How I manage aplastic anaemia in children

state of the art review How I manage aplastic anaemia in children Sujith Samarasinghe 1 and David K. H. Webb 2 1 Paediatric Haematopoietic Stem Cell Transplant Unit, Department of Adolescent and Paediatric Haematology and Oncology, Great North Children s Hospital, Royal Victoria Infirmary, Newcastle Upon Tyne, UK and 2 Department of Haematology, Great Ormond Street Hospital, London, UK Summary Aplastic anaemia (AA) is a rare heterogeneous condition in children. 15 20% of cases are constitutional and correct diagnosis of these inherited causes of AA is important for appropriate management. For idiopathic severe aplastic anaemia, a matched sibling donor (MSD) haematopoietic stem cell transplant (HSCT) is the treatment of choice. If a MSD is not available, the options include immunosuppressive therapy (IST) or unrelated donor HSCT. IST with horse antithymocyte globulin (ATG) is superior to rabbit ATG and has good long-term results. In contrast, IST with rabbit ATG has an overall response of only 30 40%. Due to improvements in outcome over the last two decades in matched unrelated donor (MUD) HSCT, results are now similar to that of MSD HSCT. The decision to proceed with IST with ATG or MUD HSCT will depend on the likelihood of finding a MUD and the differing risks and benefits that each therapy provides. Keywords: paediatric aplastic anaemia, inherited bone marrow failure syndrome, transplantation in aplastic anaemia, anti-thymocyte globulin. Incidence The incidence is 2 3 million per year (all age groups) in Europe, but higher in East Asia (Montane et al, 2008). There is a biphasic distribution, with peaks at 10 25 years and over 60 years. Aetiology This review will focus on constitutional and acquired idiopathic AA. The majority (70 80%) of cases are classified as idiopathic because their aetiology is unknown (Marsh et al, 2009). The remainder mainly consist of inherited bone marrow failure syndromes (IBMFS; 15 20%), the commonest being Fanconi anaemia (FA; see Table II). Five percent of idiopathic AA have undiagnosed IBMFS where the full disease phenotype has not become apparent (Fogarty et al, 2003; Calado & Young, 2008). The correct diagnosis of IBMFS is vital for appropriate management, education and genetic counselling. A classification of AA based on aetiology can be seen in Table II. For further information on pathophysiology please refer to a recent review (Young et al, 2010). Definition Aplastic anaemia (AA) is defined as pancytopenia with a hypocellular bone marrow in the absence of an abnormal infiltrate or marrow fibrosis. To diagnose AA, there must be at least two of the following: (i) haemoglobin <100 g/l (ii) platelet count <50 9 10 9 /l (iii) neutrophil count <15 9 10 9 /l. The modified Camitta criteria are used to assess severity (Camitta et al, 1975). The severity of AA is determined on the full blood count (FBC) and bone marrow findings (Marsh et al, 2009; Table I). Correspondence: Dr Sujith Samarasinghe, Department of Adolescent and Paediatric Haematology and Oncology and Paediatric Haematopoietic Stem Cell Transplant Unit, Great North Children s Hospital, Royal Victoria Infirmary, Newcastle Upon Tyne NE1 4LP, UK. E-mail: sujith.samarasinghe@nuth.nhs.uk First published online 20 February 2012 doi: 10.1111/j.1365-2141.2012.09058.x Clinical evaluation The distinction between inherited and acquired AA can be difficult because of the clinical and genetic heterogeneity of IBMFS (Table III). A family history should be taken for blood disorders, consanguinity, malignancy or congenital anomalies as their presence may suggest an IBMFS. Examination The physical examination in children with AA may reveal features of an IBMFS, with features unique to each of the syndromes. The commonest physical findings in FA are short stature, skin hyper/hypo pigmented areas and skeletal abnormalities, particularly affecting the thumb (Shimamura & Alter, 2010). A proportion of FA patients may be of normal stature with no apparent anomalies. Dyskeratosis congenita (DKC) has the classic diagnostic triad of nail dystrophy, reticular skin pigmentation and oral leucoleucoplakia (Shimamura ª 2012 Blackwell Publishing Ltd

Table I. Classification of aplastic anaemia based on severity. Severe aplastic anaemia (SAA) Marrow cellularity <25% (or 25 50% with <30% residual haematopoietic cells) Plus At least 2 of: (a) neutrophil count <05910 9 /l (b) platelet count <20910 9 /l (c) reticulocyte count <20910 9 /l Very severe aplastic anaemia (VSAA) As for SAA but with a neutrophil count <02910 9 /l Non-severe aplastic anaemia AA not fulfilling the criteria for SAA/VSAA Table II. Classification of aplastic anaemia based on aetiology. Constitutional Fanconi anaemia (FA) Dyskeratosis congenita (DKC) Shwachman Diamond syndrome (SDS) Congenital amegakaryocytic thrombocytopenia (CAMT) Acquired Radiation Drugs and chemicals e.g. chloramphenicol, benzene, anti-epileptics, chemotherapy Viruses e.g. Hepatitis (non-a,-b,-c,-e or -G), Epstein Barr virus Graft-versus-host disease (GVHD) Paroxysmal nocturnal haemoglobinuria (PNH) Immune-systemic lupus erythematous (rare) Idiopathic & Alter, 2010). This triad however is typically absent in early life and may remain absent even in a subset of adults. Investigations To confirm the diagnosis, assess severity and confirm/exclude constitutional AA, the following investigations are recommended (see Table III). The FBC typically shows pancytopenia, although isolated thrombocytopenia may occur early on. A macrocytic anaemia with reticulocytopenia is normally present. A bone marrow aspirate shows hypocellular particles, with increased fat cells, macrophages, mast cells, and plasma cells. Erythrocytes, megakaryocytes and granulocytes are reduced or absent. Dyserythopoeisis is very common (Marsh et al, 2009) but dysplastic megakaryocytes and granulocytes are not seen in AA. A bone marrow trephine is required to exclude abnormal fibrosis or an abnormal infiltrate. Baseline immunological investigations are required as some IBMFS are associated with immunodeficiency (Dokal, 2000; Dror et al, 2001; Knudson et al, 2005; Filipovich et al, 2010). Screening for IBMFS Table III. Suggested approach to clinical evaluation of children with aplastic anaemia. Clinical evaluation of aplastic anaemia Clinical history Drug/Toxin exposure Family history of malignancy, congenital abnormalities, blood disorders, consanguinity Recent illness/hepatitis Examination Vital signs Height/Weight Assess for congenital abnormalities Investigations Full blood count and reticulocyte count Blood film HbF% (pre-transfusion) Blood group and antibody screen Bone marrow aspirate and trephine/iron stain on bone marrow (for ring sideroblasts in Pearson syndrome) Bone marrow cytogenetics including FISH for chromosomes 5 and 7 if any MDS features present Flow cytometry of peripheral blood for PNH-FLAER or alternatively assess expression of CD55 and CD59 Vitamin B12 and folate Liver function tests Viral Serology hepatitis A,B,C, CMV, EBV, parvovirus B19, VZV, measles, HSV, HHV6, HIV, adenovirus Immunology Lymphocyte subsets, Immunoglobulins, and autoantibody screen Tissue typing patient and family- if no matched family member identified, perform unrelated donor search Chest X-ray Cardiac/Renal ultrasound-fanconi anaemia Fanconi screen- DEB or mitomycin-induced-chromosomal breakages in peripheral blood lymphocytes (mandatory) Consider flow-fish for telomere length in DKC Consider pancreatic function tests if SDS is suspected Consider gene mutation analysis for DKC/CAMT/SDS Consider mitochondrial gene deletion if Pearson syndrome suspected Lung function if DKC suspected FISH, fluorescent in situ hybridization; MDS, myelodysplastic syndrome; PNH, paroxysmal nocturnal haemoglobinuria; FLAER, fluoresceinlabelled proaerolysin; CMV, cytomegalovirus; EBV, Epstein Barr virus; VZV, Varicella zoster virus; HHV6, Human herpesvirus 6; HIV, human immunodeficiency virus; DEB, diepoxybutane; DKC, dyskeratosis congenita; SDS, Shwachman Diamond Syndrome; CAMT, congenital amegakaryocytic thrombocytopenia. A diagnosis of FA can be confirmed by demonstration of increased chromosomal breakage following exposure of peripheral blood lymphocytes to clastogens, such as mitomycin C or diepoxybutane (Table IV). All children with AA should have a clastogen stress test. Somatic reversion of the FA gene mutation can result in a false negative test (found in at least 10%; Lo Ten Foe et al, 1997). Where there is a high clinical suspicion, but a negative stress test, the diagnosis of FA can be confirmed by testing skin fibroblasts for increased chromosomal breakage. Complementation group and mutation analysis facilitates genetic counselling. ª 2012 Blackwell Publishing Ltd 27

Table IV. Characteristics of inherited bone marrow failure syndromes that predispose to aplastic anaemia. Syndrome Age at presentation (years) Haematological features Non haematological features Gene mutation/inheritance Screening/Diagnostic tests Fanconi anaemia (FA) Median age 6 5 (range 0 49) 1 M = F Progressive thrombocytopenia followed by AA. Macrocytosis Increased risk of MDS/AML Limb/thumb abnormalities, cafe-au-lait spots, short stature, microcephaly, urogenital anomalies Solid tumours (cumulative probability of malignancy by 50 years 85%) 15 genes (AR/X-linked) (FANCA, FANCB, FANCC, BCRA2 (FANCD1), FANCD2, FANCE, FANCF, FANCG, FANCI, BRIP1 (FANCJ), FANCL, FANCM, PALB2 (FANCN), RAD15C (FANCO) and SLX4 (FANCP) 2 Increased chromosomal breakage by DNA cross linkers in haematopoietic cells (90%) or fibroblasts (100%) Dyskeratosis Congenita (DKC) Median age 14 (range 0 75) M > F AA, macrocytosis, MDS/AML 90% develop AA by third decade Dystrophic nails, lacy reticular pigmentation, oral leucoplakia, solid tumours, pulmonary fibrosis, osteoporosis, cirrhosis 8 genes (AD/AR/X-linked) Flow-FISH for telomere length Molecular analysis of DKC1, TERC, TERT, NOP10, NHP2, TINF2,C16orf57 and WRAP53 (TCAB1) 3 Shwachman Diamond Syndrome (SDS) Congenital Amegakaryocytic Thrombocytopenia (CAMT) Range 0 11 M:F Range 0 5 M:F Neutropenia (77 100%), pancytopenia (10 44%), MDS/AML (13 33%) 4 Thrombocytopenia with absent megakaryocytes in bone marrow Followed by AA in majority 5 Excocrine pancreatic failure, metaphyseal dysostosis, short stature Usually no somatic abnormalities SBDS (90%) AR MPL (thrombopoeitin receptor gene) AR Decreased serum trypsinogen/ isoamylase Reduced stool elastase Imaging for fatty pancreas Molecular analysis of SBDS gene Molecular analysis of MPL gene (However not all patients have MPL mutations) M, male; F, female; AA, aplastic anaemia; MDS, myelodysplastic syndrome; AML, acute myeloid leukaemia; AD, autosomal dominant; AR, autosomal recessive. 1 Shimamura and Alter (2010), 2 Crossan and Patel (2012), 3 Dokal and Vulliamy (2010), Walne et al (2010), Zhong et al (2011), 4 Smith et al (1996), 5 Ballmaier and Germeshausen (2009). 28 ª 2012 Blackwell Publishing Ltd

Testing for other IBMFS depends on clinical suspicion. DKC is typically characterized by the detection of very short telomeres in blood leucocytes (typically less than the 1st centile for age; Alter et al, 2007). Flow cytometry with fluorescent in situ hybridization (Flow-FISH) can be used to screen for abnormal telomere length. However this test is not currently available as a routine clinical service. Furthermore, not all patients with DKC have short telomeres (Walne et al, 2010). Alternatively, blood can be sent for mutation screening but there are probably many unidentified mutations (Dokal & Vulliamy, 2010). Thus a negative genetic screen is insufficient to exclude DKC. Children with mutations in TERC, TINF2 and TERT can present with just AA, without the other manifestations of DKC; thus it is reasonable to screen all children with idiopathic AA for these mutations if the FA screen is negative. The other genes can be screened for if there are additional features of DKC (personal communication Inderjeet Dokal, Centre for Paediatrics, Blizard Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, London). If there is a clinical suspicion of Shwachmann Diamond Syndrome (SDS), exocrine pancreatic insufficiency can be screened for, though pancreatic function tends to improve with age. Molecular analysis of the SBDS gene can help confirm the diagnosis, though 10% of children with SDS do not have a mutation in the SBDS gene (Boocock et al, 2003). Congenital amegakaryocytic thrombocytopenia (CAMT) is usually diagnosed on clinical features and confirmed by molecular analysis of the MPL gene (Ihara et al, 1999; Ballmaier & Germeshausen, 2009). Differential diagnosis Around 1 2% of childhood lymphoblastic leukaemia (ALL) cases are preceded by a period of pancytopenia, often with a hypocellular marrow, which subsequently develops into overt ALL around 1 9 months later (Breatnach et al, 1981). Dysplastic granulopoeitic or megakaryocytes, increased reticulin, abnormal localization of immature precursors and increased blasts are seen in hypoplastic myelodysplastic syndrome (MDS) and not in AA (Bennett & Orazi, 2009). The detection of monosomy 7 or 5q- should be considered as MDS (Marsh et al, 2009). The presence of isolated thrombocytopenia can make distinguishing between ITP and AA (especially CAMT) difficult. ITP rather than AA is suggested by the presence of normal or increased numbers of megakaryocytes and increased reticulated platelet count. Key points AA is a rare disorder. About 70 80% of cases are idiopathic. Other potential causes of pancytopenia should be excluded. It is important to exclude IBMFS. Their presentation is heterogeneous and management different to idiopathic severe aplastic anaemia (SAA). All children should be screened for FA. Screening for other IBMFS will depend on clinical suspicion. Children with SAA and their families should be tissue typed at diagnosis. If there is no matched family member, an unrelated donor search should be undertaken. Management Transfusional support Red cell transfusions should be used only to treat definite symptoms/signs rather than maintain an arbitrary level. Leucodepleted blood products (routine in the UK) should be given to reduce the risk of human leucocyte antigen (HLA) sensitization. Cytomegalovirus (CMV)-negative blood products should be given until the patient s CMV status is known. The European Bone Marrow Transplant Severe Aplastic Anaemia Working Party (EBMT SAAWP) currently recommends that children should receive irradiated blood products following immunosuppressive therapy (IST) and that this should continue for as long as they receive ciclosporin (Marsh et al, 2010). Repeated transfusions will lead to secondary iron overload. Iron chelation should be considered when the liver iron is >7 mg/g dry weight or when the total red cell transfusion volume is >200 ml/kg. If liver iron measurement is not available, a persistently elevated ferritin level >1000 lg/l may be used as a surrogate marker of iron overload, though this is a non-specific marker (Marsh et al, 2009). Deferiprone should be avoided because of the risk of agranulocytosis. Desferrioxamine or deferasirox are however suitable iron chelators, the latter having the advantage of oral administration (Lee et al, 2010). Following successful treatment with IST or HSCT, venesection should be used to remove excess iron. Platelet transfusions can be given when the platelet count is <10 9 10 9 or <20 9 10 9 /l if pyrexial or during anti-thymocyte globulin (ATG) treatment. Prostagens can be used to prevent menstruation and tranexamic acid is useful for minor mucosal bleeds. Haematopoietic growth factors The largest prospective study assessing the efficacy of adding granulocyte colony-stimulating factor (GCSF) to IST showed no difference in overall survival (OS) or event-free survival (Tichelli et al, 2011). As prolonged use of GCSF may increase the risk of clonal disorders (Socie et al, 2007), the EBMT SAAWP recommend the use of GCSF after IST only to patients with neutropenic infection (Passweg & Marsh, 2010). ª 2012 Blackwell Publishing Ltd 29

Infection prevention and treatment Anti-fungal prophylaxis should be given when the neutrophil count is <0 5 9 10 9 /l. Prolonged courses of prophylactic antibiotics, such as ciprofloxacin, are not recommended and have a poor compliance. Febrile neutropenia requires treatment with empirical broad-spectrum antibiotics as per local antibiotic policy. A short course of GCSF (5 10 lg/kg per d) may be appropriate in severe infections. Granulocyte infusions could also be used in severe refractory sepsis, e.g. severe fungal infection. Vaccinations Live vaccines should be withheld during active treatment of AA and for at least 1 year after stopping ciclosporin. Though there is an anecdotal report of relapse of AA following influenza vaccination (Hendry et al, 2002), individual physicians need to weigh up the risks and benefits of withholding/ administering influenza vaccination. Following allogeneic HSCT, vaccinations can be administered as per current guidelines (Skinner et al, 2002). Key points Red cell transfusion should be given to prevent symptoms rather than maintain an arbitrary level. Prophylactic platelet transfusions are recommended when the platelet count <10 9 10 9 or 20 9 10 9 /l if pyrexial. Routine use of GCSF is not recommended. Antifungal prophylaxis should be given if the neutrophil count is <05 9 10 9 /l. Treatment choices in idiopathic SAA Specific therapy may be divided into haematopoietic stem cell transplantation (HSCT) and IST. IST consists of ATG and ciclosporin. Immunosuppressive therapy (IST) Horse ATG A randomized study demonstrated that IST with a combination of ATG and ciclosporin led to superior overall response (OR) rates (but not OS) compared to ATG alone (OR 65% vs. 31% at 6 months; Frickhofen et al, 2003). Thus current IST therapy consists of a combination of ATG and ciclosporin. All large paediatric studies have been performed using horse ATG (hatg) and ciclosporin (See Table V). These data show 6-month OR rates of 599% 77%, with a 10-year OS of around 80% and a relapse rate (RR) of 10 33%. There has been no improvement in survival following IST in the last two decades, suggesting that this therapy has reached a ceiling (Passweg & Tichelli, 2009). A retrospective analysis suggested that a slower tail of ciclosporin might reduce the relapse rate (RR; 76% with a slow taper and 60% with a rapid taper; Saracco et al, 2008). However data from a preliminary prospective study has cast doubt on this. The RR and risk of clonal evolution was similar in a cohort who had a gradual taper compared to a historical group with a rapid taper (Scheinberg et al, 2011a). Until further information from this study is published, all children who respond to IST should complete a minimum 12 months of therapeutic ciclosporin and then slowly taper (025 05 mg/kg per month). Following IST, there is an increased risk of clonal disorders [paroxysmal nocturnal haemoglobinuria (PNH), MDS, and acute myeloid leukaemia (AML)] of 85 15% at 10 years with no evidence of a plateau. Administration of GCSF and repeated administration of IST may increase the likelihood of clonal evolution (Saracco et al, 2008). There is no benefit of adding mycophenolate mofetil or sirolimus to ATG and ciclosporin (Scheinberg et al, 2006a, 2009). Response to IST is typically delayed and responses generally do not start before 3 4 months. For details of administration of IST and assessing response please see the review by Marsh et al (2009). Table V. Paediatric studies of immune suppressive therapy (IST) with horse ATG and ciclosporin. Study Number of patients Treatment (IST) Study period Follow up (years) Overall response Overall survival Relapse rate Clonal evolution Fuhrer et al (2005) Kamio et al (2011) Saracco et al (2008) Scheinberg et al (2008) 146 ATG, CSA, GCSF 1993 2001 41 (median) CR 69% VSAA, CR 44% SAA 93% VSAA, 81% SAA 441 ATG, CSA, 1992 2007 10 599% 82% VSAA, ±Dan ±GCSF 82% SAA, 98% NSAA 13% VSAA, NR 14% SAA 119% NR 42 ATG, CSA± GCSF 1991 1999 10 71% 83% 16% 15% 77 ATG, CSA, ±MMF, ±sirolimus 1989 2006 10 77% 80% 33% 85% ATG, Anti-Thymocyte Globulin; CSA, ciclosporin; Dan, Danazol; GCSF, granulocyte colony-stimulating factor; MMF, mycophenolate mofetil; SAA, severe aplastic anaemia; VSAA, very severe aplastic anaemia; NSAA, non severe aplastic anaemia; NR, not reported; CR, Complete remission rate. 30 ª 2012 Blackwell Publishing Ltd

Predictors of response to IST Good prognostic factors that increase the likelihood of response to IST include severity [very severe aplastic anaemia (vsaa) better than SAA; Fuhrer et al, 2005], younger age, higher pre-treatment reticulocyte count and lymphocyte count (Scheinberg et al, 2008), male gender, and a leucocyte count <2 9 10 9 /l (Yoshida et al, 2011). The time interval from diagnosis to treatment is also important; responses to IST were 66% at diagnosis, 50% at 1 month post-diagnosis and only 35% 3 6 months after diagnosis (Yoshida et al, 2011). Rabbit ATG Until 2007, in Europe the standard ATG preparation was hatg (Lymphoglobulin; Genzyme, Cambridge, MA, USA). This was withdrawn in June 2007 and is no longer available. In The USA, hatg (ATGAM; Pfizer, New York, NY, USA) is available, though it is dosed differently to Lymphoglobulin. The only alternative in Europe is rabbit ATG (Thymoglobulin; Genzyme). Historically, rabbit ATG (ratg) had previously been used for second courses of IST after failure to respond or relapse after a first course of hatg. A large prospective study comparing hatg (ATGAM) with ratg as first line therapy demonstrated inferiority of ratg (OR at 6 months 68% in hatg vs. 37% in ratg; Scheinberg et al, 2011b). There was also a superior OS at 3 years in the hatg group (96% vs. 76%). A retrospective analysis of 43 consecutive children treated with ratg/ciclosporin as first-line therapy in the UK showed an OR of 33% and an estimated 5- year failure-free survival (FFS) of 13% (Samarasinghe et al, 2012). These data indicate that ratg is effective in only around a third of patients and is clearly inferior to hatg. Retreatment with IST A prospective study of children who had failed one course of IST, demonstrated a superior FFS following matched unrelated donor (MUD) HSCT rather than a repeat course of IST (5- year FFS 839% vs. 95%, respectively; Kosaka et al, 2008). Thus a MUD HSCT is the treatment of choice for those failing one course of IST (Marsh et al, 2009). Children who have previously responded to ATG may well respond to a second course, though this should not be given any earlier than 4 6 months after the first course as it takes at least 3 months for a response to occur. The OR in relapsed patients to a second course of IST is 60 70% (Tichelli et al, 1998; Scheinberg et al, 2006b). The duration of ciclosporin should be extended if there is a response. A third course of IST should not be given if the patient has failed two previous courses (Gupta et al, 2005). Key points IST with horse ATG is superior to rabbit ATG. IST with horse ATG leads to an overall response rate of between 60% and 75%, with a long-term survival of 80%. There is a significant risk of relapse (10% at 10 years) and 10 15% risk of development of clonal abnormalities, which does not plateau. Relapse following previous successful IST has a 60 70% response to a second course of IST. Transplantation in idiopathic SAA Matched sibling donor (MSD) HSCT Haematopoietic stem cell transplant from a MSD remains the treatment of choice for children with SAA, with survival figures ranging from 85% to 97% (Kojima et al, 2000; Schrezenmeier et al, 2007). The conditioning regimen recommended by the EBMT SAAWP is cyclophosphamide (200 mg/kg), ratg (75 mg/kg) and ciclosporin and methotrexate for graft-versus-host disease (GVHD) prophylaxis. Current problems in MSD HSCT remain graft rejection (5%), acute (10 20%) and chronic GVHD (10 30%) and late effects (Kahl et al, 2005; Schrezenmeier et al, 2007). As AA is a non-malignant condition, there is no benefit to GVHD. A prospective study comparing cyclophosphamide with or without ATG showed no difference in outcome, though it was not adequately powered to detect survival differences (Champlin et al, 2007). Thus, potentially ATG may be omitted. Randomized studies confirmed the superiority of ciclosporin and methotrexate as GVHD prophylaxis compared to ciclosporin alone (Locatelli et al, 2000) or methotrexate alone (Storb et al, 1986). However neither of these studies used serotherapy. Chronic GVHD remains the most important adverse factor for long-term survival following paediatric MSD HSCT (Sanders et al, 2011). Risk factors for chronic GVHD include previous acute GVHD (Storb et al, 1983), full donor chimerism (McCann et al, 2007), high nucleated marrow cell dose ( 34 9 10 8 /kg; Kahl et al, 2005) and use of peripheral blood stem cells (PBSCs; Eapen et al, 2011). In contrast, alemtuzumab-based conditioning regimens result in low rates of chronic GVHD (Gupta et al, 2004; Marsh et al, 2011). In a retrospective analysis of 15 children who received a MSD or matched family donor HSCT (11 with idiopathic and 4 with constitutional SAA) using alemtuzumab (09 1 mg/kg), none developed GVHD (unpublished observations). Thus, alemtuzumab may be used as an alternative to ATG; due to the low rates of GVHD seen with alemtuzumab, methotrexate may be omitted. Risk factors for graft rejection include alloimmunization from previous transfusions/previous IST (Kobayashi et al, 2006), low marrow cell dose (Niederwieser et al, 1988), donor recipient gender mismatching (Stern et al, 2006) and progressive mixed chimerism (PMC; McCann et al, 2007). Monitoring donor chimerism after allogeneic HSCT can help predict impending graft failure as PMC is associated with a high rate of graft failure (McCann et al, 2007). There is a significant risk of PMC whilst tapering immuno- ª 2012 Blackwell Publishing Ltd 31

suppression. As late graft rejection is a characteristic complication of HSCT for SAA, ciclosporin should be continued for at least 9 months, even in the absence of GVHD and tailed over the following 3 months. Chimerism should be monitored at 1, 3, 6 and 12 months post-hsct and during tapering of immunosuppression. If there is an increase in recipient cells during this time period, ciclosporin should be increased to maintenance levels and a further attempt at withdrawal 3 months later (Lawler et al, 2009). Stable mixed chimerism is associated with very low rates of chronic GVHD and excellent outcome (McCann et al, 2007). Late effects remain a potential concern. In a single centre analysis, fertility was preserved in 80 90% of females and c. 60% of males with normal growth (Sanders et al, 2011). Malignancy was reported in 7 13% on long-term follow up (Kahl et al, 2005; Sanders et al, 2011). Chronic GVHD and use of total body irradiation (TBI) regimens remain the major risk factors for the development of malignancy post-msd HSCT. The conditioning regimen for MSD HSCT favoured to date in the UK is: 1 Cyclophosphamide 50 mg/kg per d 5 to 2 (total dose 200 mg/kg). 2 Serotherapy is optional. If serotherapy is used, alemtuzumab is the preferred option, 03 mg/kg per d 6 to 4 (total dose 09 mg/kg). 3 Ciclosporin and methotrexate for GVHD prophylaxis. Methotrexate may be omitted if alemtuzumab is used. In view of the potential for impaired fertility seen with high dose cyclophosphamide, an alternative approach is a fludarabine-based regimen but using a lower dose of cyclophosphamide (fludarabine 150 mg/m 2, cyclophosphamide 120 mg/kg and alemtuzumab 09 mg/kg). A small series using a similar approach showed promising results (Resnick et al, 2006). Key points MSD HSCT is first-line therapy for idiopathic SAA. Serotherapy is optional. GVHD prophylaxis consists of ciclosporin and methotrexate. Methotrexate may be omitted if alemtuzumab is used. Serial chimerism should be monitored post-hsct. Unrelated donor HSCT Children who fail IST are eligible for a MUD HSCT. Outcomes over the last two decades have improved dramatically for MUD HSCT in SAA (Perez-Albuerne et al, 2008; Viollier et al, 2008), mainly due to the use of leucodepleted blood products, improvements in tissue typing donor/recipient and development of improved conditioning regimens (Maury et al, 2007). There are currently two approaches to conditioning in MUD HSCT: a conditioning regimen incorporating low dose TBI (2 Gy; Deeg et al, 2006) and a radiation-free regimen using fludarabine (Bacigalupo et al, 2010). Deeg et al (2006) demonstrated improved outcomes using a regimen of 2 Gy TBI, cyclophosphamide and ATG compared to higher doses of TBI. With this regimen, children who were transplanted within a year of diagnosis had an 85% survival. The aim of the low dose TBI regimen was to optimize engraftment but minimize the long-term side effects associated with irradiation. There was however a relatively high incidence of acute GVHD (Grade II IV 70%) and chronic GVHD (62%). The high levels of GVHD may be partly due to the pro-inflammatory effects of TBI. Administration of TBI also remains a long-term concern in children because of the effects on fertility, growth, endocrine problems and potential for malignancy. This is all the more worrying because of the inherent risk in SAA to develop malignancy. In order to minimize these longterm side effects, a radiation-free regimen was developed by the EBMT (fludarabine 120 mg/kg, low dose cyclophosphamide 1200 mg/m 2 and ratg 15 mg/kg; Bacigalupo et al, 2005). However this regimen has been complicated by relatively high rates of graft failure in older children (5% 14 years, 32% if 15 years), post-transplant lymphoproliferative disease (PTLD) and GVHD (Bacigalupo et al, 2005, 2010). In view of these complications, a modified EBMT protocol is now proposed (aged 14 years and not sensitized, fludarabine 120 mg/kg, cyclophosphamide 120 mg/kg, ratg 75 mg/kg and prophylactic rituximab; 2 Gy TBI is added to the aforementioned regimen for those patients aged 15 years or sensitized; Kojima et al, 2011). In the UK, there has been considerable enthusiasm for using alemtuzumab (campath-1h), a monoclonal anti-cd52 antibody. A retrospective analysis of 44 consecutive children who received HLA-A, -B, -C, -DRB1, -DQ matched unrelated donor HSCTs using a fludarabine (150 mg/kg), cyclophosphamide (120 or 200 mg/kg) and alemtuzumab regimen (0 9 1 mg/kg; FCC regimen) demonstrated excellent outcome. There were no cases of graft failure, with an estimated 5 year OS/FFS of 95% (Samarasinghe et al, 2012). At a median of 2 9 years follow up, there was a low rate of severe acute GVHD (grades III IV 2 3%) and chronic GVHD (6 8%). The low rates of chronic GVHD are similar to other groups who have also used alemtuzumab-based conditioning regimens (Marsh et al, 2011). There were no cases of PTLD although two children did require rituximab because of Epstein Barr Virus reactivation. This data, along with that reported by others (Kennedy-Nasser et al, 2006), suggests that outcomes following MUD HSCT (10/10 by high resolution typing) for paediatric SAA are similar to that of MSD. As excellent engraftment can be achieved in the absence of TBI, the best approach would involve a radiation-free regimen (such as the FCC regimen) in an attempt to minimize long-term side effects. However until long-term data is available, it is difficult to be certain whether radiation-free regimens will prevent 32 ª 2012 Blackwell Publishing Ltd

long-term side effects. Therefore, post-pubertal males receiving a HSCT should have sperm cryopreserved. The conditioning regimen recommended by the UK Paediatric BMT group for 10/10 (HLA-A, -B, -C, -DRB1, -DQ matched by high resolution) MUD HSCT is (FCC regimen): 1 Fludarabine 30 mg/m 2 per d for 5 d: days 6 to 2 (Total dose 150 mg/m 2 ). 2 Cyclophosphamide 60 mg/kg per d for 2 d: days 3 to 2 (Total dose 120 mg/kg). 3 Alemtuzumab 03 mg/kg per d for 3 d: days 6 to 4 (Total dose 09 mg/kg: cap dose at 50 mg). With ciclosporin prophylaxis. Data on mismatched unrelated donor HSCT and unrelated donor umbilical cord HSCT in idiopathic SAA is limited. Retrospective data suggest that single allele mismatched unrelated donor (MMUD) HSCT have a reasonably good outcome (78% 2-year OS for 8/8 vs. 60% for 7/8; Eapen & Horowitz, 2010). Furthermore, with the advent of high resolution typing, single antigen/allele MMUD may have a similar outcome to MUD HSCT (Yagasaki et al, 2011). Due to the low cell dose in umbilical cord donations, initial reports of using unrelated donor umbilical cord HSCT for idiopathic SAA have been discouraging because of the high graft failure rate and treatment-related mortality (TRM). OS in the two largest retrospective analyses to date have ranged from 30% to 40% (Yoshimi et al, 2008; Peffault de Latour et al, 2011). Improved results were seen with higher total nucleated cell (TNC) doses (OS was 45% for TNC > 39 9 10 7 /kg vs. 18% for TNC 39 9 10 7 /kg; Peffault de Latour et al, 2011). An impressive OS was seen in a small series of adults who received unrelated umbilical cord HSCT using a fludarabine, melphalan and 4 Gy TBI conditioning regimen (3-year OS of 83%), but these results will need to be confirmed in further studies (Yamamoto et al, 2011). Selecting units to which the recipient does not have anti-hla antibodies may also improve outcomes (Takanashi et al, 2010). Key points MUD HSCT (a HLA-A, -B, -C, -DRB1, -DQ matched donor on high resolution typing) has an excellent outcome in idiopathic SAA. Radiation-free conditioning regimens are favoured in Europe. Alemtuzumab-based conditioning regimens have low rates of chronic GVHD. Stem cell choice The recommended stem source is bone marrow. PBSCs lead to an increased risk of chronic GVHD and inferior outcome (Schrezenmeier et al, 2007; Eapen et al, 2011). However, use of alemtuzumab-based conditioning regimens may minimize the effect of PBSCs on chronic GVHD (Marsh et al, 2011; Shaw et al, 2011). Umbilical cord stem cells from a MSD are also acceptable though rarely available. Key points Bone marrow is stem cell of choice. Algorithm for idiopathic paediatric SAA A recent algorithm for childhood SAA (Marsh et al, 2009) states that a MSD HSCT is the treatment of choice. Those children lacking a MSD should receive IST with ATG/ciclosporin as second choice. Should they fail IST (response assessment at 3 4 months), then they should proceed with MUD HSCT as third choice. However, with recent data demonstrating the low efficacy of ratg and the steady improvement in MUD HSCT, a new algorithm is proposed (see Fig 1). In the proposed algorithm, MSD HSCT remains the first choice. Whether IST or MUD HSCT should however be second choice is contentious. Advantages of IST with hatg include excellent long-term survival and low TRM. However, IST has a significant RR (at least 10%; Saracco et al, 2008), and a 10-year risk of developing a clonal disorder of between 10% and 15%. Furthermore, IST takes at least 3 4 months for cellular recovery, which is particularly relevant if a child develops infectious complications. MUD HSCT now has an excellent outcome with a much quicker neutrophil recovery and a marked reduction in development of secondary clonal disorders and relapse compared to IST (Socie et al, 1993). Although MUD HSCT carries a higher risk of early mortality, the subsequent survival curve is stable. In contrast, due to the increased risk of secondary clonal disorders, no such plateau in survival is seen following IST. Historically, HSCT was associated with the upfront toxicities of GVHD, graft failure and infectious complications. With improvements in supportive care, tissue typing and conditioning regimens, the TRM for a MUD HSCT in children is similar to that of MSD. The disadvantage with MUD HSCT includes the time taken to find a suitable donor (typically 3 months before HSCT can proceed) and the difficulty finding a MUD in children with more rare HLA haplotypes. In those countries where hatg is available, the choice between IST and MUD HSCT (when a 10/10 MUD donor exists) will require a careful discussion between the physician and family regarding the different risks. To help determine whether one should proceed with hatg or MUD HSCT, urgent tissue typing should be done and a judgement on the likelihood of finding a MUD can then be made. Should IST fail with hatg, the child should proceed directly to MUD HSCT (see Fig 1). Horse ATG (ATGAM) is not currently available in Europe, though the EBMT SAAWP is urgently trying to change this. Until then, in Europe due to the disap- ª 2012 Blackwell Publishing Ltd 33

Fig 1. Proposed Algorithm for children with Idiopathic SAA. SAA, severe aplastic anaemia; vsaa, very severe aplastic anaemia; HSCT, haematopoietic stem cell transplant; MSD, matched sibling donor; MUD, matched unrelated donor; MMUD, mismatched unrelated donor; IST, immunosuppressive therapy. 10/10 MUD refers to a HLA-A, -B, -C, -DRB1, -DQ matched donor by high resolution typing. A 9/10 MMUD refers to a single antigen/allele mismatched donor by high resolution typing. pointing results with ratg, a MUD HSCT should be considered as the second choice (where a suitable donor exists). The EBMT SAAWP have issued similar guidance (EBMTG SAAWP, 2011). In the proposed algorithm, IST with ratg/ciclosporin would be the third choice. The EBMT SAAWP have suggested that ratg be used at 25 mg/kg per d for 5 d rather than 375 mg/kg per d for 5 d (http://www.bcshguidelines. com/documents/use_of_rabbit_atg_july_2011.pdf) as ratg is more immunosuppressive than hatg. Single allele/antigen MMUD may be considered a suitable alternative to IST with ratg. For those children lacking a suitable unrelated donor (10/10 or 9/10) and failing IST, possible options include a second course of ATG, an alternative IST or umbilical cord/ haploidentical HSCT. Alternative IST options include high dose cyclophosphamide (Brodsky et al, 2010) or alemtuzumab (Risitano et al, 2010). However further studies are required in children to determine their long-term efficacy and optimal dosing. Non-severe aplastic anaemia (NSAA) Transfusion-independent children with a neutrophil count above 05 9 10 9 /l should be observed. If they are transfusion-dependent, or have a neutrophil count <05 9 10 9 /l, the algorithm for SAA should be followed. However, the relapse rate is much higher in NSAA than SAA following IST with horse ATG (10-year relapse rate 35% vs. 12% respectively; Kamio et al, 2011). Long-term follow up Long-term follow up is essential to document late complications. A PNH screen should be performed 3 4 months after IST and thereafter annually (Marsh et al, 2009). For management of PNH please refer to the recent article by Roth and Duhrsen (2011). A repeat bone marrow with cytogenetics should be performed if there is deterioration in blood counts. Fanconi anaemia (FA) Fanconi anaemia is characterized by a variety of congenital abnormalities, progressive bone marrow failure (BMF), and a significantly increased risk of developing leukaemia and other malignancies (Shimamura & Alter, 2010). The risk of developing BMF and haematological and non-haematological malignancies increases with advancing age with a 90%, 33%, and 28% cumulative incidence, respectively, by 40 years of age (Kutler et al, 2003). The definitive treatment to prevent haematological complications is a MSD HSCT; it does not prevent (and may exacerbate) the risks of non-haematological malignancies. MSD HSCT in FA Fanconi anaemia patients demonstrate marked sensitivity to alkylating agents and irradiation. Early results using standard 34 ª 2012 Blackwell Publishing Ltd

conditioning regimens were associated with high TRM. As such, Gluckman et al (1984) proposed the use of a conditioning regimen of low dose cyclophosphamide (20 40 mg/ kg) combined with 400 600 cgy of thoraco-abdominal irradiation or TBI with ciclosporin for GVHD prophylaxis. OS rates of between 80% and 90% have been reported using this approach (Dufour et al, 2001; Farzin et al, 2007). FA patients demonstrate increased propensity to severe GVHD (Guardiola et al, 2004). The occurrence of severe acute GVHD (grades II IV) and chronic GVHD strongly increased the risk of oropharngeal/anogenital squamous cell carcinomas in long-term FA survivors, with a 15-year incidence of head and neck cancers of 53% in one series (Guardiola et al, 2004). There is no evidence currently that low dose chemoradiotherapy conditioning regimens used in FA HSCTs increase the risk of long-term malignancy. However there has been a move to avoid irradiation-based conditioning regimens and incorporate T-cell depletion to minimize the risk of GVHD, with the aim of reducing the risk of malignancy. A retrospective comparison of irradiation-based regimens with radiation-free regimens showed no difference in OS (Pasquini et al, 2008). Prior use of androgens, age >10 years and CMV positivity in either donor/recipient were adverse factors. The optimal radiation-free conditioning regimen is unknown. The favoured conditioning regimen for FA within the UK incorporates fludarabine and low dose cyclophosphamide with serotherapy (de la Fuente et al, 2003). Whether such regimens can reduce late effects will require long-term follow up. All matched family donors should be screened very carefully for FA even if they do not show features of the disorder. Bone marrow rather than PBSCs is the stem cell of choice. Current indications for a MSD HSCT in FA are (please see www.fanconi.org.uk/clinical-network/standards-of-care/): 1 Significant cytopenia/moderate BMF (haemoglobin <90 g/ l, platelet count <40 9 10 9 /l, neutrophil count <1 9 10 9 / l). 2 Transfusion dependence (HSCT should ideally be done before 20 red cell transfusions). 3 High-risk MDS- Clonal cytogenetic changes in the marrow may be transient. However, persistent or increasing cytogenetics clones, e.g. chromosome 3q26q29 gains, persistent monosomy 7, or chromosome 7q losses or blasts >5% suggest progression to MDS. 4 AML Unrelated donor HSCT in FA Matched unrelated donor HSCT in FA has historically had a poor outcome (MacMillan & Wagner, 2010). However, with the advent of fludarabine-based conditioning regimens there has been a notable improvement. Fludarabine-based conditioning regimens are associated with a superior engraftment and better OS (3-year adjusted OS rates were 52% with fludarabine versus 13% without fludarabine P < 0 001; Wagner et al, 2007). Cord stem cells are an attractive option for patients lacking a MUD. In a retrospective EBMT analysis, the OS in 93 FA patients following unrelated umbilical cord HSCT was 40%. Outcomes were better with fludarabine-containing regimens, better HLA-match and higher TNC doses infused (Gluckman et al, 2007). Indications for a MUD HSCT are severe marrow failure (Hb < 80 g/l, platelet count <20 9 10 9 /l and neutrophil count <0 5 9 10 9 /l) or features suggestive of high risk MDS (www.fanconi.org). As MUD HSCT has a higher TRM than a MSD HSCT, the decision to proceed or use androgens will depend on donor availability and the differing risks of each therapy. The conditioning regimen recommended by The UK paediatric HSCT group for FA MSD & 10/10 MUD HSCT (HLA-A, -B, -C, -DRB1, -DQ matched by high resolution typing) is; 1 Fludarabine 30 mg/m 2 for 5 d: days 10 to 6 (150 mg/ m 2 total dose). 2 Cyclophosphamide 10 mg/kg per d for 4 d: days 6 to 2 (40 mg/kg total dose). 3 Alemtuzumab 0 3 mg/kg per d for 3 d: days 6 to 4 (total dose 09 mg/kg). With ciclosporin prophylaxis. Androgens Androgens (oxymetholone 05 mg/kg per d on alternate days) can be considered in the event of BMF and the absence of a MSD. Responses are slow (3 6 months), with the best responses seen in the red cells and the slowest in platelets. If no response has been seen by 6 months they should be stopped. The side effects include virilization, premature closure of epiphyses, jaundice, transaminitis, behavioural changes and hepatic adenomas. If used, regular ultrasounds of liver and liver function tests should be performed. Guidelines for long term follow up and surveillance of FA patients can be found at: www.fanconi.org.uk/clinical-network/standards-of-care/. Key points The treatment of choice for a child with FA and BMF is a MSD HSCT. Long-term results after a MSD HSCT indicate an OS of 80%. Severe acute and chronic GVHD strongly increases the risk of head/neck malignancy. Radiotherapy-free regimens incorporating fludarabine and T-cell depletion are increasingly favoured. If a MSD is not available the options include a MUD HSCT or androgens. ª 2012 Blackwell Publishing Ltd 35

Dyskeratosis congenita (DKC) Dyskeratosis congenita is an inherited disorder of telomere maintenance characterized by mucocutaneous abnormalities, BMF and increased predisposition to malignancy. There is considerable genetic and phenotypic heterogeneity, which can make diagnosis difficult (Vulliamy et al, 2006). For a review of the genetics please see recent article (Dokal & Vulliamy, 2010). BMF/immunodeficiency is the major cause of death, followed by pulmonary complications and malignancy (Walne & Dokal, 2009). Allogeneic HSCT is the only curative option for patients with BMF. However, high TRMs were encountered using myeloablative conditioning regimens. To counter this, reduced-intensity conditioning (RIC) regimens have successfully been used (Dietz et al, 2011). However, the numbers are too small to make any recommendations regarding the optimal RIC regimen. For those who lack a MSD or MUD, oxymetholone or growth factors can be used. The same caveats that guide use of androgens in FA apply in DKC. GCSF should not be used with oxymethalone because of the risk of splenic rupture (Shimamura & Alter, 2010). Shwachmann Diamond syndrome (SDS) Shwachmann Diamond syndrome is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency, BMF and other somatic abnormalities (especially metaphyseal dysostosis). A trial of GCSF may be considered to ameliorate infection or prevent recurrent sepsis. The lowest possible dose that maintains adequate levels of neutrophils should be used. No association between GCSF administration and malignancy has been demonstrated (Rosenberg et al, 2006). Indications for HSCT include significant cytopenias, transfusion dependence and severe recurrent sepsis secondary to persistent neutropenia (Burroughs et al, 2009). However, HSCT in SDS using myeloablative conditioning is associated with increased TRM. The optimal conditioning regimen is unknown but small case series have demonstrated the efficacy of a RIC approach (Bhatla et al, 2008). Congenital amegakaryocytic thrombocytopenia (CAMT) This is an autosomal recessive IBMFS characterized by severe thrombocytopenia at birth with a lack of or absence of megakaryocytes in the bone marrow. The disorder is characterized by a mutation in the thrombopoeitin receptor (MPL gene). The diagnosis is one of exclusion; please see the recent review by Ballmaier and Germeshausen (2009). HSCT from a MSD after development of SAA has a good outcome (Ballmaier & Germeshausen, 2009). CAMT patients do not show increased TRM like FA or DKC patients following myeloablative regimens. Increasing success has also been seen using unrelated donor HSCTs with RIC regimens, although numbers remain small (Tarek et al, 2011). Acknowledgements The authors are grateful for Professor Irene Roberts and Dr. Rod Skinner for reviewing the script. 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