review
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 challeng-
ing 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 second-
line treatment, with the exception of those with an underly-
ing diagnosis of autoimmune lymphoproliferative syndrome
who may benefit from other options such as mycophenolate
mofetil and sirolimus. Better knowledge of the immunologi-
cal 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 trans-
plantation. 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 anae-
mia, immune thrombocytopenia, children, autoimmune
lymphoproliferative syndrome, immunosuppression.
Evans Syndrome (ES) is a rare disorder that was initially
described as the presence of autoimmune haemolytic anae-
mia (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 knowl-
edge of the novel immune deregulatory syndromes underly-
ing 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 pres-
ence 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: mauriziomiano@gaslini.org
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 chil-
dren 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 paedi-
atric 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 cyto-
toxicity mechanism. More rarely, both IgA and IgM can tar-
get the RBCs as well. The maximal activity of IgG is usually
at 37°C, and for this reason they are classified as warm
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British Journal of Haematology, 2016, 172, 524–534
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antibodies. In a minority of children (25–30%), IgM destroys Table I. Causes of secondary Evans syndrome and autoimmune hae-
RBCs by activating the complement cascade, mainly in the molytic anaemia in children.
intra-vascular compartment, when exposed to cold tempera- Infections Epstein-Barr virus
tures (cold antibodies) (Petz & Garratty, 2004). Sometimes, Cytomegalovirus
both mechanisms coexist. Immune-mediated thrombocytope- Human Immunodeficiency Virus
nia is due to platelet opsonization with anti-platelet antibod- Helicobacter Pylorii
ies and/or immune-complexes (Cines & Blanchette, 2002; Hepatitis C virus
Mycoplasma pneumoniae
Provan & Newland, 2002) that leads to their premature
Parvovirus B19
removal by the reticuloendothelial system. Peripheral
Immunodeficiencies Common-Variable Immuno-Deficiency
destruction of neutrophils is usually mediated by antibodies and Lymphoproliferative (CVID)
that recognize membrane antigens, mostly located on disorders Severe Combined Immuno-Deficiency
immunoglobulin G Fc receptor type 3b (FccIIIb receptor), (SCID)
(Farruggia & Dufour, 2015). Di George syndrome
Selective IgA deficiency
Autoimmune lymphoproliferative
Secondary forms syndrome (ALPS)
AHIA and ES can be primary or secondary to other diseases, Lipopolysaccharide-responsive and
mainly infections, lymphoproliferative disorders, autoim- beige-like anchor (LRBA) deficiency
Cytotoxic T-Lymphocyte Antigen-4
mune diseases and immunodeficiencies (Table I). The recent
(CTLA4) deficiency
new findings of different genetic defects involving the critical
Phosphoinositide 3-kinase delta
pathways of immune-regulation led to the identification of (PI3KD) mutations
specific disorders, which explain some cases of ES previously Castleman disease
reported as idiopathic. Autoimmune and Systemic lupus erythematosus
Rheumatology Anti-phospholipid syndrome
Autoimmune lymphoproliferative syndrome (ALPS) and other disorders: Rheumatoid arthritis
lymphoproliferative disorders. The first studies with ES Giant-cell hepatitis
patients showed a decreased CD4/CD8 ratio and increased Malignancies Lymphoma
production of interleukin 10 and c-interferon (Wang et al, Leukemia
1983; Karakantza et al, 2000). After the scenario of ALPS Myelodysplasia
Other Drugs
emerged, some patients reported as ES were actually shown
Vaccination
to suffer from ALPS (Teachey et al, 2005), caused by a defect
Stem cell transplantation
of the FAS apoptotic pathway, resulting in abnormal lym-
phocytes survival that leads to lymphoproliferation and
autoimmunity (Bleesing et al, 2002). Cytopenia is the most through a drug-induced hypersensitivity or through its role
common manifestation of autoimmunity and may also fea- in the transcription of lymphokine genes (Clipstone & Crab-
ture the first sign of the disease. ALPS patients are character- tree, 1992).
ized by chronic lymphoproliferation, an increased number of Incomplete immune reconstitution or immune dysregula-
a specific T cell population, termed ‘double negative’ T-cells, tion may also be the cause of ACs occurring after stem cell
and an increased risk of developing malignancies. transplantation (SCT). In the largest report on post-SCT ACs
Similar clinical features overlapping with more specific in children, the use of alternative donors was reported to be
symptoms have been recently described in other disorders associated with a higher incidence, probably due to dysregu-
caused by different genetic defects involving other crucial lation caused by anti-thymocyte globulin and alemtuzumab
pathways of lymphocytes activation and survival, such as the given in the conditioning regimen. Patients who underwent
cytotoxic T-lymphocyte antigen-4 (CTLA4), lipopolysaccha- SCT because of a non-malignant disorder were also found to
ride-responsive deige-like anchor deficiency (LRBA), phos- have a higher incidence of ACs (Faraci et al, 2014).
phoinositide 3-kinase delta (PI3KD) mutations, or CTLA4
deficiencies (Seidel, 2014). Human immunodeficiency virus (HIV). Thanks to antiviral
treatment, nowadays HIV-related AC is no longer a major
Post-transplant forms. Cases of ES and AHIA have been problem in children. Although an overt haemolysis is very
reported after both solid organ and stem cell transplanta- rare, a positive DAT has been reported in 20–40% of
tions. In the first cases, autoimmune cytopenias (ACs) occur HIV-infected patients (Toy et al, 1985). This high incidence
in the early post-transplant phases, often following viral may be related to abnormal B-cell regulation by the infected
infections. In these particular patients, alloimmunity seems T-cells, a direct activation of B-cells by the virus, or an
to play an important role in the pathogenesis, as does tacro- abnormal response to other viruses or other HIV-associated
limus-based immunosuppression, which may act either opportunistic agents (Saif, 2001).

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British Journal of Haematology, 2016, 172, 524–534
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Clinical presentation
Anaemia is usually normocytic or macrocytic, often hyper-
chromic, and may develop gradually or quickly, depending
on the rate of haemolysis and on the efficacy of compen-
satory response of the bone marrow. The severity of the dis-
ease 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-cuta-
neous bleeding. The association may either develop concomi-
tantly 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 sev-
ere symptoms and/or in the case of refractory/resistant dis-
ease. 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 diag-
nosis of ES.
Diagnosis
A careful patient and family history of malignancies, infec-
tions 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 evi-
dent cases.
The diagnosis of an immune-mediated haemolysis is based
on DAT positivity, reticulocytosis, increased indirect hyper-
bilirubinaemia, 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 extravas-
cular haemolysis and/or aggregations of RBC in long chains
(‘rouleaux’). Artifactual macrocytosis due to RBC agglutina-
tion can also be present. In the event of a warm antibody-
AIHA (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 sophis-
ticated 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 rec-
ommended and, if thrombocytopenia is also present, throm-
botic 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 neu-
tropenia.
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 reticu-
locytopenia, and in ES, in order to exclude a proliferative
disorder or myelodysplasia. In very young children with neu-
tropenia, bone marrow can also be helpful to exclude a con-
genital neutropenia that usually shows a maturation arrest at
the promyelocyte stage. An immunological work-up at diag-
nosis is highly recommended, and should include the levels
of IG with subclasses, screening for other auto-immune dis-
eases, 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 diagnos-
tic 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 (Tea-
chey 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 sequenc-
ing.
Tables II and III show my suggested diagnostic work-up
and differential diagnosis for patients with ES and AIHA.
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British Journal of Haematology, 2016, 172, 524–534

Table II. Diagnostic work up for Evans syndrome and autoimmune
haemolytic anemia.
Haematological evaluation:
• 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 aminotrans-
ferase; cGT, gamma glutamyl transpeptidase; PTT, partial thrombo-
plastin 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-thyro-
peroxidase; 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 mini-
mize 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;
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British Journal of Haematology, 2016, 172, 524–534
Review
Table III. Differential diagnosis of autoimmune haemolytic anaemia
and Evans syndrome.
Autoimmune Non immune hereditary red-cell membrane
haemolytic disorders
anaemia Erythrocyte enzyme deficiency
Dyserythropoietic anaemia
Haemoglobinopathies
Wilson disease
Paroxysmal nocturnal haemoglobinuria
Monoclonal lymphoproliferation
Evans Acquired thrombotic thrombocyotpenic
syndrome 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 respond-
ing 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 hyper-
activation of the abnormal immunological process. The deci-
sion 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 dif-
ficult 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
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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 infec-
tions 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 introduc-
tion 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 back-
ground 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 steroid-
sparing tools.
Rituximab
Rituximab is a monoclonal antibody against the CD20 mole-
cule 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; Bus-
sone et al, 2009; Svahn et al, 2009; Pe~
nalver et al, 2010; Bar-
cellini & 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%, respec-
tively. 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 pre-
ferred 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 treat-
ment, which should therefore be limited to severe forms
requiring a strong approach (Rao & Oliveira, 2011).
Nonetheless, careful monitoring of B-cells and immunoglob-
ulin 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 rela-
tion with John Cunningham virus (JCV) infection in
immunocompromised patients (Bellizzi et al, 2013). How-
ever, 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 dehydroge-
nase in purine synthesis that targets B, T and NK cells. It has
been shown to be effective in AIHA and ITP in some retro-
spective 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’ dis-
order (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
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British Journal of Haematology, 2016, 172, 524–534

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 dur-
ing tapering. In general, given the good results in patients
with ALPS (Rao et al, 2005; Teachey et al, 2009; Rao & Oli-
veira, 2011; Miano et al, 2015) and the risk of rituximab-
related 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.
Sirolimus
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 abnor-
mal 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 post-
transplant 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 cytope-
nias 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,
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British Journal of Haematology, 2016, 172, 524–534
Review
and in those who need a steroid-sparing/maintenance treat-
ment.
Ciclosporin
Ciclosporin has been used at a dose of 5 mg/kg as an alter-
native 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 & Bus-
sel, 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 inten-
sive protocol could be considered in severe refractory cases
only, after failure of a more targeted option, such as
rituximab.
Other options
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 reticuloendothe-
lial 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, concomi-
tantly with steroids or after their failure (Bussel & Hilgartner,
1984; Oda et al, 1985; Otheo et al, 1997). The largest retro-
spective 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 dis-
ease, 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 side-
effects, IVIG may be a complementary tool that can be added
to treatment in severe forms where thrombocytopenia domi-
nates the clinical scenario.
Splenectomy. Splenectomy has been considered the second-
line option for the treatment of ES for many years, due to
the good response and short-term efficacy. No data is avail-
able on its efficacy compared to newer approaches, such as
rituximab. In the largest published cohort on AIHA in chil-
dren (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 consid-
529
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 damp-
ened 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 post-
splenectomy 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 vaccina-
tion 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
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.
530
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 ritux-
imab, who, however, might need for further maintenance
treatment.
New drugs
Thrombopoietin receptor agonists. In the last few years, romi-
plostim and eltrombopag have been increasingly used in
some haematological disorders. Reports of sustained remis-
sion after thrombopoietin receptor agonist discontinuation

in adults (Cervinek et al, 2015) and children (K€ uhne, 2015)
ª 2015 John Wiley & Sons Ltd
British Journal of Haematology, 2016, 172, 524–534
 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 anecdo-
tal, 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 chil-
dren with primary, secondary or post-transplant AC, includ-
ing 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 (ClinicalTri-
als.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 treat-
ments 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) data-
base, which included 7 patients with ES and 7 with AIHA.
Four out of 14 achieved a continuous remission with an
overall transplant-related mortality of 15%. SCT should be
taken into consideration for patients with relapsing/resistant
disease who experience life-threatening events.
Conclusion
The management of ES is challenging due to the lack of evi-
dence-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 treat-
ment 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 case-
by-case basis, taking into consideration the possible underly-
ing 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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