94 but a larger prospective EBMT study showed a better response rate with the combination of drugs compared with ATG alone, especially in females with severe disease, but no difference in survival.95 The use of androgens with ATG has now been superseeded by the use of CSA because of better response rates with CSA and concerns about the virilizing, hepatotoxic and behavioural side effects of androgen therapy. 3. ATG with CSA. The rationale for using CSA with ATG was based on small studies reporting efficacy of CSA alone in the treatment of AA.96,97 A German prospective, randomized study of ATG and CSA compared with ATG alone showed a significantly improved response at 3 months (65 v 39%) using the combination of ATG and CSA, but there was no significant difference for severe AA (58 v 31%). There was no difference in survival because non-responders to ATG alone were

rescued with a second course of ATG.98 However, there was a significant advantage using the combination of drugs in terms of failure-free survival, with better improvement in blood counts in the first 3 months, a shorter period of severe cytopenias, and a reduced need for salvage therapy with a second course of ATG.99 Subsequent large studies have confirmed the efficacy of combined ATG and CSA, the one from NIH demonstrating a 5-year survival of 70% and 3 month response rate of 67%.100 Is ATG better than CSA alone? There has been only one prospective study in severe AA comparing ATG with CSA.101 There was no difference in response at 3 months (12 v 16% for ATG and CSA, respectively) and survival at 2 years was similar. However, the early cross over of patients at 3 months made assessment of response difficult, and the response rates seen were surprisingly low, which may have reflected a worryingly high early mortality in patients treated with ATG. This was attributed to treatment of patients in centres inexperienced in the use of ATG. For patients with non-severe AA, a multicentre, prospective EMBT trial compared the use of CSA alone with the combination of ATG and CSA.102 This study demonstrated a significantly higher response rate (74 v 46%) at 6 months, better failure free survival, better blood counts and a lower need for a second course of ATG among those patients treated with the combination of ATG and CSA. Thus it is established that ATG is an essential drug for immunosuppressive protocols for severe and nonsevere AA which now incorporate CSA. 4. ATG and CSA with the addition of granulocyte colony stimulating factor (G-CSF). During the 1990s there has been a trend to add in a course of

166

Blood Reviews

at least 3 months of G-CSF to the combination of ATG and CSA, but there is currently much debate and uncertainty about this practice. The rationale for using G-CSF in this way was to determine whether the early mortality after ATG was reduced101 and also whether the response rate to ATG and CSA could be improved further. An uncontrolled pilot study using ATG, CSA and 3 months of G-CSF in 100 patients has shown a response rate of 77% and 5 year survival of 87%. Cytogenetic abnormalities were seen in 11%, and clonal disease (PNH, MDS or AML) in 8%.102 Preliminary analysis of a small prospective randomized study comparing ATG and CSA with or without G-CSF has shown earlier neutrophil recovery and fewer infections in the G-CSF arm, but no difference in response rates or survival.103 A second similar but larger EBMT study is now planned to further examine outcomes between these two treatment arms. Important concerns about the use of long term G-CSF with ATG and CSA are (i) whether G-CSF increases further the risk of later clonal disorders and (ii) the financial implications and cost effectiveness.41 A high incidence of MDS and AML, often associated with monosomy 7, has been reported from Japan among both children104 and adults,105 who in most cases have received particularly high doses of G-CSF and/or for prolonged period of time. Thus the concomitant use of G-CSF with ATG and CSA remains controversial. It is important that patients only receive G-CSF as part of multicentre prospective studies which incorporate formal cost effectiveness analyses and careful long-term follow-up for clonal disorders. 5. Changes in outcome with different ATG protocols with time. In conclusion, Fig. 4 summarizes data from the EBMTR showing the improvement in survival of patients treated with immunosuppressive therapy according to changes in the ATG protocol occurring with time, as discussed above.

The use of ATG in children and the elderly Results of immunosuppressive therapy in children have improved significantly with time. The EBMT has reported 29% survival for children treated before 1980, 43% between 1980 and 1989, and 59% after 1990. For those with severe disease, the survival for those treated after 1990 is now 70%.106 Thus, with time, the effect of age and the impact of disease severity have become less significant.

It is difficult to set an upper age limit for ATG treatment. There has been only one study evaluating the safety and effectiveness in elderly patients, a retrospective EBMT analysis of 127 patients over the age of 60 years.107 These patients were compared with 115 patients between 50 and 59 years and 568 between 20 and 49 years. Response, relapse and risk of clonal disorders were similar between the three groups. Age was associated with an increased risk of death due to bleeding or infection (survival figures of 50%, 57% and 72% for the three age groups, respectively). Thus, treatment is possible in the elderly, but the decision to use ATG should be carefully considered and determined particularly by the general medical condition of the individual patient. Features of the response to ATG 1. The response to ATG is delayed and rarely occurs before 3 months. A rapid response within a few weeks may indicate spontaneous recovery, especially after viral infection or a transient drug reaction. Approximately 50% respond by 3 months and 75% at 6 months (A. Bacigalupo, personal communication, 1999) although the NIH study showed that 90% of responders did so by 3 months. It appears that the combination of CSA with ATG induces earlier response compared with ATG or CSA alone.99 2. Most responders to ATG do not completely normalize their blood counts, unlike patients who have had a successful BMT, and for this reason many earlier studies chose the definition of response as transfusion independence, although a more accurate definition of haematological response has been proposed.88 In vitro studies of short-term and long-term marrow cultures have shown a delay in recovery of CFU-GM (commonly forming units – granulocyte macrophage), defective growth in long term marrow culture,108 and a prolonged deficiency of LTCIC (long-term culture initiating cell) which to date appears not to recover to normal despite normalization of CFU-GM and normal marrow cellularity, consistent with a long-lasting, severe deficiency in the stem cell compartment following successful haematological recovery.109 This may be one predisposing factor to a significant risk of relapse after ATG. 3. Relapse after ATG occurs in approximately 35% of patients,110 and this risk seems to be greatest in those who respond early and in those patients with a long interval from diagnosis to treatment. It may also occur after too early or too rapid CSA withdrawal. Withdrawal of CSA usually takes many months and often longer and some patients

Aplastic anaemia: Management

167

IS Regimen 100 90

D B

% of patients surviving

80 70 B

60

A

A

50 40 30

C

C 20 10

< 1990; IS

>1990; IS

0 12

24

36

48

60

72

84

Months from treatment

12

24

36

48

60

72

84

Months from treatment

Fig. 4 Actuarial survival of patients treated with immunosuppressive therapy stratified by (i) immunosuppressive protocol and (ii) year of treatment, before or after 1990. Left panel shows patients treated before 1990 (n = 585) and right panel after 1990 (n = 317). A = ALG alone; B = ALG + CSA; C = CSA alone; D = ALG + CSA + G-CSF. Data from EBMTR with kind permission of A. Bacigalupo.6

may need to be maintained longer-term on a small to modest dose of CSA to prevent relapse. 4. Patients may require several courses of ATG to achieve a response. A second course is successful in 40–60% of patients.99,111 For historical reasons, patients receive horse ATG for the first course, and usually rabbit for the second course. But, Tichelli and colleagues111 have reported the use of a second course of horse ATG among non-responders to the first course. In a retrospective study they showed that the incidence of acute reactions (anaphylaxis) and serum sickness was not increased on second exposure to horse protein, although serum sickness occurred earlier, and 63% responded to the second course. 5. A small proportion of patients develop later clonal disease following treatment with ATG, although this may reflect the persistence of bone marrow failure or a persistent stem cell dysfunction which becomes evident later with longer survival of patients, leading some authors to propose that AA is a pre-leukaemic condition.112,113 The probability of developing a late clonal disorder, however, appears to increase after multiple courses of ATG.12,111 Excluding studies using G-CSF with ATG, the projected incidence of MDS and AML varies from 10–20%.12–14,113 Approximately 15–20% of patients later develop PNH, on the basis of a positive Ham test. With the more recent availability of flow cytometry data to analyse

PIG-anchored protein expression on all blood cell lineages, it is clear that around one third (and up to 50% of patients in one series) of AA patients have a defect in PIG-anchored protein in the absence of evidence of haemolysis at diagnosis, and with PIG-A gene mutations, so further longterm follow-up studies of such patients will be important to assess the clinical significance of these changes.7,113 Following successful allogeneic BMT for AA, a similar risk of clonal disorders is not observed. 6. As discussed earlier, patients with otherwise typical AA may present with an abnormal cytogenetic clone.33–36 Often the abnormal clone is small in size, similar to the PNH clone when it coexists with normal cells. A small single centre study of 13 patients with AA and an abnormal cytogenetic clone showed that all patients responded to one or more course of immunosuppressive therapy or oxymetholone, and there were no cases of transformation with a median follow up of 4.1 years. Interestingly, in 4 patients the abnormal clone disappeared after immunosuppressive therapy.35 Miklaelova and colleagues have also observed the disappearance of these clones in some of their patients.36 Thus, these patients appear to show a similar response to immunosuppressive therapy as AA patients who lack an abnormal clone, and it is important not to treat these patients with chemotherapy. The clone

168

Blood Reviews

needs careful monitoring. Allogeneic BMT would be indicated if there is a suitable donor and if their AA is severe, or if there is any evidence of early transformation or increasing size of the clone.

Other therapies Cyclophosphamide is a powerful immunosuppressive drug. It is routinely used in the conditioning regimen for allogeneic BMT for AA, and in the event of graft failure it is recognized that autologous haematological recovery may occur.114 Early anecdotal reports of recovery of AA after treatment with cyclophosphamide without bone marrow infusion115 were later followed by a study of 10 patients treated with cyclophosphamide (45 mg/kg × 4), and CSA in 3 of the 10 patients. Response was reported in 7 patients, but haematological recovery was prolonged; the time to neutrophil recovery (> 0.5 × 109/l) and platelet transfusion independence was around 3 months.116 As a result of these findings, a prospective randomized study comparing cyclophosphamide with ATG as initial treatment for severe AA was initiated in the USA, but this study has been prematurely closed on account of deaths from infection during periods of severe neutropenia (N. Young, personal communication, 2000). Because it is known that patients can respond to a second or subsequent course of ATG, it would seem safer to use a well-established drug such as ATG with recognized but manageable and less serious side effects. Other possible alternative immunosuppressive drugs such as mycophenolate mofetil are being considered in the treatment of AA not responding to ATG, but to date there is no real experience of its use in AA. REFERENCES 1. International Agranulocytosis and Aplastic Anemia Study. Incidence of aplastic anemia: the relevance of diagnostic criteria. Blood 1987; 70:1718–1721. 2. Young NS. Haemopoietic cell destruction by immune mechanisms in acquired aplastic anemia. Sem Haematol 2000; 37:3–14. 3. Marsh JCW, Testa NG. Stem cell defect in aplastic anaemia. In: Schrezenmeier H, Bacigalupo A, eds. Aplastic anaemia. Pathophysiology and treatment. Cambridge: Cambridge University Press, 2000; 3–20. 4. Camitta BM, Thomas ED, Nathan DG, Santos G, Gordon S, EC, Gale RP, Rappeport JM, Storb R. Severe aplastic anaemia: a prospective study of the effect of early marrow transplantation on acute mortality. Blood 1976; 48:63–69. 5. Bacigalupo A, Hows J, Gluckman E, Nissen C, Marsh J, Van Lint MT, Congiu M, De Planque MM, Ernst P, McCann S. Bone marrow transplantation (BMT) versus immunosuppression for the treatment of severe aplastic anaemia (SAA): a report of the EBMT SAA working party. Br J Haematol 1988; 70:177–182.

6. Bacigalupo A, Brand R, Obeto R, Bruno B, Socie G, Passweg J, Locasciulli A, Van Lint M, Tichelli A, McCann S, Marsh J, Ljungman P, Hows J, Ljungman P, Marin P, Schrezenmeier H. Treatment of acquired aplastic anaemia: bone marrow transplantation compared with immunosuppressive therapy. The European Group for Blood and Marrow Transplant experience. Sem Haematol 2000; 37:69–80. 7. Heimpel H. When should the clinician suspect a drug-induced blood dyscrasia, and how should he proceed? Eur J Haematol (suppl 1) 1996; 60:11–15. 8. Young NS. Aplastic anaemia. Lancet 1995; 346:228–232. 9. Schrezenmeier H, Hertenstein B, Wagner B, Raghavachar A, Heimpel H. A pathogenetic link between aplastic anemia and paroxysmal nocturnal hemoglobinuria is suggested by a high frequency of aplastic anemia patients with a deficiency of phosphatidylinositol glycan anchored proteins. Exp Haematol 1995; 23:81–87. 10. Schubert J, Vogt HG, Zielinska-Skowronek M, Freund M, Kaltwasser JP, Hoelzer D, Schmidt RE. Development of the glycosylphosphatitylinositol-anchoring defect characteristic for paroxysmal nocturnal hemoglobinuria in patients with aplastic anemia. Blood 1994; 83:2323–2328. 11. Griscelli-Bennaceur A, Gluckman E, Scrobohaci ML, Jonveaux P, Vu T, Bazarbachi A, Carosella ED, Sigaux F, Socie G. Aplastic anemia and paroxysmal nocturnal hemoglobinuria: search for a pathogenetic link. Blood 1995; 85:1354–1363. 12. Socie G, Henry-Amar M, Bacigalupo A, Hows J, Tichelli A, Ljungman P, McCann SR, Frickhofen N, Van’t Veer-Korthof E, Gluckman E. Malignant tumors occurring after treatment of aplastic anemia. European Bone Marrow TransplantationSevere Aplastic Anaemia Working Party. N Engl J Med 1993; 329:1152–1157. 13.De Planque MM, Bacigalupo A, Wursch A, Hows JM, Devergie, Frickhofen N, Brand A, Nissen C. Long-term follow-up of severe aplastic anaemia patients treated with antithymocyte globulin. Severe Aplastic Anaemia Working Party of the European Cooperative Group for Bone Marrow Transplantation (EBMT). Br J Haematol 1989; 73:121–126. 14. Tichelli A, Gratwohl A, Nissen C, Signer E, Stebler G, Speck B. Morphology in patients with severe aplastic anemia treated with antilymphocyte globulin. Blood 1992; 80:337–345. 15. Gordon-Smith EC, Issaragrisil S. Epidemiology of aplastic anaemia. Baillieres Clinical Haematology 1992; 5:475–491. 16. Kaufman DW. The drug aetiology of agranulocytosis and aplastic anaemia. New York: Oxford University Press, 1991. 17. Issaragrisil S, Sriratanasatavorn C, Piankijagum A, Vannasaeng S, Porapakkham Y, Leaverton PE, Kaufman DW, Anderson TE, Shapiro S, Young NS. Incidence of aplastic anemia in Bangkok. The Aplastic Anemia Study Group. Blood 1991; 77:2166–2168. 18. Issaragrisil S, Kaufman DW, Anderson TE, Chansung K, Thamprasit T, Sirijirachai J, Piankijagum A, Porapakham Y, Vannasaeng S, Leaverton PE. An association of aplastic anaemia in Thailand with low socioeconomic status. Aplastic Anemia Study Group. Br J Haematol 1995; 91:80–84. 19. Brown KE, Tisdale J, Barrett AJ, Dunbar CE, Young NS. Hepatitis-associated aplastic anemia. N Engl J Med 1997; 336:1059–1064. 20. Young NS, Alter BP, eds. Aplastic anaemia; acquired and inherited. WB Saunders, 1994. 21. Philpott NJ, Marsh JC, Gordon-Smith EC, Bolton JS. Aplastic anaemia and remoxipride. Lancet 1993; 342:1244. 22. Marsh JC, Abboudi ZH, Gibson FM, Scopes J, Daly S, O’Shaunnessy DF, Baughan AS, Gordon-Smith EC. Aplastic anaemia following exposure to 3,4methylenedioxymethamphetamine (‘Ecstasy’). Br J Haematol. 1994; 88:281–282. 23. Gordon-Smith EC, Marsh JC, Geary CG. Is it time to stop using chloramphenicol on the eye? Prospective study of aplastic anaemia should give definitive answer. Br Med J 1995; 311:451.

Aplastic anaemia: Management

24. Fleming LE, Timmeny W. Aplastic anemia and pesticides. An etiologic association? J Occup Med 1993; 35:1106–1116. 25. Travis LB, Li CY, Zhang ZN, Li DG, Yin SN, Chow WH, Li, GL, Dosemeci M, Blot W, Fraumeni JFJ. Hematopoietic malignancies and related disorders among benzene-exposed workers in China. Leuk Lymphoma 1994; 14:91–96. 26. Aitchison RG, Marsh JC, Hows JM, Russell NH, GordonSmith EC. Pregnancy associated aplastic anaemia: a report of five cases and review of current management. Br J Haematol 1989; 73:541–545. 27. Howard MR, Leggat HM, Chaudhry S. Haematological and immunological abnormalities in eating disorders. Br J Hosp Med 1992; 48:234–238. 28. Roffe C, Cahill MR, Samanta A, Bricknell S, Durrant ST. Aplastic anaemia in systemic lupus erythematosus: a cellular immune mechanism? Br J Rheumatol 1991; 30:301–304. 29. Anderson KC, Weinstein HJ. Transfusion-associated graftversus-host disease. N Engl J Med 1990; 323:315–321. 30. Breatnach F, Chessells JM, Greaves MF. The aplastic presentation of childhood leukaemia – a feature of common ALL. Br J Haematol 1981; 49:387–393. 31. Tichelli A, Gratwohl A, Nissen C et al. Morphology in patients with severe aplastic anaemia treated with antilymphocyte globulin. Blood 1992; 80:337–345. 32. Barrett J, Saunthararajah Y, Molldrem J. Myelodysplastic syndrome and aplastic anaemia: distinct entities or diseases with a common pathophysiology. Sem Haematol 2000; 37:15–29. 33. Appelbaum FR, Barrall J, Storb R et al. Clonal cytogenetic abnormalities in patients with otherwise typical aplastic anaemia. Exp Haematol 1989; 15:1134–1139. 34. Tichelli A, Socie G, Marsh J et al. Cytogenetic abnormalities in aplastic anaemia. Bone Marrow Transpl 1996; 7 (suppl 1):268a. 35. Geary CG, Harrison CJ, Philpott NJ et al. Abnormal cytogenetic clones in patients with aplastic anaemia: response to immunosuppressive therapy. Br J Haematol 1999; 104:271–274. 36. Mikhailova N, Sessarego M, Fugazza G et al. Cytogenetic abnormalities in patients with aplastic anaemia. Haematologica 1986; 81:418–422. 37. Killick SB, Win N, Marsh JC, Kaye T, Yandle A, Humphries C, Knowles, SM, Gordon-Smith EC. Pilot study of HLA alloimmunization after transfusion with pre-storage leucodepleted blood products in aplastic anaemia. Br J Haematol 1997; 97:677–684. 38. Grumet FC, Yankee RA. Long-term platelet support of patients with aplastic anemia. Effect of splenectomy and steroid therapy. Ann Int Med 1970; 73:1–7. 39. Klingemann HG, Self S, Banaji M, Deeg HJ, Doney K, Slichter SJ, Thomas ED, Storb R. Refractoriness to random donor platelet transfusions in patients with aplastic anaemia: a multivariate analysis of data from 264 cases. Br J Haematol 1987; 66:115–121. 40. Kaminski ER, Hows JM, Goldman JM, Batchelor JR. Pretransfused patients with severe aplastic anaemia exhibit high numbers of cytotoxic T lymphocyte precursors probably directed at non-HLA antigens. Br J Haematol 1990; 76:401–405. 41. Marsh JCW. Haematopoietic growth factors in the pathogenesis and for the treatment of aplastic anaemia. Sem Haematol 2000; 37:81–90. 42. Marsh JC, Socie G, Schrezenmeier H, Tichelli A, Gluckman, Ljungman P, McCann SR, Raghavachar A, Marin P, Hows JM. Haemopoietic growth factors in aplastic anaemia: a cautionary note. European Bone Marrow Transplant Working Party for Severe Aplastic Anaemia. Lancet 1994; 344:172–173. 43. Storb R, Etzioni R, Anasetti C et al. Cyclophosphamide combined with antithymocyte globulin in preparation for allogeneic marrow transplants in patients with aplastic anaemia. Blood 1994; 84:941–949. 44. Storb R, Leisenning W, Anasetti C et al. Long term follow up of allogeneic marrow transplants in patients with aplastic

169

anaemia conditioned with cyclophosphamide with antithymocyte globulin. Blood 1997; 89:3890–3891. 45. Storb R, Yu C, Wagner JL et al. Stable mixed haemopoietic chimerism in DLA identical littermate dogs given sublethal total body irradiation before and after pharmacological immunosuppression after marrow transplantation. Blood 1997; 89:3048–3054. 46. Storb R, Prentice RL, Thomas ED. Marrow transplanted for treatment of aplastic anaemia. An analysis of factors associated with graft rejection. N Engl J Med 1977; 296:61–66. 47. McCann SR, Passweg JR, Storb R, Deeg HJ. HLA identical sibling bone marrow transplantation to treat severe aplastic anaemia. In: Schrezenmeier H, Bacigalupo A, eds. Aplastic anaemia. Pathophysiology and treatment. Cambridge: Cambridge University Press, 2000; 230–257. 48. Storb R, Doney KC, Thomas ED et al. Marrow transplantation with or without donor buffy coat cells for 65 transfused aplastic anaemia patients. Blood 1982; 59:231–246. 49. Storek J, Gooley T, Siadak M et al. Allogeneic peripheral blood stem cell transplantation may be associated with a high risk of chronic graft versus host disease. Blood 1997; 90:4705–4709. 50. Solano C, Martinez C, Brunet S et al. Chronic graft versus host disease after allogeneic peripheral blood progenitor cell or bone marrow transplant from matched related donors: a case control study. Bone Marrow Transplant 1998; 22:1129–1135. 51. Passweg JR, Socie G, Hinterberger W, Bacigalupo A, Biggs JC, Camitta BM, Champlin RE, Gale RP, Gluckman E, Gordon-Smith EC, Hows JM, Klein JP, Nugent ML, Pasquini R, Rowlings PA, Speck B, Tichelli A, Zhang MJ, Horowitz MM, Bortin MM. Bone marrow transplantation for severe aplastic anemia: has outcome improved? Blood 1997; 90:858–864. 52. McCann SR, Bacigalupo A, Gluckman E, Hinterberger W, Hows J, Ljungman P, Marin P, Nissen C, van’t Veer K, Raghavachar. Graft rejection and second bone marrow transplants for acquired aplastic anaemia: a report from the Aplastic Anaemia Working Party of the European Bone Marrow Transplant Group. Bone Marrow Transplantation 1994; 13:233–237. 53. Deeg HJ, Socie G. Malignancies after haemopoietic stem cell transplantation: many questions, some answers. Blood 1998; 91:1833–1844. 54. Deeg HJ, Leisenring W, Storb R et al. Long term outcome after marrow transplantation for severe aplastic anaemia. Blood 1998; 91:3637–3645. 55. Sanders JG, Hawley J, Levy W et al. Pregnancies following high dose cyclophosphamide with or without high dose busulphan or total body irradiation and bone marrow transplantation. Blood 1996; 87:3045–3052. 56. Sanders JE, Whitehead J, Storb R et al. Bone marrow transplant experience for children with aplastic anaemia. Paediatrics 1986; 77:179–186. 57. Gluckman E, Socie G, Devergie A et al. Bone marrow transplantation in 107 patients with severe aplastic anaemia using cyclophosphamide and thoracoabdominal irradiation for conditioning: long term follow up. Blood 1991; 78:2451–2455. 58. Deeg HJ, Socie G, Schoch G et al. Malignancies after marrow transplantation for aplastic anaemia and Fanconi anaemia: a joint Seattle and Paris analysis of results in 700 patients. Blood 1996; 87:386–392. 59. Bacigalupo A, Hows J, Gordon-Smith EC et al. Bone marrow transplantation from donors other than HLA identical siblings: a report of the EBMT Working Party. Bone Marrow Transplant 1988; 3:531–535. 60. Wagner JL, Deeg HJ, Seidel K et al. Bone marrow transplantation for severe aplastic anaemia from genotypically HLA non identical relatives. Transplantation. 1996; 61:54–61. 61. Hows J, Stone JV, Camitta BM. Alternative donor bone marrow transplantation for severe acquired aplastic anaemia. In: Schrezenmeier H, Bacigalupo A, eds Aplastic anaemia.

170

Blood Reviews

Pathophysiology and treatment. Cambridge: Cambridge University Press, 2000; 258–274. 62. Beatty PG, Dahlberg S, Michelson EM et al. Probability of finding HLA matched unrelated donors. Transplantation 1986; 45:714–721. 63. Hows JM, Yin JL, Marsh J et al. Histocompatible unrelated volunteer donors compared with HLA non-identical family donors in marrow transplantation for aplastic anaemia and leukaemia. Blood 1986; 68:1322–1328. 64. Storb R, Longton G, Anansetti C et al. Changing trends in marrow transplantation for aplastic anaemia. Bone Marrow Transplant 1992; 10:45–52. 65. Hinterberger W, Rowlings PA, Hinterberger-Fischer M et al. Results of transplantating bone marrow from genetically identical twins into patients with aplastic anaemia. Ann Intern Med 1997; 126:116–122. 66. Hows J, Downie T, Nunn A et al. Fate of patients undergoing unrelated donor searches in the UK. Bone Marrow Transplant 1996; 17:579a. 67. Kernan NA, Bartsch G, Ash RC, Beatty PG, Champlin R, Filipovich A, Gajewski J, Hansen JA, Henslee-Downey J, McCullough J et al. Analysis of 462 transplantations from unrelated donors facilitated by the National Marrow Donor Program. N Engl J Med 1993; 328:593–602. 68. Margolis D, Camitta B, Pietoyga D et al. Unrelated donor bone marrow transplantation to treat severe aplastic anaemia in children and young adults. Br J Haematol 1996; 94: 65–72. 69. Kodera Y, Morishima Y, Kato S et al. Analysis of 500 bone marrow transplants from unrelated donors (UR-BMT) facilitated by the Japan Marrow Donor Program: confirmation of UR-BMT as a standard therapy for patients with leukaemia and aplastic anaemia. Bone Marrow Transplant 1999; 24:995–1003. 70. Consensus document of a group of international experts. Guidelines for treating aplastic anaemia. In: Schrezenmeier H, Aplastic anaemia. Pathophysiology. Bacigalupo A, eds. Cambridge: Cambridge University Press, 2000; 308–315. 71. Deeg HJ, Amylon M, Harris R et al. Marrow transplantation from unrelated donors for patients with aplastic anaemia (AA) – optimisation of a conditioning regimen. Blood 1999; 94, suppl 1 (part 1 of 2):3147a. 72. Deeg HJ, Seidel K, Casper J et al. Marrow transplantation from unrelated donors for patients with severe aplastic anaemia who have failed immunosuppressive therapy. Biol Blood Marrow Transplant 1999; 5:243–252. 73. Mathe G, Amiel JL, Schwarzenberg L, Choay J, Trolard P, Schneider M, Hayat M, Schlumberger JR, Jasmin C. Bone marrow graft in man after conditioning by antilymphocytic serum. Br Med J 1970; 2:131–136. 74. Speck B, Gluckman E, Haak HL, van Rood JJ. Treatment of aplastic anaemia by antilymphocyte globulin with and without allogeneic bone-marrow infusions. Lancet 1977; 2:1145–1148. 75. Rebellato LM, Gross U, Verbanac KM, Thomas JM. A comprehensive definition of the major antibody specificites in polyclonal rabbit antithymocyte globulin. Transplantation 1994; 57:685–694. 76. Bonnefoy-Berard N, Vincent C, Revillard JP. Antibodies against functional leukocyte surface molecules in polyclonal antilymphocyte and antithymocyte globulins. Transplantation. 1991; 51:669–673. 77. Gascon P, Zoumbos NC, Scala G et al. Lymphokine abnormalities in aplastic anaemia: implications for the mechanism of action of antithymocyte globulin. Blood 1985; 65:407–413. 78. Nimer SD, Golde DW, Kwan K et al. In vitro production of granulocyte-macrophage colony stimulating factor in aplastic anaemia: possible mechanisms of action of antithymocyte globulin. Blood 1991; 78:163–8. 79. Tong J, Bacigalupo A, Piaggio G et al. Effect of antilymphocyte globulin (ALG) on bone marrow T/non-T cells for aplastic anaemia patients and normal controls. Br. J Haematol 1989; 73:546–550.

80. Genestier L, Fournel S, Flacher M et al. Induction of Fas (Apo-1, CD95) – mediated apoptosis of activated lymphocytes by polyclonal antithymocyte globulins. Blood 1998; 91:2360–2368. 81. Hunter RF, Mold NG, Mitchell RB, Huang AT. Differenetiation of normal marrow and HL60 cells induced by antithymocyte globulin. Proc Nat Acad Sci USA 1985; 82:4823–4827. 82. Huang AT, Mold NG. The role of CD45RO in antithymocyte globulin’s stimulation of primitive haemopoietic cells. Br J Haematol 1994; 88:643–646. 83. Killick SB, Marsh JCW, Gordon-Smith EC, Sorlin L, Gibson FM. Effect of antithymocyte globulin on bone marrow CD34+ cells in aplastic anaemia and myelodysplasia. Br J Haematol 2000; 108:582–591. 84. Heimpel H. Therapy of aplastic anaemia: is a consensus possible? In: Schrezenmeier H, Frickhofen N, eds. Aplastic anaemia: Current perspectives on the pathogenesis and treatment. Blackwell-MZV, 1993; 154–159. 85. Alter BP, Potter NU, Li FP. Classification and aetiology of the aplastic anaemias. Clin Haematol 1978; 7:431–466. 86. Mir MA, Geary CG. Aplastic anaemia: an analysis of 174 patients. Postgrad Med J 1980; 56:322–330. 87. Keisu M, Heit W, Lambertenghi-Deliliers G, Parcells K, Polliack A, Heimpel H. Transient pancytopenia. A report from the International Agranulocytosis and Aplastic Study. Blood 1990; 61:240–244. 88. Camitta BM. What is the definition of cure of aplastic anaemia? Acta Haematologica 2000; 103:16–18. 89. Champlin R, Ho W, Gale RP. Antithymocyte globulin treatment in patients with aplastic anemia: a prospective randomized trial. N Engl J Med 1983; 308:113–118. 90. Bacigalupo A, Podesta M, van Lint MT et al. Severe aplastic anaemia: correlation of in vitro tests with clinical response to immunosuppression in 20 patients. Br J Haematol 1981; 47:423–432. 91.Gluckman E, Devergie A, Doros A et al. Results of immunosuppression in 170 cases of severe aplastic anaemia. Report of the European Group of Bone Marrow Transplantation (EGBMT). Br J Haematol 1982; 51:541–550. 92. Marsh JCW, Zomas A, Hows JM et al. Avascular necrosis after treatment of aplastic anaemia with antilymphocyte globulin and high dose methyl prednisolone. Br J Haematol 1993; 84:731–734. 93. Marsh JCW. Results of immunosuppression in aplastic anaemia. Acta Haematologica 2000; 103:26–32. 94. Champlin RE, Ho W, Feig SA et al. Do abdrogens enhance the response to antithymocyte globulin in patients with aplastic anaemia? A prospective randomised trial. Blood 1985; 66:184–188. 95. Bacigalupo A, Chaple M, Hows J, Van Lint MT, McCann S, Milligan D, Chessells J, Goldstone AH, Ottolander J, van’t Veer ET et al. Treatment of aplastic anaemia (AA) with antilymphocyte globulin (ALG) and methylprednisolone (MPred) with or without androgens: a randomized trial from the EBMT SAA working party. Br J Haematol 1993; 83:145–151. 96. Leonard EM, Raefsky E, Griffith P, Kimball J, Nienhuis, AW, Young NS. Cyclosporine therapy of aplastic anaemia, congenital and acquired red cell aplasia. Br J Haematol 1989; 72:278–284. 97. Hinterberger-Fischer M, Hocker P, Lechner, Seewann H, Hinterberger W. Oral cyclosporin-A is effective treatment for untreated and also for previously immunosuppressed patients with severe bone marrow failure. Eur J Haematol 1989; 43:136–142. 98. Frickhofen N, Kaltwasser JP, Schrezenmeier H, Raghavachar, Vogt HG, Herrmann F, Freund M, Meusers P, Salama A, Heimpel H. Treatment of aplastic anemia with antilymphocyte globulin and methylprednisolone with or without cyclosporine. The German Aplastic Anemia Study Group. N Engl J Med 1991; 324:1297–1304.

Aplastic anaemia: Management

99. Frickhofen N, Rosenfeld SJ. Immunosuppressive therapy of aplastic anaemia with antithymocyte globulin and cyclosporine. Semin. Haematol 2000; 37:56–68. 100. Rosenfeld SJ, Kimball J, Vining D, Young NS. Intensive immunosuppression with antithymocyte globulin and cyclosporine as treatment for severe acquired aplastic anemia. Blood 1995; 85:3058–3065. 101. Gluckman E, Esperou-Bourdeau H, Baruchel A, Boogaerts M, Briere J, Donadio D, Leverger G, Leporrier M, Reiffers J, Janvier M et al. Multicenter randomized study comparing cyclosporine-A alone and antithymocyte globulin with prednisone for treatment of severe aplastic anemia. Blood 1992; 79:2540–2546. 102. Marsh J, Schrezenmeier H, Marin P, Ilhan O, Ljungman P, McCann S, Socie G, Tichelli A, Passweg J, Hows J, Raghavachar A, Locasciulli A, Bacigalupo A. Prospective randomized multicenter study comparing cyclosporin alone versus the combination of antithymocyte globulin and cyclosporin for treatment of patients with nonsevere aplastic anemia: a report from the European Blood and Marrow Transplant (EBMT) Severe Aplastic Anaemia Working Party. Blood 1999; 93:2191–2195. 103. Gluckman E, Rokicka-Milewska R, Gordon-Smith EC, Hann I, Nikiforakis E, Tavakoli F, Cohen-Scali S. Results of a randomised study of glycosylated rHuG-CSF Lenograstim in severe aplastic anaemia. Blood 1998; 92:376a, (abstr.) 104. Ohara A, Kojima S, Hamajima N et al. Myelodysplastic syndrome and acute myeloid leukaemia as late complications in children with acquired aplastic anaemia. Blood 1997; 90:1009–1013. 105. Kaito K, Kobayashi M, Katayama T et al. Long term administration of G-CSF for aplastic anaemia is closely related to the early evolution of monosomy 7 in adults. Br J Haematol 1998; 103:297–303. 106. Locascuilli A. Treatment of children with acquired aplastic anaemia. In: Schrezenmeier H, Bacigalupo A, eds. Aplastic anaemia. Pathophysiology and treatment. Cambridge: Cambridge University Press, 2000; 275–287.

171

107. Tichelli A, Socie G, henry-Amar M et al. Effectiveness of immunosuppressive therapy in older patients with aplastic anaemia. Ann Intern Med 1999; 130:193–201. 108. Marsh JCW, Chang J, Testa NG, Hows JM, Dexter TM. The haemnatopoietic defect in aplastic anaemia assessed by long term marrow culture. Blood 1990; 76:1–10. 109. Podesta M, Piaggio G, Frassoni F et al. The assessment of the haemopoietic reservoir after immunosuppressive therapy or bone marrow transplantation in severe aplastic anaemia. Blood 1998; 91:1959–1965. 110. Schrezenmeier H, Marin P. Raghavachar A, McCann S, Hows, Gluckman E, Nissen C, van’t Veer-Korthof ET, Ljungman P, Hinterberger. Relapse of aplastic anaemia after immunosuppressive treatment: a report from the European Bone Marrow Transplantation Group SAA Working Party. Br J Haematol 1993; 85:371–377. 111. Tichelli A, Passweg J, Nissen C, Bargetzi M, Hoffmann T, Wodnar-Filipowicz A, Signer E, Speck B, Gratwohl A. Repeated treatment with horse antilymphocyte globulin for severe aplastic anaemia. Br J Haematol 1998; 100:393–400. 112. Marsh JCW, Geary CG. Is aplastic anaemia a pre-leukaemic disorder? Br J Haematol 1991; 77:447–452. 113. Socie G, Rosenfeld S, Frickhofen N, Gluckman E, Tichelli A. Late clonal diseases of treated aplastic anaemia. Sem Haematol 2000; 37:91–101. 114. Hamblin M, Marsh JC, Lawler M et al. Campath-1G in vivo confers a low incidence of graft versus host disease associated with a high incidence of mixed chimerism after bone marrow transplantation for severe aplastic anaemia using HLA identical sibling donors. Bone Marrow Transplant 1996; 17:819–824. 115. Baran DT, Griner PF, Klemperer MR. Recovery from aplastic anaemia after treatment with cyclophosphamide. N Engl J Med 1976; 295:1522. 116. Brodsky RA, Sensenbrenner LL, Jones RJ. Complete remission in severe aplastic anaemia after high dose cyclophosphamide without bone marrow transplantation. Blood 1996; 87:491–494.