Stem cell transplantation for non-malignant disorders

BaillieÁre's Clinical Haematology Vol. 13, No. 3, pp. 343±363, 2000

doi:10.1053/beha.2000.0082, available online at http://www.idealibrary.com on

2 Stem cell transplantation for non-malignant disorders Colin G. Steward*

MA, BM, BCh, MRCP, FRCPCH, PhD

Consultant Senior Lecturer in Bone Marrow Transplantation of Metabolic and Genetic Disease Bristol Royal Hospital for Sick Children and Department of Pathology & Microbiology, Bristol University Medical School, Bristol, UK

Stem cell transplantation (SCT) can be used to cure or ameliorate a wide variety of nonmalignant diseases. These range from inherent defects of haemopoietic cell production or function, through metabolic diseases (where blood cells are providing in vivo enzyme therapy to solid organs), to severe autoimmune diseases. However, although transplantation has revolutionized the treatment of many of the diseases discussed, severe toxicities remain. In some cases these are inherent to the disease concerned but frequently they relate to the conditioning regime or post-transplant complications such as graft-versus-host disease (GvHD). This chapter concentrates on the indications for transplant, outcome statistics and problems inherent in particular conditions, seen in the light of technological improvements during the 1990s and the potential impact of enzyme and gene therapies. Key words: adrenoleukodystrophy (ALD); bone marrow transplantation; congenital erythropoietic porphyria (CEP); globoid cell leukodystrophy (GLD); haematopoietic stem cell transplantation; haemoglobinopathies; haemophagocytic lymphohistiocytosis (HLH); malignant infantile osteopetrosis (MIOP); metachromatic leukodystrophy (MLD); mucopolysaccharidoses (MPS).

Bone marrow transplantation (BMT) ®rst became feasible with the discovery of the human leukocyte antigen (HLA) system by Dausset and others in the mid-1960s. Soon afterwards (1968) the ®rst successful matched sibling donor (MSD) transplant was performed for severe combined immune de®ciency (SCID).1 Technical improvement has been constant ever since. Notable among these are (1) improved tissue typing and widening of the prospective donor pool through large registries of unrelated donors (UD), cord blood (CB) banks and the use of haploidentical parental donors, (2) improvements in GvHD prophylaxis using immunosuppressive drugs or T-cell depletion strategies, (3) the discovery of haemopoietic growth factors and, hence, transplantation using high doses of peripheral blood stem cells (PBSC), and (4) major developments in supportive care. Other exciting innovations are taking place at the time of writing: in manipulation of chimaerism (donor/recipient haemopoietic balance), immunotherapy of viral infections and `non-myeloablative' conditioning * Address for correspondence: BMT Unit, Royal Hospital for Sick Children, St Michael's Hill, Bristol BS2 8BJ, UK. 1521±6926/00/030343+21 $35.00/00

c 2000 Harcourt Publishers Ltd. *

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therapy. These now allow cure or massive amelioration of most of the diseases discussed in this chapter. Yet many patients still reject their grafts, die of complications induced by chemoradiotherapy or from infections, develop debilitating or fatal GvHD and su€er late e€ects of their transplant conditioning. Fortunately, there seems much ground for hope that the next decade will see quantum improvements in care, with changes in conditioning treatment ± so that most patients are not rendered infertile ± and major reductions in hospital inpatient stay. However, these advances in transplantation must be weighed against alternative approaches such as enzyme and gene therapy, especially in those diseases where alternative donor transplants have poor results (e.g. DNA repair disorders, malignant infantile osteopetrosis (MIOP) and adrenoleukodystrophy (ALD)). For this reason standardization of transplant indications and audit of outcome are now major thrusts of European Blood and Marrow Transplantation Group (EBMT) activity. SPECIAL CONSIDERATIONS IN STEM CELL TRANSPLANTATION FOR GENETIC DISEASES There are several di€erences of philosophy between transplants for malignant and genetic diseases. These apply to the following areas: Conditioning therapy In transplantation for malignancy side-e€ects such as infertility, hormonal function, growth, neuropsychological function and a risk of second malignancy take second place to the aim of eradicating chemoresistant malignant cells. However, in many genetic diseases, for example, thalassaemia major, patients would have a good quality of life for many years even without transplantation. Therefore, avoidance of late e€ects is a primary consideration. It was this which stimulated Hobbs in 1980 to use Santos' protocol of busulphan and cyclophosphamide conditioning for transplantation of Hurler's disease, thereby avoiding irradiation. This protocol was highly successful and is still widely used today as conditioning therapy for genetic disease transplants.2 Unfortunately, busulphan has proved to have major drawbacks, particularly in the causation of hepatic veno-occlusive disease (VOD, a rather unpredictable and dose-limiting complication) and gonadal damage ± many patients develop primary gonadal failure and will be infertile, especially those transplanted after infancy.3,4 This explains major current interest in the use of `minimally myeloablative' protocols, although many contain the drug melphalan, which is likely to impair fertility at the doses currently used. Chimaerism Graft rejection may occur as either a primary event (never reaching a sustained neutrophil count) or a secondary event. In secondary rejection graft failure may render the patient aplastic, but more commonly this occurs as a silent process ± wherein the blood count is maintained but recipient cells gradually replace those of the donor. This is a recurrent phenomenon after chemotherapy-only conditioning and is particularly common in patients who have received T-cell-depleted transplants and in those who do not develop GvHD (e.g. ref. 5). Most often, secondary graft rejection is a partial

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phenomenon in which chimaerism gradually swings back in favour of the recipient in the early months after SCT but then stabilizes by the end of the ®rst year posttransplant. In some malignant diseases, for example acute lymphoblastic leukaemia (ALL), complete silent secondary rejection may be compatible with sustained remission, and perhaps even with permanent cure. This does not hold true for genetic diseases. In some instances mixed chimaerism may be adequate for cure, for example haemophagocytic lymphohistiocytosis (HLH), MIOP. In others, mixed chimaerae tend to convert to full donor chimaerism because recipient cells are at a competitive disadvantage to donor cells, for example Fanconi anaemia. However, in many diseases substantial mixed chimaerism or full rejection of donor cells are disastrous. Good examples are the metabolic diseases where enzyme production tends to be proportional to the percentage of donor engraftment. Practical chimaeric analysis is best performed by either PCR ampli®cation of DNA microsatellite regions, FACS analysis of HLA-antigens discrepant between patient and donor or (in sex mismatched transplants) cytogenetic analysis. In Bristol, for instance, we use a technique which allows analysis of granulocyte, T-, B- or NK-cell chimaerism throughout the ®rst month of transplant using 1±5 ml of peripheral blood.6 This has many uses, including (1) early detection of primary graft rejection, (2) monitoring of secondary graft rejection, for example, allowing safer withdrawal of cyclosporin therapy after transplant for severe aplastic anaemia (SAA), and (3) comparison of chimaerism in cells of di€erent lineage. Graft-versus-host disease In many types of transplant GvHD can be advantageous, provided that it is of limited severity and easily controlled. For example, in chronic myeloid leukaemia (CML) cure rates are higher for those who develop limited GvHD than for patients with none, due to a graft-versus-leukaemia e€ect. However, in genetic transplantation the risks frequently outweigh the gains. In many genetic diseases outcome is worsened. For example, although full donor engraftment is more likely after GvHD, neurodevelopmental outcome for patients with Hurler's disease and adrenoleukodystrophy (ALD) is de®nitively worse in those who sustain signi®cant GvHD.7 Furthermore, in SCID patients both the complication itself and the immunosuppression used for control worsen immune reconstitution and handling of infections. CLASSES OF DISEASE TREATED Stem cell transplantation will alleviate any disease which is due to an intrinsic defect of haemopoietic cells. In the descriptive section which follows these disorders are grouped into disorders of red cells, white cells, platelets or stem cells and those which commonly present with acute haemophagocytosis. This is for convenience only and generally corresponds to the cell line which is most clearly disturbed. Hence, autoimmune diseases are categorized under white cell disorders, and Schwachman±Diamond syndrome under stem cell disorders (due to the propensity for development of aplastic anaemia). The role of transplantation in Diamond±Blackfan anaemia is discussed in Chapter 4. The other major class of diseases treated are metabolic disorders which can bene®t from enzyme donation from donor blood cells. These are all lysosomal storage

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disorders, with the exception of ALD. The management and SCT of these conditions is complex. Because few haematologists would encounter them, and numbers of transplants performed are relatively small, they are discussed only brie¯y. RED CELL DISORDERS Thalassaemia major Thalassaemia major represents by far the commonest indication for SCT among the non-malignant diseases, more than 1500 patients having been transplanted from matched siblings since the ®rst successful procedure in 1981.8 Very few alternative donor transplants have been performed. However, with the success of rigorous iron chelation regimes in increasing life expectancy and the development of oral iron chelators, the role of SCT in managing thalassaemia remains highly contentious. The best results are obtained in those transplanted early in life before iron overload and HLA sensitization have become established problems. Lucarelli et al demonstrated the ecacy of BMT in this setting in a large group of children (416 years) treated with a conditioning protocol based on busulphan 14 mg/kg total dose and cyclophosphamide 200 mg/kg total dose. They showed that outcome could be strati®ed according to three risk factors: (1) hepatomegaly greater than 2 cm, (2) portal ®brosis on pre-transplant liver biopsy, and (3) failure to achieve chelation with desferrioxamine subcutaneously for 8±10 hours on at least 5 days per week commencing within 18 months of starting hypertransfusion therapy. Patients with none of these poor prognosis features were assigned to class 1, those with one or two adverse features to class 2, and patients with all three to class 3.9 The probabilities of survival and event-free survival are 93 and 91% in class 1, 87 and 83% in class 2, and 61 and 53% in class 3 patients.10 The poor results in class 3 patients prompted the use of a smaller dose of cyclophosphamide (160 mg/kg), a protocol also used in adults aged 17±35 years. This produced death rates in class 3 children of 14% but in class 3 adults of 35% together with rejection rates of 28 and 4% respectively. Since 1997 the same investigators have turned to a protocol for poor-risk patients based on pre-conditioning therapy with hydroxyurea, azathioprine and ¯udarabine. Conditioning therapy in these patients comprises busulphan 14 mg/kg for all patients followed by cyclophosphamide 160 mg/kg for those aged less than 17 years and 90 mg/kg for older patients. Early results suggest much lower rejection rates and less overall toxicity. Mixed chimaerism is a frequent event in the ®rst year after transplant. Many patients convert eventually to full donor chimaerism, suggesting some competitive advantage for the donor cells. As many as 30% of patients become stable mixed chimaerae but they usually have normal haemoglobin levels.11 Following successful transplant, recurrent venesection is frequently used to reduce iron overload and this may result in reversal of early hepatic ®brosis.12 Sickle cell anaemia In marked contrast to thalassaemia major, SCT is relatively rarely employed in severe sickle cell disease ± less than 200 transplants having been performed worldwide. In Britain, current guidelines restrict the use of transplant to those with severe sickle cell anaemia aged less than 16 years who have an MSD and one of the following (1) preexisting sickle-related neurological defect, cerebrovascular accident or subarachnoid haemorrhage (but not severe neurological de®cit such as moya-moya13), (2) more than

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two episodes of acute chest syndrome, (3) stage I chronic sickle lung disease, (4) recurrent severe vaso-occlusive crises ± at least three per year for 3 or more years, or (5) imminent return to a country where sickle care will be problematic. The only major series comprises children transplanted in Belgium or France from African republics where primary healthcare is of poor quality. Of 42 children, 38 were alive with donor engraftment (in two following two procedures) 1±75 months after SCT, and only one child had died.14 Perplexingly, several children who have rejected grafts have subsequently developed increased fetal haemoglobin levels (22±33%) which have rendered them symptom-free15 ± a phenomenon not seen following SCT for thalassaemia. The future role of transplantation in sickle cell anaemia is dicult to evaluate. Already it is interesting to query the discrepancy in the use of SCT in thalassaemia major and sickle cell anaemia when both diseases share poor long-term prognoses. For example, a recent large cohort study from Los Angeles showed that the average life expectancy of sickle cell African-Americans was only 40 years for men and 49 years for women, approximately a 30-year reduction over una€ected peers.16 However, this debate may become irrelevant if drugs which can increase the HbF concentration (e.g. hydroxyurea, sodium phenylbutyrate) are shown to be safe for prolonged use in children. Congenital erythropoietic porphyria (CEP, Gunther's disease) This rare autosomal recessive disease is characterized by severe, mutilating photosensitivity, excretion of red urine, discoloration of teeth and chronic haemolysis. Severely a€ected patients have an acceptable life only by avoidance of sun and use of total sunblocks. The author is aware of ®ve transplants for this disease: four have survived long term (one after two grafts) and were free of photosensitivity and haemolysis at the last follow-up (four reviewed in ref. 17). WHITE-CELL DISORDERS Severe combined immune de®ciency (SCID) The cure or amelioration of SCID by SCT constitutes one of the great success stories of non-malignant transplantation, especially when considering the poor clinical status and high infective load of many patients. Since the ®rst successful MSD procedures in 1968 the donor pool has been widened to include other related donors, UD, CB and haploidentical parental donors. As patients lack signi®cant T-cell function they often accept even mismatched grafts without conditioning therapy and with relatively little GvHD. Indeed, this last complication is rare in unconditioned transplants from MSDs, elegantly demonstrating the role of conditioning therapy, tissue damage and resultant infection in precipitating GvHD. Currently, cure rates run at greater than 90% for those having HLA-id donors. Tand B-cell reconstitution are generally rapid (with the exception of adenosine deaminase de®ciency [ADA]-SCID). Results are less good following T-cell depleted HLA-non identical transplants: in 1999 the EBMT reported disease-free survival of 60% for those with SCID with functioning B-cells (B‡ SCID) compared with only 35% for Bÿ SCID. Follow-up was in excess of 52 months for all patients.18 Perhaps the most remarkable results came from a series of 77 patients transplanted from haploidentical donors at Duke University between 1982 and 1998.19 These grafts were T-cell depleted by soybean agglutination and two rounds of rosetting with sheep

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erythrocytes, producing 10 000-fold (`4 log') depletion of T-cells. Although grafted with neither conditioning therapy nor GvHD prophylaxis, 78% of these patients remain alive long term. GvHD developed in 28 patients but was only of grade III in eight patients and no patient died of this complication. Unfortunately, these results would be hard to reproduce in Europe where almost one quarter of patients have the phenotype T-BNK‡ , characterized by high rates of rejection and chronic GvHD ± no such patients were present in the Duke series. The evolution of chimaerism after SCID transplantation remains one of the most fascinating enigmas of transplantation. After unconditioned SCT, from either siblings or other donors, it is very common to ®nd that the T-cell compartment is entirely reconstituted by donor cells, yet the myeloid and B-cells are largely or exclusively of recipient origin.20 This implies that either donor stem cells preferentially di€erentiate into T-cells or that lymphoid progenitors di€erentiate in the patient's thymus. If the latter is true we may see gradual loss of naõÈ ve T-cells in these patients long after SCT.21

T-cell immunode®ciencies SCT has also been attempted for a variety of T-cell disorders which cause death in childhood, including cartilage hair hypoplasia, MHC class II de®ciency, Omenn's syndrome and purine nucleoside phosphorylase (PNP) de®ciency. In general, results are less good than for SCID, with only about half of the patients surviving. This may be attributable to the less severe phenotype so that patients tend to present later when already signi®cantly infected. Transplant should therefore be performed as soon as possible after diagnosis for these diseases.

Wiskott±Aldrich syndrome Partial cure of this disease by SCT using immunosuppressive chemotherapy only as conditioning therapy was ®rst reported in 1968; the ®rst true correction followed in 1978 with full conditioning.22 Now, more than 80% of patients can be cured by HLA-id SCT23 although transplantation early in life is crucial for best results. In addition to high rates of non-engraftment and severe GvHD24, non-identical SCT is complicated commonly by EBV-driven post-transplant lymphoproliferative disease (PTLD) (39% compared to 12% in T-cell de®ciencies and 3% in SCID).

CD40 ligand de®ciency (hyper-IgM syndrome) This disease results in susceptibility to opportunistic infection, especially with Pneumocystis, Toxoplasma and Cryptosporidium, and early death ± less than 40% of patients will survive to the age of 25 years.25 Cryptosporidial infection causes chronic cholangitis and eventual cirrhosis in many patients, which favours early transplantation. However, the decision to proceed is dicult because many patients have a good quality of life for many years without SCT, and prediction of prognosis is dicult. The outlook for patients may be helped by non-myeloablative methods of transplantation, which have already yielded promising early results in several patients with cholangitis (P. Veys, personal communication).

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Autoimmune disease The past 5 years have seen steadily increasing interest in SCT as a means of controlling autoimmune disorders refractory to conventional immunosuppression.26 This follows occasional reports of sustained remission of autoimmune disorders following allogenic or autologous transplantation for other concomitant disorders, but is also due to the increasing sophistication of T-cell depletion manoeuvres and better supportive care of immunosuppressed patients. So far, these e€orts have concentrated entirely on the use of autologous PBSC transplants because these result in death in less than 2% of recipients ± much less than that seen following allogeneic SCT. The primary diseases so far treated have been rheumatoid arthritis and systemic lupus erythematosus (SLE), although multiple sclerosis, systemic sclerosis and other severe autoimmune diseases are also candidates. T-cell depleted, mobilized PBSC are generally used (aiming for a target CD34 dose of 42  106/kg recipient weight and T-cell dose of 51  105/kg). Promising results have been obtained from a number of institutions in adult and juvenile SLE, with some remissions sustained for more than 1 year and evidence of improvement in severe renal and respiratory complications. However, the largest single body of experience is in children treated for juvenile chronic arthritis (JCA, 10 systemic, two polyarticular) in Utrecht (four reported in ref. 27). All had progressive disease despite maximal immunosuppression. Disease responses were good with six patients staying in sustained remission without any drug therapy, two in partial remission and one with mild disease relapse at follow-up of 6±29 months. However, three children died of `macrophage activating syndrome' (MAS) at 12 days, 17 days and 5 months post-transplant. This complication, which may occur spontaneously in JCA28, constitutes a fulminant haemophagocytosis with many of the characteristics of HLH. The future of this therapy therefore remains in doubt unless this complication can be avoided.

DISORDERS OF MONONUCLEAR PHAGOCYTES The major phagocyte disorders amenable to transplantation include leukocyte adhesion de®ciency (LAD), chronic granulomatous disease (CGD) and severe congenital neutropenia (SCN). These diseases all require myeloablative conditioning therapy for successful engraftment. In the case of CGD and SCN alternative therapies are usually used which are safer in the short to medium term. This lies at odds with the observation that SCT is best performed at a young age (preferably less than 4 years) when the infective load and organ damage are at a minimum.29,30 Careful assessment of each patient in terms of quality of life, disease status and donor considerations are therefore required. Leukocyte adhesion de®ciency Leukocyte adhesion de®ciency is due to defective expression of the b2-integrin subunit of the leukocyte adhesion proteins. In its most severe form bacterial infections cause death in the ®rst few years of life. SCT can cure more than 70% of children with this disease, but aggressive conditioning with total body irradiation (TBI) or etoposide-based protocols may be required.

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Chronic granulomatous disease CGD results from abnormality of one of the four subunits of the NADPH oxidase complex. In the 1980s and early 1990s prophylactic g-interferon formed a mainstay of therapy31, but similar results can now be achieved with a careful regime of cotrimoxazole and itraconazole prophylaxis (D. Goldblatt, personal communication). However, complications such as abscesses, colitis, cystitis and pneumonia impair quality of life, and many patients will have a reduced life expectancy. With meticulous management, SCT may cure more than 75% of patients with HLA-id donors but patients are at risk from GvHD and MAS due to their nascent infective load (R. Seger, personal communication). Transplant therefore tends to be reserved for those who have developed stubborn or serious infection. Severe congenital neutropenia Severe congenital neutropenia is now routinely treated by G-CSF administration.32 However, 5% of patients fail to respond, even to high doses (up to 150 mg/kg/day), and approximately 2% of patients per year transform to MDS or AML (half of whom have monosomy of chromosome 7). Many of these patients have developed clonal mutation of the G-CSF receptor. SCT therefore probably represents the treatment of choice for patients requiring high G-CSF doses, acquiring clonal mutations or in transformation.33 However, as yet, relatively few transplants have been reported. Malignant infantile osteopetrosis This disease is thought to result primarily from abnormal development or function of osteoclast precursors. However, despite knowledge of genetic and biochemical mechanisms in numerous naturally occurring and gene knockout animal models, the human disease is poorly understood. Two major forms are recognized currently: that due to carbonic anhydrase isoenzyme II de®ciency (which results in less aggressive osteosclerosis, renal tubular acidosis and cerebral calci®cation with mental retardation) and a severe neuronopathic form (resembling a neuronal ceroid lipofuscinosis). Although CAII de®ciency was the ®rst in which transplant was attempted34 it remains unclear whether SCT can prevent cerebral calci®cation and intellectual impairment, transplantation is certainly completely ine€ective in preventing neurodegeneration in the neuronopathic form.35 SCT can be curative for other children with MIOP, normalizing bone density in the ®rst year after transplantation and preventing further nerve compression (see Figure 1). Unfortunately, vision seems to be preserved only if atrophy is not fully established before transplantation. Cure rates in Europe run at almost 80% where HLA-id donors are available but less than 40% using alternative donors.35 The latter is principally due to high rates of rejection following T-cell depletion (especially that involving CAMPATH antibodies) and poor handling of post-transplant viral infections. However, outlook may be improving for children with MIOP. Technical advances in T-cell depletion by immunomagnetic means now enable successful high-dose haploidentical SCT without GvHD using PBSC + bone marrow.36 Furthermore the recognition that CD34 cells spontaneously circulate in the peripheral blood of MIOP patients in equivalent numbers to that of G-CSF mobilized PBSC donors has allowed (1) collection of back-up material before transplant (personal experience), and (2) detailed in vitro modelling of osteopetrotic osteoclasts.37

Figure 1. Bone clearing after stem cell transplantation for malignant infantile osteopetrosis. This series of radiographs of the lower femur show the rapid reduction of bone density and remodelling which occur in the ®rst 2 years after successful transplantation. At the time of BMT the bone shows complete loss of cortico-medullary di€erentiation; by 22 months post-transplant appearances have normalized.

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DISORDERS PRESENTING WITH ACUTE HAEMOPHAGOCYTOSIS Haemophagocytic lymphohistiocytosis Haemophagocytic lymphohistiocytosis (HLH) is a disease which presents mainly in early infancy and is characterized by uncontrolled immune activation of macrophages and T-lymphocytes.38 CNS involvement occurs in 73%, either in the form of lymphocytic meningitis or parenchymal disease.39 Where there is a positive family history (usually following autosomal recessive (AR) inheritance) the disease is termed familial erythrophagocytic lymphohistiocytosis (FEL). Virus-induced forms also occur (virusassociated haemophagocytosis, VAHS), although presentations of FEL are also often triggered by viruses. In addition, a child may have true FEL but be the ®rst to present in the family. This causes considerable diagnostic diculties. In practice, children who present early in infancy have a very poor prognosis. Remissions can be induced by immunosuppressive therapy + chemotherapy in many patients but this is durable in only 10%.40 Older children are more likely to have VAHS, and cure rates without recourse to transplantation are higher. The critical role of BMT was ®rst reported in 198641, and up to 80% of children will stay in sustained remission after HLA-id BMT. Results have been generally poorer following transplant from other related or unrelated donors. However, these now appear to be improving with the use of conditioning regimes in which VP16 + ATG therapy is added to busulphan/cyclophosphamide. Further promising approaches are cord blood transplants or the use of highly T-cell-depleted haploidentical transplants. Two particular problems lie in (a) curing those with parenchymal CNS disease42 and (b) avoiding the use of sibling donors who themselves actually harbour the causative gene/protein defect (this can result in later HLH in donor cells). Hope for the latter may come from genetic tests: defects of candidate genes on chromosomes 9 and 10 may be responsible for approximately 50% of cases.43 HLH is easily confused with several other rarer disorders. In each case patients are prone to fulminant haemophagocytic episodes, often triggered by viral infections. All of these diseases can only be cured by initial control of haemophagocytosis and subsequent SCT. These include (1) X-linked lymphoproliferative disease, in which EBV infection triggers uncontrolled proliferation of T- and B-cells, killing more than 75% of a€ected males by the age of 10 years, (2) Chediak±Higashi syndrome44, and (3) Griscelli syndrome.45 PLATELET DISORDERS Glanzmann thrombasthenia Successful SCT has been reported in three patients (reviewed in ref. 46) but this should be reserved for those patients with severe disease who have failed alternative therapies such as intravenous administration of 1-deamino-8-D-arginine vasopressin (DDAVP) or recombinant activated Factor VIIa.47 STEM CELL DISORDERS Aplastic anaemia The latest decade has seen great advances in the treatment of severe aplastic anaemia (SAA) by immunosuppressive therapy with cyclosporin, ATG + G-CSF, with initial response rates in the range 70±90%.48,49 However, responses to such therapy often

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take 2±3 months to develop, and children under 5 years of age tend to respond poorly to immunosuppression.50 Furthermore, responses are frequently partial, aplasia may return, and some patients develop MDS, AML or premalignant clones.51 For children with SAA who have HLA-id donors, SCT therefore remains the treatment of choice, and long-term survival rates exceed 90% in some paediatric series.49 Conditioning therapy for HLA-id transplants usually comprises cyclophosphamide 50 mg/kg for four doses. This results in graft rejection in less than 10% of cases, although the risk of this is greater in those who have become HLA-sensitized by frequent transfusions (a reducing problem with widespread leukodepletion of blood products). Rates of acute and chronic GvHD can be reduced by the use of peritransplant serotherapy with ATG or CAMPATH-1G. Typically, chronic GvHD will occur in less than 20% of paediatric patients. Graft rejection can occur following cessation of the cyclosporin administered as GvHD prophylaxis, and this therapy is often maintained for 1 year after SCT and then withdrawn cautiously, accompanied by careful monitoring of chimaerism. Recurrence of aplastic anaemia is recorded up to 14 years after transplant and may be caused by autoreactive donor cells recognizing epitopes on maturing marrow precursor cells52 or by late graft rejection.53 Patients may be salvaged by further immunosuppressive therapy or re-transplantation from the original donor. Results from alternative donor SCT are much less satisfactory, with less than half of the patients being long-term survivors, and are especially poor from mismatched related donors. Due to higher rates of graft rejection, TBI is often included in conditioning regimes, increasing both toxicity and rates of GvHD. T-cell-depletion strategies reduce the incidence of GvHD but only at the expense of greater graft rejection. These poor results are undoubtedly aggravated by the fact that many patients have endured prolonged pancytopenia during trials of immunosuppression, increasing infective load and HLA sensitization. However, most centres would currently reserve SCT for patients with no sign of response to two courses of immunosuppression. This may change if protocols combining lower-dose TBI and ATG with conventional-dose cyclophosphamide ful®l early promise. Fanconi anaemia (FA) Safe transplantation in this disease became possible only with the recognition by Gluckman et al that most patients with HLA-id donors could be engrafted using lowdose total abdominal irradiation (5 Gy in a single fraction) and cyclophosphamide 20 mg/kg total dose as conditioning therapy. Eight-year survival of almost 60% can be achieved from MSD transplants with this protocol. Unfortunately, chronic GvHD developed in 70%.54 A worrying recent trend has been the development of squamous carcinomas of the mouth and pharynx more than 5 years after transplant in many of those who sustained signi®cant GvHD (to which Fanconi patients are excessively prone). Current studies are therefore examining the use of pre- and post-transplant ATG55 and substitution of irradiation with reduced-dose busulphan in an attempt to reduce the incidence of GvHD (E. Gluckman, personal communication). Results are much more disappointing from unrelated donors, probably because (a) HLA discrepancy increases the risk of rejection, placing much more emphasis on e€ective ablation, and (b) there is a greater risk of GvHD. A recent EBMT report detailed 69 patients transplanted from matched UDs: the 3 year probability of survival was 33%, and 34% of the patients had developed grade III±IV acute GvHD.56 Signi®cant

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adverse factors (in descending order of signi®cance) were malformations involving three or more sites, elevated serum transaminases before transplant, use of female donors, recipient CMV seropositivity and use of androgens pre-transplant. Others have found mosaicism, with more than 10% of lymphocytes being diepoxybutane (DEB) resistant, to be another factor favouring graft rejection (A. Auerbach, personal communication). It is to be hoped that long-term results with CB or nonmyeloablative transplantation will improve outcome for these patients. Dyskeratosis congenita (DC) BMT has been used in a small number of cases of DC but with poor results, for example, only four of 11 patients who received HLA-identical BMT in one series were alive 8 months±6 years post-BMT.57 Unusual complications after MSD transplantation include renal microangiopathy, veno-occlusive disease and idiopathic pneumonitis.58 Because these results echo those of Fanconi patients given high-dose chemotherapy, it is interesting to note the relatively successful grafting of two siblings from HLA-id donors using reduced-dose conditioning therapy.59 It should be stressed that SCT appears not to alleviate systemic problems, other than AA, and indeed, may well increase the likelihood of malignancy. Schwachman±Diamond syndrome From only a small number of transplants performed, one patient has died of multiorgan toxicity and two of cardiac failure.60,61 The latter is extraordinary in patients who have not received previous chemotherapy and in a disease not noted for cardiac complications. These poor results, coupled with the propensity of a quarter of patients to develop aplastic anaemia62, raise the possibility of inherent DNA fragility, as in Fanconi anaemia. Transplantation avoiding the use of cyclophosphamide (a cardiotoxic drug) and careful assessment of cardiac status should be considered. METABOLIC DISEASES With the exception of ALD this group of disorders are caused by de®ciency of lysosomal enzymes. These enzymes can be substituted from donor white cells by a mechanism of release into plasma and re-uptake via mannose-6-phosphate receptors. Animal studies show that CNS microglia are replaced slowly (approximately 20% within 3±4 months of transplant63) by cells from donor marrow, thereby donating enzyme to brain cells.64 Results appear best when normal donors are employed, because carriers will usually have only half of the normal enzyme levels. In general, those who present earliest have the most severe enzyme de®ciencies and rapid disease progression, and do least well with transplantation.65 Mucopolysaccharidoses: Hurler's disease (MPS type I) and Maroteaux± Lamy syndrome (MPS VI) MPS I varies from a mild disease (Scheie) through intermediate forms (Hurler±Scheie) to classical Hurler's disease, the most severe of all the MPS disorders. The ®rst successful BMT for Hurler's disease was performed by Hobbs et al in 1980.66 This was stunningly successful, transplantation reversing many of the worst aspects of disease, and has been

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followed by over 100 further transplants. Major improvements are that breathing diculties are alleviated rapidly, corneal clouding largely clears, hepatosplenomegaly reduces and hydrocephalus is prevented. However, IQ may take up to 1 year to stabilize after transplant. As a result, transplants before 2 years of age yield the best results.7 Sadly, there appears to be little bene®t to the severe bone disease (dysostosis multiplex) and patients will usually require orthopaedic procedures on their knees, hips and spine as well as carpal tunnel releases. Unfortunately, Hurler patients have a unique propensity to reject transplants from HLA-id siblings (e.g. complete donor engraftment occurred in only 54% of HLA-id transplants in the largest series reported).7 This may result from abnormal handling of busulphan but renders UD transplantation particularly fraught. This may be helped by the use of alternative conditioning regimes presently under development. MPS VI has been a much rarer indication for SCT because most patients have normal intelligence. However, many patients die from pulmonary or cardiac insuciency in the second to third decade, and those with severe disease can now be predicted by assessing enzyme activity against natural substrate. Favourable responses, including resolution of pulmonary hypertension and cardiomyopathy, have been described.67 Adrenoleukodystrophy (ALD) This X-linked disease typically presents as either isolated adrenal hypofunction (Addisonian presentation), as a severe progressive leukodystrophy with childhood onset (childhood cerebral ALD, CCER) or with spastic paraparesis or psychiatric problems between 20 and 50 years of age (adrenomyeloneuropathy, AMN). These forms a€ect approximately 10, 25 and 65% of patients respectively. A€ected males, whether symptomatic or not, are characterized by having abnormal very-long-chain fatty acids (VLCFA). Lorenzo's oil and special diets can rapidly normalize the ratio of VLCFA (24 and 26 carbon backbone fatty acids) to shorter fatty acids, but there is little evidence of any bene®cial e€ect. A majority of patients develop neurological problems by the sixth decade and have reduced life expectancy, but children with CCER will often die within 5 years of diagnosis. SCT can undoubtedly arrest disease progression68, yet the mechanism is completely obscure (see Figure 2). Microglial replacement may be involved, or an immune e€ect, although pharmacological immunosuppression does not slow progression. The aim of transplant has been to treat early CCER; no attempt has been made to alleviate isolated AMN. Relatively small numbers of transplants have been performed, but Krivit and Peters (Minneapolis) have shown that SCT is best attempted when boys show progressive impairment of performance IQ (but not to below 80) or more severe magnetic resonance imaging (MRI) changes. Those with frank neurological presentations do poorly. This approach avoids performance of SCT in boys not destined to develop CCER. However, once a decision has been taken to transplant this must be performed swiftly because the disease can progress rapidly and deterioration (albeit at a slower rate) may continue for 1±2 years after transplant. Full donor chimaerism is not essential for virtual normalization of VLCFA (personal observation). Globoid cell leukodystrophy (GLD) GLD is an AR disorder which results in progressive CNS deterioration. Depending on the severity of the galactocerebrosidase de®ciency the disease may present in infancy (classical Krabbe disease) or as juvenile or adult forms. Responses are very poor in

356 C. G. Steward

Figure 2. MRI improvement following stem cell transplantation for adrenoleukodystrophy. The active in¯ammatory front of the leukodystrophy process is shown in the pre-transplant ®lm as an area of ring enhancement (arrowed) following gadolinium injection (T1-weighted image). Four months after transplant this appearance had greatly improved.

infantile disease because patients typically present at 3±6 months of age when severely symptomatic. However, one child diagnosed at birth because of a positive family history and transplanted at 2 months of age from an HLA-id donor was showing favourable developmental progress when last reported at 16 months post-BMT.69 In the same paper, four patients with juvenile disease were also reported: two were transplanted when pre-symptomatic, and two others 3±6 years after the ®rst recognized symptoms. All showed signs of CNS improvement (based on a combination of clinical, MRI and CSF protein assessments) at follow-up ranging from 3.2 to 8.9 years. Metachromatic leukodystrophy (MLD) Three common variants of this AR leukodystrophy are recognized from a severe late infantile (LI) form, through juvenile disease to an adult form. Numbers of transplants are very limited. However, there is very little evidence that SCT has any bene®t in LI disease (although one possible exception would be in a child recognized soon after birth because of a previous index case). By contrast, juvenile disease can bene®t if transplanted early, and several adults with psychiatric presentations have also done well.65 Gaucher's disease This disease is not currently considered an indication for SCT where enzyme therapy with mannose-terminated glucocerebrosidase (Ceredase/Cerezyme2) is freely available. However, long-term follow-up of type III disease (Norbottnian, subacute neuronopathic) may yet con®rm superior results for SCT. Initial attempts to transplant Gaucher patients were dogged by rejection secondary to massive hepatosplenomegaly.70 After the introduction of pre-transplant splenectomy all seven subsequent patients engrafted successfully (although one required two successive UD transplants). All parameters of disease improved dramatically after BMT, including parenchymal lung disease in one patient. A further six patients were later transplanted in Stockholm.71 One rejected the graft, and two became 30 and 80%

Stem cell transplantation 357

stable donor chimaerae. Three patients developed worsening or new spinal kyphosis despite full engraftment. Of particular importance, four patients had type III disease, of whom three have 80±100% donor engraftment. All three have shown a neuropsychological pro®le on long-term follow-up better than expected in the natural history of the disease (one has an IQ of 116 at 21 years of age). Other metabolic diseases After encouraging results in a dog model of fucosidosis, a single patient has been transplanted. BMT was performed at 8 months (early in the disease course, following identi®cation in an older brother) and the child had only mild developmental delay at 18 months when last reported.72 Of the remaining lysosomal storage diseases there have also been promising reports in MPS VII (Sly syndrome) and aspartylglucosaminuria.68 NEW DIRECTIONS Donor selection The choice of donor material is constantly widening with rapidly expanding UD registries and CB banks. The next decade is likely to see improved typing, including that of some minor antigens, and vastly reduced search to transplant times. The advent of PBSC collection and extreme T-cell depletion (to less than 5  104 T-cells/kg recipient) have brought the potential of haploidentical transplantation for those lacking well-matched family donors or UDs.36 These also abolish the delay of searching for a donor, ideal in rapidly progressive diseases such as HLH or MIOP. Minimally myeloablative transplants For some time it has been hypothesized that (1) partial myeloablation and heavy immunosuppression may be preferable to full myeloablative transplants (as these would reduce tissue injury and cytokine release they should engender less GvHD), (2) this, in turn, would allow infusion of more T-cells, leading to immunological ablation of residual recipient cells, and (3) where patients engrafted as mixed chimaerae, their marrow could subsequently be forced towards full donor chimaerism by graded donor leukocyte infusions (DLI) after transplant. During the past 4 years these predictions have been tested in patients un®t for conventional SCT. Early signs are promising, with some protocols appearing to induce high rates of full donor chimaerism with minimal toxicity and GvHD, although follow-up is very short and numbers limited.73 If these results are sustained, many haematological diseases currently considered of insucient severity to risk SCT will become candidates for transplantation. Improving GvHD prevention and immune reconstitution Recent e€orts in reducing GvHD have centred on the development of better drugs (e.g. FK506, mycophenolate mofetil) and more profound methods of T-cell depletion. However, all these methods result in immunosuppression. For example, of 18 children transplanted in Paris for various genetic diseases using extremely T-cell-depleted highdose SCT, only three patients developed GvHD but ®ve died of viral infection within 3 months post-transplant (A. Fischer, personal communication). This is stimulating

358 C. G. Steward

trials with graded DLI after transplant using T-cells physically depleted of alloreactive cells or anergized after challenge with recipient cells in an MLR (e.g. ref. 74). In utero and cord blood transplantation Attempts at in utero BMT have been disappointing, with the exception of a small number in fetuses with SCID (who arguably may have done equally well with early postnatal transplants).75,76 The reason is that fetuses become immunocompetent very early in gestation (by 12±14 weeks) and are therefore able to reject grafts by the time they are physically large enough to receive stem cell injections. CB transplantation appears much more promising. Early results show that GvHD is signi®cantly less common than following equivalently matched UD transplant, allowing the use of relatively more mismatched CB donations than would be considered acceptable from a UD.77 Unfortunately the inherently low cell doses result in delayed neutrophil and platelet recovery, giving poorer results in larger children and adults. Cord transplants should be carefully considered as the stem cell source of choice in any small child (520 kg) who is CMV-negative (CMV status also worsens outcome). Infection control Despite the advent of better preventative strategies small numbers of patients still die from infections, notably with Aspergillus, CMV, adenovirus, RSV and EBV-posttransplant lymphoproliferative disease (PTLD). While an increasing number of drugs are available ± for example, acyclovir, ganciclovir, foscarnet and cidofovir are now available to combat CMV infection ± there are promising improvements in both preventative and immunotherapeutic strategies. Our understanding of CMV biology after transplant is being sequentially improved by detection systems based on expression of the CMV phosphoprotein antigen pp65. This can be detected by conventional ELISA, by DNA-PCR or by monitoring speci®c cytotoxic lymphocyte (CTL) numbers using tetramers to peptide nonamers derived from pp65. These tests allow better guidance of therapy using toxic agents such as ganciclovir and foscarnet. Speci®c anti-CMV CTL can also be cultured and administered prophylactically; this may abolish the risk of CMV re-activation in at-risk patients.78 PCR assays may also be used to assess the viral load of EBV genomes in serum. This can give early warning of developing EBV-PTLD and allow prophylactic use of donorderived EBV-speci®c CTL in at-risk patients.79 Unfortunately the culture process is long and expensive and therefore outwith the scope of most units. However, there have been several very promising recent reports of the use of anti-CD20, 21 or 24 monoclonal antibodies.80,81 Immunotherapy may be increasingly vital in the future if viral resistance patterns worsen. One problem in the future may be HSV resistance, exacerbated by prolonged and continuous use of acyclovir as prophylaxis against CMV and other Herpes group virus infections. Acyclovir-resistant strains may also show resistance to other agents such as valaciclovir and foscarnet, although cidofovir may have a valuable role in their control. Mesenchymal stem cells Mesenchymal stem cells (MSC) extracted from marrow can di€erentiate under appropriate stimuli into osteoblasts, chondrocytes, myocytes or adipocytes and even into

Stem cell transplantation 359

oligodendria, astrocytes and neurones. In addition they can suppress alloreactive T-cell responses, support stromal cell growth and express proteins such as iduronidase at much higher levels than lymphocytes.82 Unfortunately, cells of mesenchymal origin appear to engraft very poorly, especially after chemotherapy conditioning, so that these usually remain exclusively of recipient origin.83 Some groups are now attempting to supplement stem cell infusions with cultured MSC in an attempt to improve donor contributions to stroma. If successful, these could facilitate treatment of diseases such as severe (type III) osteogenesis imperfecta. Enzyme/gene therapy In future it will be vital to balance the relative roles of enzyme, gene and other developing therapies (e.g. `substrate deprivation' therapy in the glycolipid diseases) with that of SCT. For gene therapy to become widely employed it will be necessary to develop safe vectors capable of eciently transducing haemopoietic stem cells and giving stable gene expression. It is not dicult to foresee eventual success in those diseases where donor (and therefore gene-corrected autologous) cells enjoy competitive advantage. For other diseases the need to displace untransfected stem cells without recourse to conditioning therapy will be a major problem. By contrast, relatively successful enzyme treatments are already available for ADA-SCID, Gaucher's and Hurler's diseases and others are already in development ± for example, for MPS II, VI and VII. Problems with these treatments include the likelihood of poor CNS penetration, the necessity for lifelong administration, and expense. However, such treatments will always have a role while SCT carries signi®cant short- and long-term risks. CONCLUSION SCT has the potential to cure almost any patient with a genetic disease of the blood system. In the past this has usually been at great risk and discomfort to patients. However, recent technical advances are destroying many hallowed tenets of transplantation, such as the necessity for profound myeloablation. This is opening new possibilities in treatment which may radically a€ect the future safety, scope and acceptability of SCT. Acknowledgements The author gratefully acknowledges the support of the COGENT Trust, of his medical, nursing and laboratory colleagues at the Bristol Royal Hospital for Sick Children and Bristol University Medical School, and of many valued colleagues in the Inborn Errors Working Party of the EBMT. Particular thanks also go to Drs Nick Goulden, Irene Roberts and Ed Wraith for advice on preparation of this manuscript.

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