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How I manage Evans Syndrome and AIHA cases in children Maurizio Miano Clinical and Experimental Haematology Unit, Department of Haematology/Oncology, IRCCS Istituto Giannina Gaslini, Genoa, Italy

Summary The management of Evans Syndrome in children is challenging due to the lack of evidence-based data on treatment. Steroids, the first-choice therapy, are successful in about 80% of cases. For children who are resistant, relapse or become steroid-dependent, rituximab is considered a valid secondline treatment, with the exception of those with an underlying diagnosis of autoimmune lymphoproliferative syndrome who may benefit from other options such as mycophenolate mofetil and sirolimus. Better knowledge of the immunological mechanisms underlying cytopenias and the availability of new immunosuppressive drugs can be helpful in the choice of more targeted therapies that would enable the reduction of the use of long-term steroid administration or other more aggressive options, such as splenectomy or stem cell transplantation. This manuscript provides an overview of the pathogenic background of the disease, and suggests a clinical approach to diagnosis and treatment with a particular focus on the management of relapsing/resistant disease. Keywords: Evans syndrome, autoimmune haemolytic anaemia, immune thrombocytopenia, children, autoimmune lymphoproliferative syndrome, immunosuppression. Evans Syndrome (ES) is a rare disorder that was initially described as the presence of autoimmune haemolytic anaemia (AIHA) and immune thrombocytopenia with unknown aetiology (Evans et al, 1951). Over time, the definition of the diseases has changed and currently ES is defined as the destruction of at least two blood cell lineages in the absence of other diagnoses (Wang, 1988; Mathew et al, 1997; Savasßan et al, 1997). However, due to the recently acquired knowledge of the novel immune deregulatory syndromes underlying most cases, ES might also be considered as a manifestation of different immune-mediated disorders. Red blood cell involvement might also be expressed by the presence of laboratory signs of the disease, such as an isolated

Correspondence: Maurizio Miano, Clinical and Experimental Haematology Unit, IRCCS Istituto Giannina Gaslini, Largo G. Gaslini 5, 16148-Genoa, Italy. E-mail: [email protected]

First published online 2 December 2015 doi: 10.1111/bjh.13866

direct antiglobulin test (DAT) showing positivity without haemolysis. Front-line treatment is based on steroid administration, the first reported therapy (Dameshek et al, 1951), although it has never been validated by any clinical trial. Very little data is available on second and later-line approaches for the relapsing/resistant disease, which is a very challenging issue, particularly in children who are at risk of severe long-term steroid-related side effects. In the last few years, new immunosuppressive drugs have provided encouraging results in controlling the immune cytopenias and/or in acting as steroid-sparing tools. We focus here on the diagnosis and management of children with ES with particular attention to second and third line treatment.

Incidence and prevalence Data on the incidence and prevalence of ES is scarce and is essentially derived from cohorts of patients with AIHA in which cell lineages additional to red cells was involved. AIHA has an estimated incidence of 0
4 cases/100 000 children per year. No data is available on the exact prevalence in paediatric patients. Between 13% and 73% of patients with AIHA are reported to have multi-lineage involvement (Pui et al, 1980; Wang, 1988; Mathew et al, 1997). In the largest available study of 265 children with AIHA, ES, defined as the involvement of both red cells and platelets, was reported to account for 37% of cases (Aladjidi et al, 2011). In the same study, the mortality rate was reported to be as high as 10%.

Pathogenesis Although the diagnosis of ES may not necessarily include the immune destruction of red cells, AIHA remains the most important component of the disease. Immune-mediated haemolysis can be driven by different mechanisms. In most (60–70%) cases (Aladjidi et al, 2011), auto-reactive immunoglobulin (Ig) G binds red blood cell (RBC) antigens and causes their destruction, mainly in the extra-vascular compartment, through the antibody-dependent cellular cytotoxicity mechanism. More rarely, both IgA and IgM can target the RBCs as well. The maximal activity of IgG is usually at 37°C, and for this reason they are classified as warm ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2016, 172, 524–534

Review antibodies. In a minority of children (25–30%), IgM destroys RBCs by activating the complement cascade, mainly in the intra-vascular compartment, when exposed to cold temperatures (cold antibodies) (Petz & Garratty, 2004). Sometimes, both mechanisms coexist. Immune-mediated thrombocytopenia is due to platelet opsonization with anti-platelet antibodies and/or immune-complexes (Cines & Blanchette, 2002; Provan & Newland, 2002) that leads to their premature removal by the reticuloendothelial system. Peripheral destruction of neutrophils is usually mediated by antibodies that recognize membrane antigens, mostly located on immunoglobulin G Fc receptor type 3b (FccIIIb receptor), (Farruggia & Dufour, 2015).

Table I. Causes of secondary Evans syndrome and autoimmune haemolytic anaemia in children. Infections

Immunodeficiencies and Lymphoproliferative disorders

Secondary forms AHIA and ES can be primary or secondary to other diseases, mainly infections, lymphoproliferative disorders, autoimmune diseases and immunodeficiencies (Table I). The recent new findings of different genetic defects involving the critical pathways of immune-regulation led to the identification of specific disorders, which explain some cases of ES previously reported as idiopathic. Autoimmune lymphoproliferative syndrome (ALPS) and other lymphoproliferative disorders. The first studies with ES patients showed a decreased CD4/CD8 ratio and increased production of interleukin 10 and c-interferon (Wang et al, 1983; Karakantza et al, 2000). After the scenario of ALPS emerged, some patients reported as ES were actually shown to suffer from ALPS (Teachey et al, 2005), caused by a defect of the FAS apoptotic pathway, resulting in abnormal lymphocytes survival that leads to lymphoproliferation and autoimmunity (Bleesing et al, 2002). Cytopenia is the most common manifestation of autoimmunity and may also feature the first sign of the disease. ALPS patients are characterized by chronic lymphoproliferation, an increased number of a specific T cell population, termed ‘double negative’ T-cells, and an increased risk of developing malignancies. Similar clinical features overlapping with more specific symptoms have been recently described in other disorders caused by different genetic defects involving other crucial pathways of lymphocytes activation and survival, such as the cytotoxic T-lymphocyte antigen-4 (CTLA4), lipopolysaccharide-responsive deige-like anchor deficiency (LRBA), phosphoinositide 3-kinase delta (PI3KD) mutations, or CTLA4 deficiencies (Seidel, 2014). Post-transplant forms. Cases of ES and AHIA have been reported after both solid organ and stem cell transplantations. In the first cases, autoimmune cytopenias (ACs) occur in the early post-transplant phases, often following viral infections. In these particular patients, alloimmunity seems to play an important role in the pathogenesis, as does tacrolimus-based immunosuppression, which may act either ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2016, 172, 524–534

Autoimmune and Rheumatology disorders: Malignancies

Other

Epstein-Barr virus Cytomegalovirus Human Immunodeficiency Virus Helicobacter Pylorii Hepatitis C virus Mycoplasma pneumoniae Parvovirus B19 Common-Variable Immuno-Deficiency (CVID) Severe Combined Immuno-Deficiency (SCID) Di George syndrome Selective IgA deficiency Autoimmune lymphoproliferative syndrome (ALPS) Lipopolysaccharide-responsive and beige-like anchor (LRBA) deficiency Cytotoxic T-Lymphocyte Antigen-4 (CTLA4) deficiency Phosphoinositide 3-kinase delta (PI3KD) mutations Castleman disease Systemic lupus erythematosus Anti-phospholipid syndrome Rheumatoid arthritis Giant-cell hepatitis Lymphoma Leukemia Myelodysplasia Drugs Vaccination Stem cell transplantation

through a drug-induced hypersensitivity or through its role in the transcription of lymphokine genes (Clipstone & Crabtree, 1992). Incomplete immune reconstitution or immune dysregulation may also be the cause of ACs occurring after stem cell transplantation (SCT). In the largest report on post-SCT ACs in children, the use of alternative donors was reported to be associated with a higher incidence, probably due to dysregulation caused by anti-thymocyte globulin and alemtuzumab given in the conditioning regimen. Patients who underwent SCT because of a non-malignant disorder were also found to have a higher incidence of ACs (Faraci et al, 2014). Human immunodeficiency virus (HIV). Thanks to antiviral treatment, nowadays HIV-related AC is no longer a major problem in children. Although an overt haemolysis is very rare, a positive DAT has been reported in 20–40% of HIV-infected patients (Toy et al, 1985). This high incidence may be related to abnormal B-cell regulation by the infected T-cells, a direct activation of B-cells by the virus, or an abnormal response to other viruses or other HIV-associated opportunistic agents (Saif, 2001). 525

Review

Clinical presentation Anaemia is usually normocytic or macrocytic, often hyperchromic, and may develop gradually or quickly, depending on the rate of haemolysis and on the efficacy of compensatory response of the bone marrow. The severity of the disease can thus vary from mild to acutely life-threatening forms. Symptoms usually include pallor, fatigue, dyspnoea, tachycardia and often fever. Hepato-splenomegaly, jaundice and haematuria/hemoglobinuria may also be present. The most frequent association with AIHA is thrombocytopenia, which may present with petechiae, bruising and muco-cutaneous bleeding. The association may either develop concomitantly or separately after a median interval of 3 years (Aladjidi et al, 2011). For this reason, the diagnosis of ES is challenging and should also be potentially considered at the onset of a unilineage cytopenia, especially in cases with severe symptoms and/or in the case of refractory/resistant disease. Neutropenia can be asymptomatic or present through infection-related symptoms. Its combination with other ACs (for example thrombocytopenia and neutropenia), although less frequent, is still compatible with the phenotypical diagnosis of ES.

Diagnosis A careful patient and family history of malignancies, infections and immunological disorders is important to identify other underlying diseases. It is also useful to identify recent exposure to drugs or vaccinations that might trigger the cytopenia. Clinical examination should focus on the search for lymphadenopathy and hepato-splenomegaly. Although the most recent definition (Wang, 1988; Mathew et al, 1997; Savasßan et al, 1997) also enables a diagnosis of ES in the absence of immune destruction of red cells, AIHA is an important element for the diagnosis of the disease that should also be looked for in less clinically evident cases. The diagnosis of an immune-mediated haemolysis is based on DAT positivity, reticulocytosis, increased indirect hyperbilirubinaemia, increased lactate dehydrogenase (LDH) level and reduced serum haptoglobin. Sometimes, DAT may be the only sign of RBC involvement. A peripheral blood film may show spherocytes that suggest the presence of extravascular haemolysis and/or aggregations of RBC in long chains (‘rouleaux’). Artifactual macrocytosis due to RBC agglutination can also be present. In the event of a warm antibodyAIHA (WA-AIHA), DAT is usually IgG+ or IgG/C3d+. Patients with cold antibody-AIHA usually have a negative or C3d+ DAT, however, a negative DAT should not exclude the diagnosis of WA-AIHA, as about 10% of patients (Petz & Garratty, 2004) may have an undetectably low number of Ab molecules on the RBC surface thus requiring a more sophisticated laboratory work-up. DAT can also be negative when 526

‘warm’ IgM or IgA, which should be identified by monospecific anti-IgA sera or by low-affinity antibodies, mediates haemolysis. In all these cases, the exclusion of other non immune-mediated causes of haemolysis is strongly recommended and, if thrombocytopenia is also present, thrombotic thrombocytopenic purpura and atypical haemolytic uraemic syndrome should be excluded by investigating ADAMS13 activity and complement levels. About 40% of children (Aladjidi et al, 2011) can present with a normal/low count of reticulocytes (Liesveld et al, 1987; Jastaniah et al, 2004) that can also be the target of the self-directed RBC antibodies. Earlier intra-medullary precursors may also be involved through the destruction process thus explaining cases that can initially present as pure red cell anaemia (Miano et al, 2014a). Although the sensitiveness of anti-platelets and anti-neutrophils is very low, their identification can be helpful to confirm the immunological background of thrombocytopenia and neutropenia. Bone marrow aspirate is not considered essential for the diagnosis of AIHA, as it usually presents normal features or increased cellularity (Pui et al,1980; Mathew et al, 1997). Nonetheless, it should be performed in all cases with reticulocytopenia, and in ES, in order to exclude a proliferative disorder or myelodysplasia. In very young children with neutropenia, bone marrow can also be helpful to exclude a congenital neutropenia that usually shows a maturation arrest at the promyelocyte stage. An immunological work-up at diagnosis is highly recommended, and should include the levels of IG with subclasses, screening for other auto-immune diseases, and lymphocytes subset count with double-negative T-cells, in order to detect other underlying disorders that might be masked by the subsequent immunosuppressive treatment. Serological and molecular search for previous and/or on-going infections is also necessary at the onset of the disease to identify any infective trigger. It is of note that severe acute onset, chronic/relapsing phenotypes and ES as an expression of multi-lineage cytopenia, are more frequently associated with secondary forms and may represent an epiphenomenon of an underlying immune disorder (Teachey & Lambert, 2013). It is worth noting that, thanks to more advanced diagnostic tools and more prompt consideration by clinicians, about half of the patients that show the clinical phenotype of ES are nowadays recognized to have an underlying ALPS (Teachey et al, 2005; Seif et al, 2010). For the other patients, the search for gene defects related to recently identified novel immunodysregulatory syndromes and known to underlie cases of ES should also be considered, using, when available, both traditional DNA assays or more modern techniques, such as next generation sequencing or whole exome sequencing. Tables II and III show my suggested diagnostic work-up and differential diagnosis for patients with ES and AIHA. ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2016, 172, 524–534

Review Table II. Diagnostic work up for Evans syndrome and autoimmune haemolytic anemia.

Table III. Differential diagnosis of autoimmune haemolytic anaemia and Evans syndrome.

Haematological evaluation:

Autoimmune haemolytic anaemia

• • • • • • • • • •

Full Blood Count* Reticulocytes* Blood smear*,† Blood group* Red cell antigen typing including: C, c, D, E, e, K, Jka, Jkb, Fya, Fyb, S, s Haemolysis indexes (LDH, haptoglobin, bilirubin)* Direct and Indirect Antiglobulin Tests* BUN, creatinine ALT, AST, cGT, C-reactive protein* PTT, Fibrinogen, d-Dimer Bone marrow aspirate (if reticulocytopenia and/or ES)

Infective screening:

• HIV, HCV, HBV serological evaluation† • EBV, CMV, Parvovirus B19, serological † and molecular evaluation • Helicobacter Pylori Immunological work-up:

• Lymphocytes subsets: CD3*, CD4*, CD19*, CD16*, CD56*, B naive (CD19+IgD+CD27 ), B memory (CD19+CD27+) • Immunoglobulin serum levels*,† • IgG subclasses (patients aged >2 years)† • C3, C4, CH50 • Autoantibodies: anti-nuclear, anti-extractable nuclear, anti-DNA, anti-phospholipid, anti-Sm, anti-TG, anti-TPO† • ALPS screening: double-negative T-cells, Vitamin B12, IL10, IL18, circulating FAS, Fas-apoptosis functional test Radiological evaluation:

• Chest X-ray • Abdominal sonography (spleen, liver, lymph nodes) ES, Evans syndrome; LDH, lactate dehydrogenase; BUN, blood urea nitrogen; ALT, alanine aminotransferase; AST, aspartate aminotransferase; cGT, gamma glutamyl transpeptidase; PTT, partial thromboplastin time; HIV, Human Immunodeficiency virus; HCV, Hepatitis C virus; HBV, Hepatitis B virus; EBV, Epstein–Barr virus; CMV, Cytomegalovirus; anti-TG, anti-thyroglobulin; anti-TPO, anti-thyroperoxidase; ALPS; Autoimmune lymphoproliferative syndrome; IVIG, intravenous immunoglobulin; IL, interleukin. *Mandatory first-line evaluation. †Before transfusion/IVIG administration.

First-line treatment The aim of the treatment is to control cytopenias, of which AIHA is by far the most frequent expression, and to minimize toxicity. Steroids are the first-choice treatment, although the first successful experiences (Dameshek et al, 1951; Pui et al, 1980; Wang, 1988) have never been validated by any clinical trial. Patients are usually treated with prednisolone at a dose of 1–6 mg/kg/d (Allgood & Chaplin, 1967; Pui et al, 1980; ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2016, 172, 524–534

Evans syndrome

Non immune hereditary red-cell membrane disorders Erythrocyte enzyme deficiency Dyserythropoietic anaemia Haemoglobinopathies Wilson disease Paroxysmal nocturnal haemoglobinuria Monoclonal lymphoproliferation Acquired thrombotic thrombocyotpenic purpura Inherited ADAMTS13 deficiency Haemolytic uraemic syndrome Kasabach-Merrit syndrome Disseminated intravascular coagulation Monoclonal lymphoproliferation

Mathew et al, 1997; Meyer et al, 1997; Naithani et al, 2007; Ware, 2009). This approach induces remission in 80–85% of patients with most children responding to 1–2 mg/kg/d. I start with 1–2 mg/kg prednisolone and give higher doses (4–6 mg/kg/d for the first 72 h) in the case of severe clinical symptoms in patients with more aggressive forms. Although remission is obtained in most cases, the risk of relapse is high, especially during the tapering off. This is why a full dose of steroid should be given for at least 3–4 weeks. Patients should be evaluated after 3 weeks to determine whether to continue full dosage for another week in responding subjects and then slowly taper off over not less than 6 months. In patients who show a partial response after the first 3 weeks, the full dosage might be given for 2 further weeks, however, children who do not show any response after 3 weeks probably require a different strategy and should be shifted to a second-line treatment. Another reported scheme includes mega-doses of methylprednisolone: 30 mg/ kg/d for 3 d, 20 mg/kg/d for the following 4 d and then tapering by half of the dose each week (Meytes et al, 1985; Nanan et al, 1995; Ozsoylu, 2004). Packed red blood cell (PRBC) transfusions should be reserved for very symptomatic patients, who may suffer from life-threatening events. In fact, transfused PRBC may be destroyed by the auto-antibody, resulting in a further hyperactivation of the abnormal immunological process. The decision to transfuse should be driven by the aim to correct the clinical symptoms, rather than the haemoglobin level. In fact, the usual wide range of auto-antibody reactivity makes it difficult to find a truly compatible donor, and the choice of the ‘best available’ matched unit should be carefully made on the basis of extensive RBC phenotyping. Small (3 ml/kg) amounts of leucodepleted PRBC should be given slowly under careful supervision. In the case of life-threatening events not responding to transfusions, plasma-exchange may be an option to remove circulating antibodies that, however, do not look fully established. In this respect, because little 527

Review data is available (McCarthy et al, 1999; Roy-Burman & Glader, 2002; Beretta et al, 2009; Lucchini et al, 2009), the American Society for Apheresis (ASFA) states that its optimal role is not established for WA-AHIA (category III) even if it may have some efficacy in cold-AHIA (Category II) (Schwartz et al, 2013). Therefore, the cost/benefit ratio of this procedure should also be considered, especially for small weighted children. Platelet transfusions should be reserved only for cases with life-threatening haemorrhages. In cases of severe neutropenia, granulocyte colony-stimulating factor (5 lg/kg/dose) might be used ‘on demand’ if severe infections coexist, but only for the duration of the infection (Fioredda et al, 2012).

Second and further-line treatment Most children with ES experience a relapsing/resistant cytopenia, or a chronic disease, often requiring prolonged treatment with steroids, which may lead to severe side effects on the bone and the endocrine systems. Before the introduction of new therapeutic options, splenectomy represented the only choice for these patients. Due to its risks, this option should be now considered only after the failure of other alternative drugs available for refractory patients and should not be performed in patients with ALPS (Rao, 2015). The choice of the most appropriate second/further line treatment should take into consideration the immunological background of the disease and the severity of clinical symptoms. Another crucial point is the time required to achieve efficacy, which has to be short in severe and life-threatening events, but could be more delayed in the case of steroid-dependency or when a maintenance treatment is indicated. Monoclonal antibodies, multi-agent approaches including chemotherapy and splenectomy can be used more as ‘attack’ therapy to obtain a fast response. Immunosuppressive agents, such as mycophenolate mofetil (MMF), sirolimus and ciclosporin, can be more suitable for response maintenance or as steroidsparing tools.

Rituximab Rituximab is a monoclonal antibody against the CD20 molecule that causes B-cell depletion and is usually given at a dose of 375 mg/m2 on days 1, 8, 15 and 22. Most available data comes from retrospective studies of patients with AHIA (Quartier et al, 2001; Zecca et al, 2003; Garvey, 2008; Bussone et al, 2009; Svahn et al, 2009; Pe~ nalver et al, 2010; Barcellini & Zanella, 2011) and immune thrombocytopenic purpura (ITP) (Bennett et al, 2006; Patel et al, 2012) with overall response rates between 77–93% and 39–68%, respectively. Experience in ES patients is limited to case reports and to a series of adults (Michel et al, 2009) and children (Bader-Meunier et al, 2007) where rituximab, alone or in combination with steroids, showed a response rate of 82% and 75%, respectively. 528

In the absence of prospective trials and based on the above data, at Istituto Giannina Gaslini, rituximab is preferred to splenectomy as second line treatment for children with AHIA due to the higher risk of infective complications following this practice in younger children. The time to response to the treatment, described as a median of 3–6 weeks but also after up to 12 weeks, must be considered for the eventual choice to temporarily overlap the steroid treatment according to the severity of the disease. In patients with ES, however, the use of rituximab as a second-line option should be carefully weighed, taking into consideration a potential underlying diagnosis of ALPS. Indeed, ALPS patients were reported to have lower response rates, higher risk of infection and prolonged hypogammaglobulinaemia (Teachey et al, 2009; Rao & Oliveira, 2011) after this treatment, which should therefore be limited to severe forms requiring a strong approach (Rao & Oliveira, 2011). Nonetheless, careful monitoring of B-cells and immunoglobulin levels after treatment should be performed in all patients receiving intravenous immunoglobulin (IVIG) until their recovery. Apart from ALPS patients, severe B-cell depletion and the consequent need for long-term IVIG replacement mainly occurs in patients undergoing repeated doses and/or in the setting of other underlying immune-disorders. (Cooper et al, 2009). The most important side-effect of rituximab is multifocal leucoencephalopathy, which has often been described in relation with John Cunningham virus (JCV) infection in immunocompromised patients (Bellizzi et al, 2013). However, this complication is very rare in children, perhaps due to the lack of exposure to the virus, and has been mostly reported after chemotherapy and SCT or in patients with systemic lupus erythematosus (Carson et al, 2009).

Mycophenolate mofetil MMF is an inhibitor of inosine monophosphate dehydrogenase in purine synthesis that targets B, T and NK cells. It has been shown to be effective in AIHA and ITP in some retrospective studies on adult patients that occasionally included children (Howard et al, 2002; Hou et al, 2003; Zhang et al, 2005; Provan et al, 2006). In those studies, response rates ranged between 62% and 82%. Other anecdotal studies reported efficacy in children with ES (Guirat-Dhouib et al, 2010) and ALPS-associated cytopenia (Rao et al, 2005). In a recent study of primary and secondary AC (Miano et al, 2015), MMF was safe and effective, mainly as a second line treatment, in all 11 patients with ES associated with an underlying diagnosis of ALPS (n = 9) or ‘ALPS-related’ disorder (n = 2), defined as the presence of at least one absolute or primary additional diagnostic criterion for ALPS (Oliveira et al, 2010), suggesting a potential role for this drug in patients with ES who may suffer from a broader underlying immunological disorder than ALPS. I use MMF at a dose of 600 mg/m2 twice a day (maintaining serum levels between ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2016, 172, 524–534

Review 1–3
5 lg/ml) and evaluate the response after 3 months. Due to the risk of relapse after discontinuation and the absence of data on MMF long-term toxicity, the treatment is continued in responding children, with a full dose for a total of 2 years, then the drug is tapered off over 6 months before stopping. Although further studies are required to understand its real efficacy and safety, current results seem to suggest that MMF may play a role as second/third-line treatment. Given that MMF needs rather a long time to generate a response, ideally it should be overlapped early with more aggressive therapy, such as high-dose steroids or rituximab, to enable the achievement of therapeutic plasma levels, and given as a ‘maintenance treatment’. This scheme seems to be applicable to relapsing patients, steroid-dependent children requiring drug sparing or those who experience flare-up episodes during tapering. In general, given the good results in patients with ALPS (Rao et al, 2005; Teachey et al, 2009; Rao & Oliveira, 2011; Miano et al, 2015) and the risk of rituximabrelated complications, I administer MMF to children with ES secondary to ALPS in a more up-front approach soon after the failure of first-line treatment.

and in those who need a steroid-sparing/maintenance treatment.

Ciclosporin Ciclosporin has been used at a dose of 5 mg/kg as an alternative option in adults and children with refractory isolated AIHA (D€ undar et al, 1991) or ES (Emilia et al, 1996; Ucßar et al, 1999; Williams & Boxer, 2003). Ciclosporin has also been used as an alternative treatment in the context of a multi-agent approach including danazol (Scaradavou & Bussel, 1995) or in protocols including weekly vincristine and methylprednisone. Due to the side effects and the need for frequent clinical and serum level monitoring, ciclosporin alone might be an option as a steroid-sparing/maintenance treatment only after the failure of newer and more tolerable agents, such as MMF and sirolimus. Its use in the context of a multi-agent intensive protocol could be considered in severe refractory cases only, after failure of a more targeted option, such as rituximab.

Sirolimus

Other options

Sirolimus is an inhibitor of the mammalian (mechanistic) target of rapamycin (mTOR), which is known to increase T-regulatory cells (T-regs) and to induce apoptosis in abnormal lymphocytes (Zhan et al, 2013). The drug has been used for over 20 years (Hartford & Ratain, 2007) in the setting of autoimmune diseases (Brusko et al, 2008) and solid organ transplantation (Karim & Giles, 2008) and has a safe profile. Very limited data is available on sirolimus use in ES and most information comes from AIHA and ALPS settings. Its mechanism of action accounts for successful results in posttransplant AIHA (Valentini et al, 2006; Acquazzino et al, 2013; Loar et al, 2013) and in some paediatric patients with primary refractory AIHA (Miano et al, 2014a,b). Sirolimus has also been described as useful in ALPS patients with or without cytopenia (Teachey et al, 2009) in which it also reduced the count of double-negative T-cells. In a recent study from my institution (Miano et al, 2015), sirolimus was effective in all five children with refractory AIHA, in 6/10 with ITP, and also in the only patient affected with ES. All these children previously failed MMF, suggesting a potential role for sirolimus in patients with primary refractory cytopenias or with underlying defects other than ALPS. At the Istituto Giannina Gaslini, sirolimus is given at the dose of 2–3 mg/m2 once a day, maintaining serum levels between 4–12 ng/ml, with an optimal target of 9 ng/ml, for at least 3 months before evaluating its efficacy. As with MMF, I continue the treatment in responders for a total of 2 years followed by another 6 months of tapering off before finally stopping. On the basis of the available data so far, sirolimus may be used as a rescue treatment after MMF failure in patients with chronic/refractory ES and AIHA,

IVIG. Although the role of IVIG for patients with ITP has been clearly established, their use in the setting of AIHA and ES is still controversial and little data is available for these diseases. Their potential efficacy seems to be related to the saturation of Fc phagocyte receptors in the reticuloendothelial system. A few reports have been published on their use in AIHA at the dose of 0
4–0
5 g/kg for 4–5 d, concomitantly with steroids or after their failure (Bussel & Hilgartner, 1984; Oda et al, 1985; Otheo et al, 1997). The largest retrospective report on 73 patients with AIHA showed around 40% of responses, which reached 55% in children (Flores et al, 1993). The recently published Canadian guidelines do not recommend the routine use of IVIG in patients with AIHA and suggest their use only in patients with severe disease, however, in cases of symptomatic thrombocytopenia, IVIG are a recommended option (Anderson et al, 2007). Based on this evidence and on the low risk of severe sideeffects, IVIG may be a complementary tool that can be added to treatment in severe forms where thrombocytopenia dominates the clinical scenario.

ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2016, 172, 524–534

Splenectomy. Splenectomy has been considered the secondline option for the treatment of ES for many years, due to the good response and short-term efficacy. No data is available on its efficacy compared to newer approaches, such as rituximab. In the largest published cohort on AIHA in children (Aladjidi et al, 2011), splenectomy was performed in 13
9% of cases. Although it is difficult to evaluate its real efficacy, children seem to have a better response than adults mainly because of a reduced need for steroid treatment. Splenectomy-related complications are still a crucial consid529

Review eration for the choice of the right treatment, although in recent years they have been significantly reduced thanks to the introduction of both laparoscopy, which has diminished the surgical risk compared to traditional surgery (0
5–1
6% vs. 6%) (Casaccia et al, 2006), and pre-operative vaccination (American Academy of Pediatrics, 2000), which has dampened the incidence of overwhelming infections (Pilishvili et al, 2010) previously reported in 3
3–5% of cases (Bisharat et al, 2001; Davidson & Wall, 2001). Mortality due to postsplenectomy sepsis remains an important problem, however, and can be as high as 50% (Rubin & Schaffner, 2014). For this reason, the use of prophylactic antimicrobial therapy is recommended, especially in younger children. Moreover, despite the quick response, splenectomy has proven to induce a rather short term (few months) remission (Coon, 1985) in patients with ES (Mathew et al, 1997) and it is not recommended in children under 6 years of age, due to immaturity of the immune system and incomplete vaccination programmes. Overall, based on the data described above, splenectomy should be considered after the failure of other treatments in patients with severe forms who require a prompt response. Cyclophosphamide. Little data is available on the use of cyclophosphamide in ES. First experiences, at an oral dose of

1–2 mg/kg for 2–3 months (Oda et al, 1985; Wang, 1988; Gombakis et al, 1999) have proved to be effective. Higher i.v. doses (200 mg/kg in 4 d) without stem-cell rescue were also given in patients with AIHA, ITP and ES (Brodsky et al, 1998; Moyo et al, 2002) with some partial remissions reported. Alemtuzumab. Alemtuzumab is a humanized anti-CD52 monoclonal antibody that targets T and B lymphocytes, monocytes and eosinophils. It has been successfully used in some reported adult and paediatric patients with AIHA or AC (Willis et al, 2001; Ru & Liebman, 2003; Cheung et al, 2006; Miano et al, 2014a) at a dose of 10 mg/m2/d for 10 d, or of 0
2 mg/kg for 5 d. After the initial response, most patients experience relapse. For this reason, I see some space for alemtuzumab in severe forms of ES after failure of rituximab, who, however, might need for further maintenance treatment.

New drugs Thrombopoietin receptor agonists. In the last few years, romiplostim and eltrombopag have been increasingly used in some haematological disorders. Reports of sustained remission after thrombopoietin receptor agonist discontinuation  in adults (Cervinek et al, 2015) and children (K€ uhne, 2015)

Fig 1. Therapeutic approach to patients with Evans syndrome at the Istituto Giannina Gaslini. CR, complete remission; PR, partial remission; NR, no repsonse; IVIG, intravenous immunoglobulin; MMF, mycophenolate mofetil; ALPS, Autoimmune Lymphoproliferative Syndrome.

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Review suffering from ITP have been reported, and encouraging results have been published in some ES children, (Pasquet et al, 2014; Seidel et al, 2014). Its use in ES remains anecdotal, however, and might be clarified by future studies. Bortezomib. Bortezomib is an inhibitor of 26S proteasome that has been successfully used in AC following SCT (Waespe et al, 2014) and in a case of IgM-driven AIHA secondary to a monoclonal gammopathy (Carson et al, 2010). Seven children with primary, secondary or post-transplant AC, including one with ES, treated with bortezomib, have also been recently reported (Khandelwal et al, 2014) and a clinical trial is on-going in the setting of post-transplant AC (ClinicalTrials.gov identifier: NCT01930253). The idea to target plasma cells in a context where an antibody-mediated attack cannot be controlled by anti-CD20 may be based on the speculation that a cluster of plasma cells not targetable by previous treatments might be a cause of the persistence of the disease.

Stem cell transplantation There is little data, and mainly from small series/case reports, on both autologous and allogeneic haematopoietic SCT in patients with ES and AIHA. Overall, these studies reported complete remission of around 50% (Petz & Garratty, 2004; Urban et al, 2006). Passweg and Rabusin (2008) published a study on a cohort of patients with AC from the European Society of Blood and Marrow Transplantation (EBMT) database, which included 7 patients with ES and 7 with AIHA. Four out of 14 achieved a continuous remission with an

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Conclusion The management of ES is challenging due to the lack of evidence-based data on treatment. An accurate immunological work-up is necessary in all newly diagnosed children in order to promptly identify the underlying disorders that may require specific treatment. After the failure of up-front treatment with steroids, I give rituximab to patients with ES, apart from children with an underlying diagnosis of ALPS with no severe phenotype for whom I would rather suggest the use of MMF and sirolimus. In my opinion, these drugs may also be useful as a maintenance treatment in patients responding to rituximab or in the case of steroid-dependent disease. Further alternative treatments should be chosen on a caseby-case basis, taking into consideration the possible underlying disorder and the response to the previous therapies. In fact, a partial/short response to anti-CD20 would prompt the concomitant involvement of a T-cell component in the pathogenesis and then I would suggest a T-cell targeting drug. Figure 1 shows the therapeutic approach adopted at my institution for ES, which is inevitably based on data from retrospective studies and therefore should be regarded with caution. The need to obtain evidence-based treatment data urgently requires the design of international clinical trials that would enable stronger indications to be derived.

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