Expert Review of Hematology

ISSN: 1747-4086 (Print) 1747-4094 (Online) Journal homepage: http://www.tandfonline.com/loi/ierr20

Updates on the pathophysiology and treatment of

aplastic anemia: a comprehensive review

Prajwal Chaitanya Boddu & Tapan Mahendra Kadia

To cite this article: Prajwal Chaitanya Boddu & Tapan Mahendra Kadia (2017): Updates on the
pathophysiology and treatment of aplastic anemia: a comprehensive review, Expert Review of
Hematology, DOI: 10.1080/17474086.2017.1313700

To link to this article: http://dx.doi.org/10.1080/17474086.2017.1313700

Published online: 28 Apr 2017.

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EXPERT REVIEW OF HEMATOLOGY, 2017
https://doi.org/10.1080/17474086.2017.1313700

REVIEW

Updates on the pathophysiology and treatment of aplastic anemia: a

comprehensive review
Prajwal Chaitanya Boddu and Tapan Mahendra Kadia
Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

ABSTRACT ARTICLE HISTORY

Introduction: The past decade or longer has witnessed an acceleration in our understanding of Received 22 December 2016
previously developed immune system and clonal evolution mechanisms, and the genesis of more Accepted 28 March 2017
novel concepts of telomere attrition. Many of these concepts are steadily finding their way into KEYWORDS
translation in various aspects of clinical practice, and provide prospects to improve AA management Aplastic; telomere;
and inform therapeutic strategy development. In this review, we intend to discuss the pathophysiology immunosuppressive; clonal
and treatments with an emphasis on most recent developments to provide an update on our under- evolution; eltrombopag;
standing of disease mechanisms. alemtuzumab;
Areas covered: A literature search was undertaken addressing various aspects of pathophysiology with antithymocyte globulin;
a focus on the role of immune system repertoire, telomeres and mutational events surrounding AA. We transplant
also reviewed upon the temporal evolution of treatment strategies in AA to the contemporary manage-
ment of today and commented briefly upon the more recently investigated novel therapies and their
expanding niche especially in the transplant and salvage setting.
Expert commentary: Immune-mediated destruction of hematopoietic stem and progenitor cells,
leading to a marrow devoid of hematopoietic elements, and peripheral pancytopenia are the hallmarks
of AA. Recent studies have shed light on another facet of the disease – as a clonal disorder character-
ized by karyotypic abnormalities, genomic instability, telomere attrition, and recurrent somatic muta-
tions reminiscent of myeloid malignancies. Further understanding of this underlying pathophysiology
can help in improving prognostication and treatment of this disease.

1. Introduction transplantation regimens, intensifying immunosuppressive strate-

gies and enhancing supportive care, over several decades of
The first formal description dates back to 1888 when Paul Ehrlich
treatment, has transformed AA into a disease now typified by
described a case of a young pregnant woman who had developed
greater than 80% 10-year survival expectancy.
profound anemia, bleeding, and high fever that eventually proved
Aplastic anemia, a misnomer, is an acquired marrow failure
fatal. The term aplastic anemia (AA) was introduced, 16 years later,
syndrome characterized by findings of peripheral pancytopenia
by Chauffard to describe this condition, characterized morpholo-
and a hypoplastic bone marrow [2]. Aplastic anemia shares clinical
gically by a yellow fatty bone marrow. Since its earliest descriptions
manifestations and some of its marrow morphological findings
until mid-1960s, the inexorably pernicious, uniformly fatal clinical
with other acquired (hypoplastic myelodysplastic syndrome
picture of this disease was accurately depicted in Wintrobe’s
(MDS), hypoplastic leukemias such as hairy cell leukemia, large
description ‘. . .more or less rapidly fatal. . . death ensues in the
granular lymphocytosis, paroxysmal nocturnal hemoglobinuria)
course of a few weeks or life may linger as long as six months.’
and congenital (Fanconi anemia, dyskeratosis congenita) bone
The advent of allogenic bone marrow transplantation in the late
marrow failure syndromes which is why diagnosis requires a care-
1960s and immunosuppressive therapy with antilymphocyte sera
fully constructed history and exhaustive exclusion of potential
in the 1970s proved to be major therapeutic successes leading to a
alternative etiologies – a rather difficult task of morphologic dis-
significant alteration in the natural history of the disease and
tinction based on subtleties in marrow findings, sometimes fre-
markedly improved survival outcomes. Early observations
quenting repeat marrow investigations.
spawned a series of investigations clarifying the autoimmune
Both AA and hypoplastic MDS share histological character-
nature of the disease and were subsequently followed by intensi-
istics of a hypocellular marrow and distinction relies heavily
fication efforts on previously existing immunosuppressive strate-
upon other histologic and flow cytometric characteristics such
gies. In this context, various studies observed that the addition of
as megakaryocytic and CD34+ blast numeration which are low
cyclosporine (Cys) to antithymocyte horse sera furnishes as a
in aplastic anemia and high in hypoplastic MDS, respectively
highly effective immunosuppressive strategy establishing the
[3]. Also, h-MDS is characterized by the presence of dysmega-
combination’s role as the current immunosuppressive treatment
karyocytopoiesis while their absence favors the diagnosis of
of choice [1]. The collective progress made in refining
aplastic anemia. A positive family history, abnormal

CONTACT Tapan Mahendra Kadia tkadia@mdanderson.org Department of Leukemia, UT MD Anderson Cancer center, 1515 Holcombe Blvd., Unit 428,
Houston, TX 77030, USA
© 2017 Informa UK Limited, trading as Taylor & Francis Group
2 P. C. BODDU AND T. M. KADIA
clinicomorphologic features, and laboratory findings showing
increased chromosomal fragility or telomere shortening sug-
gest a congenital form of AA [4,5]. Additional diagnostic clues
may be obtained from blood film examination such as the
presence of monocytopenia and hairy cells (in hairy cell leu-
kemia) [6]. Aplastic anemia may be associated with other
disease entities such as paroxysmal nocturnal hemoglobinuria,
hepatitides, and autoimmune disorders and patients with pre-
sumptive AA should be evaluated for the same [7–9].
Pertinently, prompt diagnosis and treatment are essential to
optimal management of AA given that short diagnosis to
treatment interval times is known to positively affect overall
survival [10,11].
Severity grading of AA, originally developed in the 1970s
by study groups to select patients most appropriate for bone
marrow transplant, has since undergone minor refinements
and continues to find great prognostic and therapeutic rele-
vance in current day management. Aplastic anemia is graded
primarily on peripheral blood count values into (1) severe
aplastic anemia (SAA): (a) bone marrow cellularity (BMC)
<25% (or) <50% normal cellularity in which <30% are hema-
topoietic and (b) two of three additional criteria i.e. absolute
reticulocyte count <20k/µL, absolute neutrophil count (ANC)
<500/µL, or platelet count <20k/µL; (2) very severe aplastic
anemia (VSAA): SAA criteria + ANC <200/µL; (3) moderate/non
severe: BMC <30% with absence of severe pancytopenia but
decrease of at least two of three blood counts below normal
[12–14].
2. Pathophysiology
Bone marrow hematopoiesis constitutes a well-orchestrated
developmental process of proliferation and maturation of mar-
row cellular elements from pluripotent hematopoietic stem cell
(HSC) precursors. HSCs have the remarkable property of robust
self-renewal and a potential to differentiate along multiple line-
age pathways. It has been estimated that about 1 × 104 HSCs are
present in the human bone marrow of which only one-tenth are
actively contributing to hematopoiesis at any given time while
the remaining fraction dwell in quiescence [15]. Progenitor and
stem cells, although not identifiable morphologically in a bone
marrow, can be distinguished flow cytometrically by the pre-
sence of CD34, a glycosylated transmembrane protein involved
in stem-cell stromal adhesion and maintenance of a phenotypi-
cally undifferentiated state [16]. In this regard, there also exists a
minor fraction of CD34 negative HSC population with marrow
repopulating capacity. Flow cytometric and immuno-phenotypic
numeration reveal a profound deficit of the hematopoietic and
progenitor cell compartment in AA [17]. These numbers may
drop to lower than 1% of the original HSC population in severe
AA [17]. The most profound deficits occur in the mitotically active
CD34+ Kit+ and uncommitted progenitor (CD34+CD38−) subset
of HPCs but can also involve the more lineage-committed CD34+
CD38+ fractions [17]. Surrogate in-vitro assays, such as the long-
term culture-initiating cells assay (LTC-IC), have been developed
to study the properties of primitive human marrow progenitors
and correlate well with the kinetic properties of CD34+ stem cells
and their primary colony formations [18]. Data available through
these assay studies confirm marked quantitative and qualitative
defects in the progenitor cells. These defects remain sustained
even after marrow recovery and reconstitution of blood counts
[19]. The low LTC-IC numbers, as determined by these assays, do
not correlate with marrow recovery and this may be due to the
massive compensatory reservoir of hematopoietic progeni-
tors [19].
HSCs have been observed to be true first responders, either
through direct or indirect signaling, to various inflammatory
and infectious insults. Activated HSCs rapidly switch after with-
drawal of the inciting stressor to a quiescent state, a requisite
for preservation of self-renewal potential [20]. It is for this
reason that a tight regulatory control of inflammatory cascade
signaling is essential for long-term maintenance of the HSC
populations [21]. Prolonged activation consequent to dysre-
gulated signaling induces functional fatigue and impairs HSCs’
self-renewal potential [22]. This eventually leads to depletion
of HSC populations, as is observed in acquired AA. The inflam-
matory milieu in the setting of immune destruction of stem
and progenitor cells leads to depletion of HSCs. These defec-
tive HPCs have been found to be insensitive to growth factor
treatments and this therapeutic approach may prove detri-
mental as they are expected to relatively increase the apop-
tosis-susceptible proliferative fraction.
Apart from the more obvious quantitative deficits, HPCs
may also suffer intrinsic qualitative defects. Stem cells in AA
patients have been demonstrated to show reduced clono-
genic potential in short-term primary and secondary colony
assays [23]. Detailed transcriptome analyses have identified
upregulation of several cell-cycle inhibitory and proapoptotic
genes and decreased expression of cell cycle promoting genes
and antiapoptotic genes in AA samples compared with
healthy controls [24]. Additionally, sublethal DNA damage
can manifest as chromosomal aberrations such as mutations
and unbalanced translocations and pave the way for clonal
evolution toward more malevolent complications of marrow
injury including myelodysplasia and leukemia.
Various etiological drivers mediating HSC destruction have
been identified including radiation, chemicals/toxins, drugs,
viruses, among others. Radiation causes HSC injury through a
multitude of mechanisms including direct lethal damage of
DNA through induction of double-stranded breaks and free
radical species formation, acceleration of HSC senescence
leading to decrease clonogenesis, induction of HSC differen-
tiation at the expense of compromising self-renewal capacity,
and via destruction of the highly supportive niche surrounding
the HSCs [25,26]. Unlike most other mechanisms, radiation-
mediated HSC depletion represents a more permanent form of
stem-cell injury [26,27].
The list of drugs, toxins, and chemicals implicated in caus-
ing bone marrow aplasia is exhaustive and the interested
reader may refer to other comprehensive texts for a detailed
review. Chemical-mediated HSC injury may be due to (a) direct
dose-dependent or idiosyncratic toxicity mediated by parent
drug or its metabolites, or (b) immune-mediated HSC destruc-
tion by drug–antigen formation [2]. Chloramphenicol, a pro-
totype for drug-related AA, exhibits two distinct modes of
marrow aplasia – (a) a dose-dependent reversible type and
(b) a dose-independent idiosyncratic type from toxic metabo-
lite injury [28]. Benzene, a ubiquitous and highly volatile

petrochemical i.e. used extensively in the hydrocarbon/auto-
mobile industry, has a more consistent marrow aplastic effect
compared with other well-studied agents. Models of drug-
immune system interaction include immune activation via
drug–hapten–antigen formation and direct stimulation of
immune receptors through non-covalent pharmacological
interactions. Drug-induced AA responds to immunosuppres-
sive treatments and the distinction does not confer a different
prognosis or therapy response compared with other etiologi-
cal types of AA. Infections with EBV, CMV, VZV, HHV-6, parvo-
virus B19, HIV, hepatitis A and C have been implicated in
aplastic anemia, with only human parvovirus B19 and HHV-6
demonstrated to have a direct cytotoxic effect on the progeni-
tors [29].
2.1. The T-cell repertoire
A large body of evidence supports the proposition of AA
being an immune-mediated phenomenon. In this regard, AA
serves as a bone marrow prototype of misdirected immune
response. Response to immunosuppressive therapies in
majority of AA cases provides compelling clinical evidence
attesting to the immune-mediated nature of the disease.
Clinical evidence is corroborated by multiple studies con-
firming clonal expansions of activated T cells directed
against marrow progenitors. Although plenty remains to
be clarified regarding the exact nature of the pathogenic
immune response and of putative antigens involved, it
appears that both adaptive and innate immune system
machineries are involved in contributing to hematopoietic
progenitor injury (Figure 1).
Available evidence suggests a gross homeostatic dysre-
gulation of the T-cell repertoire. Among the T-lymphocyte
subsets, CD8+ T cells have the best characterized role in AA-
immune pathogenesis. CD8+ T cells are increased in AA
Figure 1. A model of T-cell immune mediated stem cell progenitor cell injury. T-cells are actively involved in the disease process with CD8 T cells orchestrating a
direct cellular mediated cytotoxicity. IFN-γ and TNF-Alpha are major cytokine mediators responsible for activating cellular death/apoptotic machinery including Fas/
FasL pathway (highlighted in yellow). NK-cells are also involved, as demonstrated by upregulation of bound NKG2D ligands on the stem cell progenitor and
decrease in soluble NKG2D ligands. Full color available online.
EXPERT REVIEW OF HEMATOLOGY 3
patients and skewed toward oligo clonal patterns with
highly restricted Vbeta chain sequences [30,31]. In fact, the
presence, disappearance, and return of these highly skewed
immune-dominant clonotypes may help predict, follow
responses to, and detect relapse after immunosuppressive
therapy, respectively [31,32]. The putative autoantigens
against which these T cells react are not clearly known
but serum autoantibody detection studies suggest multiple
potential candidates. Autoantibodies against antigens
namely diazepam-binding inhibitor-related protein 1 (DRS-
1), kinectin (KTN1), postmeiotic segregation increased 1
(PMS1), heterogeneous nuclear ribonucleoprotein K (hnRNP
K) among others have been identified [33]. The potential
pathogenic and predictive role of these antibodies in AA
pathophysiology and immune therapy response, respec-
tively, may have clinical relevance and warrants further
investigation.
The role of T helper (CD4+) cells in AA is less well
characterized compared with their CD8+ T counterparts.
All subsets of T helper cells i.e. IFN-γ-producing Th1 cells,
IL-4-producing Th2 cells, T-regulatory (T-reg) cells, and Th17
cells have been implicated to play a role in AA. Similar to
CD8+ T cells, T helper cells show clonal expansion with
restriction in their TCR sequences [34]. Activated and resting
T-reg cells are reduced and are functionally defective with a
suboptimal ability to suppress effector T-cell activity [34].
Kordasti et al. were able to characterize a distinct T-reg
subsets of predictive value to immunosuppressive therapy
(IST) response in AA. The investigators identified a particular
T-reg subset, with phenotypic expression of CD95, CCR4,
and CD45RO within FOXP3high, CD127low, to predominate
among treatment responders [35]. In addition, there exists
a polarization shift in favor of Th-1 over Th-2 response
resulting in increased production of IFN-γ, a potent stimu-
lator of CD8+ T cells [36]. Finally, although the contribution
4 P. C. BODDU AND T. M. KADIA

of Th17 cells in AA pathogenesis remains contentious, regulatory factor-1) through a complex network of signal
increased numbers in blood samples of AA patients with intermediates via the Fas/FasL and TRAIL pathways [37,45]
an inverse relationship to counter regulatory T-reg cells (Figure 1). Cytokine polymorphisms involving TNF-α, IFN-γ,
suggest a potential role early in the course of the dis- and other cytokines like TGF-β are overrepresented in cer-
ease [34]. tain populations of AA and may have an influence on dis-
Natural Killer cells are key mediator cells in innate immu- ease expression independent of HLA [46].
nity responses. Liu et al. demonstrated that NK cell subset
of lymphocytes is severely reduced in active AA and
increases dramatically with responses to immunosuppres- 2.3. The role of telomeres
sive therapy [37]. The NKG2D protein–ligand system repre-
Telomeres are short guanine-rich sequences at the ends of
sents one of the best characterized pathways in NK-cell-
linear chromosomes in all eukaryotic cells, varying in size
mediated immunity. Hanaoka et al. demonstrated increased
from 5 to up to 20 kilobases in length depending upon the
expressions of various stress-inducible NKG2D ligands and
age of the organism, activity of preservative telomerases
were able to preserve hematopoietic colony formations by
and the cell’s proliferation history [47]. Telomeric DNA is
using antibodies against these ligands [38]. These observa-
critical for maintenance of chromosome stability and is
tions may have clinical implications in the evaluation of IST
progressively lost with each replication cycle. Telomeric
responses and developing targeted therapies against
DNA loss leads progressively to cellular senescence.
NKG2D ligands.
Telomere loss can also destabilize the genome by promot-
ing unbalanced chromosome translocations and aneuploidy.
2.2. Effector mediators and their role in apoptosis Telomeric length is preserved by telomerases, RNA–protein
complexes which add lost sequences to DNA ends at each
The apoptotic fraction of CD34+ stem cells is increased in
replication cycle. Telomerases are heavily expressed in HSCs
the aplastic marrow and the proportion of apoptotic cells
and their activity is upregulated during critical periods of
relate to the severity of the disease [39]. Various pathologic
rapid stem-cell proliferation. Aplastic anemia may be viewed
and genetic correlates to apoptosis have been identified
as an acquired form of telomere disease. Telomere attrition
and have been demonstrated to play a crucial role in AA
is observed in peripheral granulocyte and lymphocyte cells
pathogenesis. There is an increased expression of Fas, one
in AA patients and the degree of shortening correlates with
of the receptors in the death receptor signaling pathway, on
the severity of the disease [48,49]. Telomere length has also
CD34+ cells in AA [40]. Bone marrow studies on AA samples
been correlated to increased risk for relapse and inferior
have confirmed CD34+ progenitor cells to be more apopto-
survival [50]. It is not entirely clear as to the mechanism
tic in AA patients when compared with normal donors.
behind telomeric shortening in AA but may be the culmina-
Although this is primarily related to the significantly higher
tion of occult DNA damaging effects in the setting of
proportion of Fas+ CD34+ cells in AA, observation of
reduced telomerase expression and activity due to various
increased apoptosis even in CD34+ Fas− cells in a sizeable
poorly understood genetic determinants. In this context,
fraction of AA patients suggests that the Fas system is not
mutations in TERC and TERT, genes encoding the compo-
an indispensible pathway for apoptosis and there might
nents of the telomerase complex, have been identified in
exist alternate, hitherto unknown mechanisms operating
AA cells [51,52]. These have also been identified rarely in
[41]. Fas antigen surface levels on progenitor cells are clo-
healthy adults suggesting that a complete phenotypic
sely regulated by IFN-γ and TNF-α, and its expression is
expression of their deleterious effects may require an appro-
enhanced as a result of exposure of these cells to the two
priate environmental setting. Telomeric length serves as a
pathogenic cytokines which are constitutively secreted by
marker for DNA damage and reflects underlying ongoing
activated T-effector cells [40,42]. IFN-γ expression is
replicative stress on stem cells [53]. Progressive telomere
increased in the marrow of patients with AA. Intracellular
attrition characteristically occurs before emergence of chro-
levels of IFN-γ in marrow-infiltrating T cells decrease with
mosome 7 loss in acute myeloid leukemia (AML) after SAA
response to IST but increase at onset of relapse [43]. Dufour
[54]. Unlike in other congenital telomeropathies where there
et al. reported on a series of 53 AA patients, who had flow
exist constitutional defects in telomere–telomerase com-
cytometry profiling performed on their bone marrow speci-
plexes, it remains to be clarified if telomere attrition plays
mens, before and after IST administration. While in-vitro
an active role in AA pathogenesis or is merely a passive
blockade of either TNF-α or IFN-γ significantly increased
epiphenomenon of underlying disease progression. This will
blast forming unit populations, only TNF-α blockade signifi-
have future therapeutic implications in the development
cantly increased stem-cell colonies over normal controls, in
and role of highly novel therapies targeting this mechanism
the IST responder patients. Nevertheless, levels of both
in AA.
these cytokines were significantly lower in responders
when compared with nonresponders, providing further evi-
dence to the important role of these cytokines in AA patho-
2.4. Clonal evolution
genesis [44].
These two cytokines transduce their potent apoptotic As mentioned before, direct DNA damaging and telomere
signals to activate the downstream caspase cascade and attritive effects can promote genomic instability by creating
upregulate transcription factors (such as interferon mutations, unbalanced translocations, and aneuploidy. In fact,

accelerated telomerase attrition has been shown to precede
clonal evolution and may thus serve as a predictive biomarker
for future clonal evolution [54]. Chromosomal aberrations can
eventually lead to the formation of dysplastic clones which
may evolve over time. These clones develop unique mechan-
isms of T-cell autoimmune escape, such as by losing their AA-
overrepresented HLA allele loci [55]. Additionally, these clones
may acquire apoptosis-resistance mechanisms and hypersen-
sitive growth factor signaling receptors thereby gaining a
survival advantage that selects for their clinical emergence in
a conducive environmental setting. Clonal evolution rates in
AA occur at the rate of 10–15% over a 10-year duration
[56,57]. Numerous karyotype defects with abnormalities of
chromosome 7, trisomy 8, structural and numerical abnormal-
ities of chromosome 13, deletion of Y chromosome, and com-
plex cytogenetics have been identified; the most stereotypical
defects involve chromosome 6, 7, and 8 [56,57]. The type of
karyotype defect has a prognostic role in response and clinical
outcomes, with chromosome 7 abnormalities portending
worse outcomes while trisomy 8 signals a favorable prognosis.
Importantly, not all cytogenetic abnormalities lead to clonal
evolution; for example, uniparental disomy of the 6p has not
been described in association with AA-related MDS/AML [58].
In addition, somatic mutations involving various bone mar-
row failure and myeloid cancer-associated genes such as
ASXL1, DNMT3A, PIGA, BCOR have been identified in up to
one-third of AA patients and their presence has been corre-
lated with longer disease duration and increased risk of trans-
formation to myelodysplasia/AML [59]. Yoshizato et al. were
able to detect clonal hematopoiesis in up to 47% of their 439
AA study patients and demonstrated the process to be highly
variable with little value in predicting responses and long-term
outcomes. However, certain mutations in PIGA, BCOR, and
BCOR1 did correlate with a better response to IST, while
mutations in DNMT3A and ASXL1 associated themselves with
worse outcomes [59]. The early detection of the deleterious
clones argues for consideration for stem-cell transplantation
(SCT) in favor of IST due to the very high risk of disease
transformation.
3. Treatment
Response criteria have been developed to assess and compare
effectiveness of therapies: complete response (CR) defined by
normalization of the blood counts i.e. neutrophil
count > 1.5 × 109/L, platelet count > 150 × 109/L, and
Hb > 12 g/dL; partial response (PR) defined by transfusion
independency in patients who needed transfusions before
treatment, with either improvement of the severity degree of
the AA, or neutrophil counts increasing >0.5 × 109/L and
platelet count >20 × 109/L, in case the values were lower
before treatment. Nonresponse is generally defined by the
persistence of transfusion dependency or of values lower
than those mentioned above [14]. There are minor variations
in defining response criteria in various studies (Table 1).
In the pretreatment era, the diagnosis of severe and very
severe aplastic anemia called for a dismal prognosis with 2-
year mortality rates approaching 80% on supportive care [77].
The grim picture of aplastic anemia dramatically transformed
EXPERT REVIEW OF HEMATOLOGY 5
in the 1970s with the advent of bone marrow transplantation
and non-transplant immunosuppressive therapies. Prognosis
varies markedly depending on the severity of aplastic anemia.
The natural history of non-severe AA, although variable, fol-
lows a fairly indolent course and does not generally warrant
specific treatment apart from supportive care. However, Kwon
et al. in their evaluation of 96 NSAA patients, two-thirds of
whom received some form of immunosuppressive treatment,
suggested that NSAA treated with immunosuppressives may
decrease its risk of progression to SAA or VSAA [78]. They also
identified lower baseline white blood cell (WBC) and ANC
counts, and treatment non-responsiveness to be predictive
of an increased risk of evolution to SAA or VSAA. Unlike in
SAA patients in whom baseline absolute reticulocyte and
absolute lymphocyte counts predict treatment responsiveness
to IST, no predictive identifiers were identified for NSAA in this
study. This study also reported progression to SAA/VSAA in
20% of the study population. Taking these findings into con-
sideration, it is reasonable to treat NSAA patients with more
severe cytopenias, especially those with transfusion depen-
dence. It is important to identify and clearly define these
‘higher risk’ NSAA patients for early institution of immunosup-
pressive treatment since a delay in treatment from the time of
diagnosis can negatively impact survival.
3.1. Bone marrow transplant-general considerations
The year 1972 marked the beginning of bone marrow trans-
plant era with Donnall Thomas performing the first successful
procedure in a patient with aplastic anemia [79]. Evidence of
prolonged disease-free survival post engraftment with HLA-
matched sibling marrow grafts soon established the impor-
tance of marrow transplantation in severe aplastic anemia.
Trends in 5-year survival post bone marrow transplant have
considerably improved from 1976 to 1992 (66% vs. 48%,
respectively) and primarily reflect an improvement in post-
transplantation anti-graft versus host disease (GVHD) prophy-
laxis with agents like cyclosporine [67]. A European bone
marrow transplant study analysis on 2002 patients, engrafted
between 1976 and 1998, identified patient’s age, donor type
(HLA-identical sibling vs. alternate donor), interval between
diagnosis and transplant, and female donor for a male recipi-
ent to be important factors predicting survival outcome after
BMT [80]. Infection pre-transplant, a heavy transfusion history
are additional risk factors associated with worsened survival
[81]. Alternatively, younger age, engraftment from a HLA-
matched sibling, transfusion and/or treatment naivety before
transplantation, higher baseline ANCs associate with over 80%
long-term survival [80,82].
The type of graft also has an effect on the risk of chronic
GVHD (cGVHD) and mortality. The EBMT group retrospectively
reviewed outcomes in 692 patients, 134 of whom had periph-
eral blood progenitor cell grafts, and 558 had bone marrow
grafts. Disparity in outcomes by the type of graft was observed
in patients younger than 20 years of age in whom cGVHD and
overall mortality (5-year OS 85% vs. 73%, respectively) were
higher after PBC grafts than after bone marrow transplanta-
tion [83]. A later study on outcomes in 1886 AA patients, by
Bacigalupo et al., showed the advantages of bone marrow
 6
P. C. BODDU AND T. M. KADIA

Table 1. Frontline immunosuppressive therapies in aplastic anemia.

Median
Overall Time to response follow-up
Group Study type Regimen Study population Age Response criteria response (%) (months) (years) Relapse Clonal evolution Survival
Frickhofen Prospective, single center, hATG + Cys + P NAA/SAA/VAA 32 CR: Hg ≥ 13 g/dL, ANC ≥ 1.5 × 109/L, 11 years ORR: Median at 2 months 11.3 38% 25% (both arms 58% at 11.3 years
et al. [60] randomized pt ≥ 150 × 109/L. 70 combined)
N = 84 PR: TI (or) ↑ in 1/3 (Hg by 3 g/dL, ANC by
0.5 × 109/L, pt by 20 × 109/L)
Rosenfeld Prospective, single center hATG + Cys + P SAA N = 122 35 R = 2/3 (ANC ≥ 0.5 × 109/L; 1 year ORR: 60% by 3 months 7 35% 11% 55% at 7 years
et al. [61] pt ≥ 200 × 109/L; and reticulocyte count 58
≥40 × 109/L)
Bacigalupo Prospective, multicenter hATG + Cys + P + G-CSF SAA N = 100 16 CR: Hg ≥ 11 g/dL, ANC ≥ 1.5 × 109/L, 5 years ORR: Median at 3 months 5 12% 11% 87% at 5 years
et al. [1] pt ≥ 100 × 109/L. PR: TI + Hg ≥ 8 g/dL, 77
ANC ≥ 0.5 × 109/L, p ≥ 30 × 109/L
Tichelli et al. Prospective, multicenter, hATG + Cys ± G-CSF SAA N = 192 46 CR: Hg ≥ 11 g/dL, ANC ≥ 1.5 × 109/L, 6 years ORR: – 6 33% 19% 76% at 6 years
[62] randomized pt ≥ 150 × 109/L. PR: TI + not meeting CR 70
criteria
Kojima et al. Prospective, multicenter, hATG + Cys + Danazol ± GCSF VSAA N = 50 9 CR: Hg ≥ 11 g/dL, ANC ≥ 1.5 × 109/L, 3 years ORR: 47% ORRs in 3 months 3 22% 6% 88% at 3 years
[63] randomized MAA/SAA N = 69 pt ≥ 100 × 109/L. PR: TI + Hg ≥ 8 g/dL, 68 for VSAA
ANC ≥ 0.5 × 109/L, p ≥ 20 × 109/L
Kadia et Prospective, single center rATG + Cys + P + GCSF SAA N = 24 55 CR: ANC ≥ 1 × 109/L, Hgb ≥ 100 g/L, 3 years ORR: Median at 3 months 3 – – 70% at 3 years
al. [64] pt ≥ 100 × 109/L. 64
MDS N = 24 PR: TI + ANC ≥ 0.5 × 109/L, pt ≥ 20 × 109/L,
Hgb ≥ 8 g/dL
Scheinberg Single-center, prospective, Cys + P + (hATG vs. rATG) SAA N = 120 31–37 Hematological response by improvement in 6 months 62% and 33% at 2.3 28% hATG, 11% 21% hATG, 14% hATG: 96% at 3 years
et al. [65] randomized blood counts ORR: 68 3 months for Hatg and rATG rATG rATG: 76% at 3 years
hATG; 37 rATG, respectively
rATG
Marsh et Multicenter, prospective rATG + CyS + P NAA N = 9 36 CR: Hg ≥ 12 g/dL, ANC ≥ 1.5 × 109/L, pt 2 years bRR: 34% at 3 months 1.1 34% at 3 months – 68% at 2 years
al. [66] ≥150 × 109/L 60
SAA N = 20 PR: TI (or) ↑ in 1/3(Hb by 3 g/dL, ANC by
VSAA N = 6 0.5 × 109/L, pt by 20 × 109/L)

Only studies involving ATG (either hATG or rATG) + cyclosporine backbone therapy are included in the table. Cys: cyclosporine; ATG: antithymocyte globulin; P: steroids; NAA/SAA/VSAA: non-severe/severe/very severe aplastic
anemia, respectively; CR: complete remission; PR: partial remission; ORR: overall response rates.

transplant to maintain even in patients above 50 years ago.
Similar to previous study, acute GVHD (aGVHD) and cGVHD
rates were more frequent in peripheral blood transplan-
tees [84].
The risk of graft failure and GVHD remains significant pro-
blem, which can be partially mitigated by appropriate pre-
and posttransplant immunosuppressive regimens. Historically,
graft failure occurred in 11–32% of AA patients who receive
bone marrow transplant. Factors associated with a reduced
risk of graft failure include pretransplant irradiation and use of
cyclosporine prophylaxis against GVHD [85]. Factors favoring
rejection include a prior transfusion history, low number of
marrow cells, and lack of transfused donor buffy coat cells
[86–88]. Unfortunately, addition of buffy coat cells improves
graft failure rates but may worsen GVHD. Shorter disease-
transplant time, increasing the number of marrow cells for
transfusion, and using cyclosporine for post transfusion anti-
GVHD prophylaxis may offer the best chance of decreasing
graft failure rates [10,86,87]. Acute severe GVHD and sympto-
matic cGVHD occur in 11–40% and 21–32% of bone marrow
transplant recipients, respectively, depending of various fac-
tors [85,88]. aGVHD has decreased considerably with the use
of potent posttransplant immunosuppressives, while cGVHD
continues to be a potentially lethal long-term complication.
Studies on anti-GVHD prophylaxis regimens favor the use of
cyclosporine mainly due to a comparatively lower risk of
interstitial pneumonia and cGVHD compared to other agents
[81,89].
Cyclophosphamide (CY) with or without antithymocyte glo-
bulin (ATG) has been used to condition AA patients in pre-
paration for a bone marrow transplant. The addition of ATG to
CY has not been demonstrated to have an additional benefit
in improving transplant outcomes [85]. However, fludarabine
in combination with CY conditioning allows engraftment even
in heavily transfused patients and has minimal treatment
related mortality [1]. Failure of engraftment may not always
be detrimental due to facilitation of autologous marrow recov-
ery by CY conditioning. Furthermore, there is mounting evi-
dence to suggest that the further addition of alemtuzumab to
the fludarabine and CY provides a highly effective condition-
ing regimen associated with significantly lower graft failure
and GVHD rates [90,91]. This becomes especially significant
and merits further evaluation in the era of MUD and haplo-
SCTs.
Matched sibling donor transplant offers the best choice for
long-term reconstitution of hematopoiesis with relatively
favorable transplant-related mortality and GVHD risks when
compared with other donor-type transplants. Treatment with
upfront HSCT is currently recommended in younger (18–
40 years) patients with an available HLA-matched sibling
[92]. Long-term follow-up in this population, across various
studies, have documented 5-year survival rates in excess of
80% [69,70,73,93]. Transplant survivorship is complicated by
long-term side effects including infertility, cataracts, hypothyr-
oidism, secondary solid malignancies among others [94]. The
impressive outcomes in the younger age population, however,
do not apply to older patients, who fare poorly after trans-
plantation [80,95]. A retrospective CIBMTR study assessing the
effect of age on transplant outcomes found mortality risks to
EXPERT REVIEW OF HEMATOLOGY 7
be significantly higher in patients above 40, as compared with
patients aged 20–40 years [96]. Also, investigators determined
the projected 5-year survival rate in this age group to be
similar to that after IST (in responders) [61,96]. Important
reasons identified to negatively affect outcomes included a
higher proportion of older patients with prior use of IST, poor
performance score, increased time between diagnosis and
HSCT, and use of PBSC grafts [96]. The optimal treatment
strategy in older patients awaits a randomized study compar-
ing upfront immunosuppressive and transplantation therapy.
There exists further prognostic heterogeneity within this age
group with age >50 years predictive of inferior survival when
compared with 40–50 years of age [95]. Fludarabine + CYP, as
opposed to conventional ATG + CYP used in younger patients,
offers superior outcomes and hence advocated for older
patients. With improvements in conditioning regimens, appro-
priate patient selection, and supportive care, it is likely the age
eligibility for HSCT will continue to expand.
One of the significant limitations of bone marrow trans-
plant consideration is the availability of HLA-matched sibling
donor. Historically, the risk for transplant-related mortality and
GVHD is almost doubled in patients engrafted from a mis-
matched/unrelated donor as compared with a matched-
related donor transplant [82], which is why BMT from unre-
lated/mismatched donors was traditionally reserved as a sal-
vage option in patients who fail frontline ISTs (Table 2). This
historical notion should be reconsidered in light of emerging
evidence showing an encouraging trend in outcomes over the
years, after unrelated bone marrow transplant, with much
lower graft failure, acute and cGvHD rates, and this relates
primarily to improved donor matching. Dufour et al. reported
on equally favorable outcomes in a pediatric AA patient popu-
lation receiving matched unrelated donor (MUD) transplant as
opposed to matched sibling donor transplant, while also
demonstrating that MUD as initial therapy was superior to
MUD post-IST failure [97]. Appropriate pretransplant condi-
tioning such as fludarabine-based therapy may partly mitigate
the detrimental effect of post-IST failure on MUD out-
comes [91].
Haploidentical (haplo-SCT) transplant offers a viable option
in patients who have failed previous ISTs and lack a suitable
matched donor. Miao et al. reported on outcomes in a series of
39 SAA patients who received haplo-SCT after being condi-
tioned with Busulfan + CY + ATG. GVHD prophylaxis constituted
CsA, MMF, and methotrexate. aGVHD rates were about 9%;
transplant-related mortality occurred in six patients, with overall
survival rate of 83% [98]. A head-to-head comparison between
haplo-HSCT and MRD type showed no significant difference in
failure-free or overall survival rates, although aGVHD and
cGVHD rates were significantly higher in the former [99].
Along these lines, GVHD and graft failure remain legitimate
concerns with mismatched transplants. Non-myeloablative con-
ditioning with posttransplant high-dose CY offers an efficacious
strategy with acceptable graft failure and severe aGVHD or
cGVHD rates [100]. Anti-GVHD prophylaxis may be improved
upon by the addition of high-dose CY in patients at higher risk
of GVHD, such as those with mismatched transplants [100].
Novel strategies involving in-vitro depletion of CD3 T cells
from grafts prior to transplant have demonstrated to have
 8
P. C. BODDU AND T. M. KADIA

Table 2. Bone marrow transplantation in aplastic anemia.

Group/ Median Conditioning
Reference Study population age regimen Post-graft prophylaxis Graft failure/rejection Acute GVHD CGVHD Survival, %
[67] MRD = 471 20 Cy ± TBI/ MTX ± other, Cys ± other, At 5 years, 16% At 100 days, 19% At 5 years, 32% At 5 years, 66%
LFI ± ATG, other MTX + CSA, T-cell depletion,
other
[68] MRD = 71 19 Cy ± others ± TBI Cys ± MTX – At 100 days, 30% At median 4.5 years, 35% At 10 years, 73%
[69] MRD = 64 28 – – 12.5% At 100 days, 31% 18.8% At 6 years, 79%
[70] MRD = 81 24 BU + CY Cys + MTX Primary and At 100 days, 37% At median 8 years, 39% At 8 years, 56%
secondary = 22%
[71] MRD = 79 22 CY + TBI ± ATG Cys + MTX Primary and At 100 days, 7% At median 3.2 years, 35% At 5 years, 74%
secondary = 5.1%
[72] MRD = 113 28 CY + PB + ATG Cys + MTX Primary and secondary at At 100 days, 11% At 1.3 years, 12% At 6 years, 89%
1.3 years, 15%
[73] MRD = 81 26 CY Cys + MTX 11% At 100 days, 24% At median 9.2 years, 26% At 6 years, 88%
[74] MUD = 181, 16 CY ± others, MTX ± Cys other, CsA ± other, Primary and secondary at At 100 days, 48% = MUD, At 5 years, At 5 years,
MMRD = 86, TBI ± irradiation T-depletion ± other 1 year, 15–35% = MMRD, 29% = MUD, 39% = MUD,
MMUD = 51 15% = MUD, 37% = MMUD 19–23% = MMRD23% = MMUD 30–
21–25% = MMRD, 49% = MMRD,
18% = MMUD 36% = MMUD
[75] MUD = 79; 17 CY ± TBI/LFI; CCy/ Cys + MTX, Tac + MTX Primary and At 100 days, 29% At 2 years, 30% At 5 years, 56%
MMUD = 75 ATG ± TBI/LFI secondary = 11%
[76] MUD = 62; 19 TBI + CY ± ATG Cys + MTX Primary and At 100 days, 70% = MUD, At median of 7 years, At 5 years, 61%
MMUD = 25 secondary = 5% 75% = MMUD 52% = MUD, 57% = MMUD
Studies with more than 50 patients included in the table. First seven studies were allogenic sibling transplant, and the bottom 3 studies were mismatched/unrelated donor transplants. MRD: matched-related transplant;
MUD: matched unrelated transplant; MMRD: mismatched-related donor transplant; MMUD: mismatched unrelated donor transplant.

excellent outcomes with lower GVHD rates [101]. Additionally,
tacrolimus/methotrexate combination regimen has been shown
to be more effective in preventing GVHD in those who receive
transplant from unrelated donors [102]. With posttransplant
immunosuppressive strategies improving, this transplant
should be explored more intensively in future clinical trials.
Another alternative for HSCT in the absence of suitable
donor is umbilical cord blood transplantation (UCBT), although
experience is limited. Latour et al. reported on outcomes with
this procedure in 13 marrow failure patients (SAA = 5). In
multivariate analysis, only factor associated with engraftment
and survival was total nucleated cell dose (>3.9 × 107/kg,
P = .007). A Japanese retrospective study comparing alternate
donor transplants demonstrated similar survival of UCBT to be
similar to after MUD, in patients younger than 40 years of age
[103]. While high engraftment failures limit the utility of UCBT,
it certainly offers an alternative in patients without available
UBMT donors.
3.2. Immunosuppressive therapy
Bone marrow transplant and immunosuppressive treatment
differ vastly with respect to treatment-associated side effects
with complications vary depending on the choice of therapy;
graft rejection and GVHD being significant risks after BMT,
while relapse, post-therapy malignancies, and secondary clo-
nal disorders occur more commonly after immunosuppressive
therapy [10,86,88,104]. While the two treatment choices were
historically reported to be comparable in efficacy, improve-
ments in HLA matching, conditioning and posttransplant
GVHD prophylaxis protocols, and supportive care have, over
time, translated to comparatively superior efficacy of frontline
transplant over frontline IST, both in terms of event-free survi-
val and overall survival [68]. An EBMT study comparing out-
comes in AA, adolescent (12–18 years) patients treated with
frontline MRD transplant versus immunosuppressive therapy
demonstrated superior 3-year event-free survival in patients
receiving transplant over IST (83% vs. 37%, P < .001) [10]. The
‘immune’ hypothesis of aplastic anemia received credibility
with Speck et al. demonstrating the value of immunosuppres-
sive therapy when administered alone in AA patients consid-
ered ineligible for transplantation [105]. Study investigators
concluded that horse ATG (hATG) was comparable to hATG
followed by allo-HSCT in terms of hematological response
(40% vs. 42%, respectively) and 1-year survival (58% vs. 54%,
respectively) rates. The therapeutic value of hATG in AA was
further established, by Champlin et al, in a prospective study
randomizing AA patients to either hATG or best supportive
care [106]. Eleven of 21 patients randomized to receive hATG
had sustained improvements in blood counts within 3 months
of treatment while none of the 21 control patients receiving
supportive care alone improved. Since survival was not the
primary end point of this study, most AA patients in the
supportive care arm were allowed to crossover and receive
ATG after the 3-month timepoint. Duration of pancytopenia
prior to treatment correlated inversely with the robustness of
response to IST. It was further observed that actuarial survival
outcomes after responding to hATG were not determined by
the pretreatment severity of AA. Conversely, neutrophil and
EXPERT REVIEW OF HEMATOLOGY 9
platelet counts before treatment with ATG were found to be
highly predictive of survival in another study in SAA [107].
However, most of the patients in this other study had long
duration of aplasia, and only 40% had response to ATG. Data
were not available on the predictive value of cytopenia sever-
ity in ATG responders [107]. These and multiple other studies
recognized the potentially serious side effects concerning ATG
treatment. This gradually led to steroids being combined with
ATG monotherapy, which not only helped abrogate serious
side effects but also were also found to have an additive
beneficial effect in SAA treatment response.
Frickhofen et al. first explored the combination of ATG sera
and cyclosporine, as a part of ongoing intensification efforts,
in a prospective trial of 84 BMT ineligible patients randomized
to receive either hATG + methylprednisolone or
hATG + methylprednisolone + cyclosporine [108]. The latter
regimen proved superior in terms of 3- (39% vs. 65%, P < .03)
and 6-month (46% vs. 70%, P < .05) overall remission (CR + PR)
rates, and there was a trend toward superior survival in the
cyclosporine group. Superiority in the 6-month response (65%
vs. 31%, P < .02) and actuarial survival (at 41 months, 80% vs.
44%, P = .077) rates was most pronounced in the SAA/VSAA
category of patients. The superior efficacy of this combination
was later confirmed in other studies [109]. Similarly, androgens
improved responses when added to ATG and 6-methylpredni-
solone [110].
3.2.1. Growth factor support in AA management
Growth factors have been explored extensively in AA to has-
ten recovery of blood counts, thereby potentially averting AA-
related complications. A 4-drug regimen involving hATG, ster-
oids, cyclosporine, and G-CSF was explored by Bacigalupo
et al., in a cohort of 100 SAA patients [1]. Response rates
were 77% (48 CR, 29 PR) and 5-year actuarial survival prob-
ability was 87%. Severity of baseline neutropenia (less than vs.
greater than 0.2 × 109/L) determined 5-year survival (76% vs.
98%; P = .001). This regimen was tested in a phase III rando-
mized study comparing standard immunosuppressive therapy
with or without growth factor support. The addition of leno-
grastim to the combination resulted in a higher response rate
(83% vs. 44%; P < .0001) and shorter median time to complete
neutrophil response (6 vs. 16 weeks; P < .0001). There was no
difference in survival, response rates, or secondary leukemia
rates at a follow-up of 5 years [111]. This was later confirmed
in another randomized prospective study in which the addi-
tion of G-CSF to hATG, Cys, and prednisolone for the
treatment of SAA shortened hospital stay and decreased infec-
tion rates but had no effect on overall and event-free survi-
val [62].
3.2.2. Eltrombopag
Eltrombopag (E-PAG) is a synthetic thrombopoetin (TPO)-
mimetic that binds to and activates the TPO receptor present
on stem progenitor cells and megakaryocytes. Eltrombopag
was initially studied in refractory setting where it was shown
to be effective in producing trilineage hematopoiesis. Olnes
et al. first conducted a study on 25 refractory SAA patients
with an objective to assess platelet count recovery [112]. Dose
was started at 50 mg daily and titrated based on platelet
10 P. C. BODDU AND T. M. KADIA
response to a maximum of 150 mg daily. Overall response
rates were seen in 44% of the patients, of whom 8 maintained
a response at 10 months. Important clinical responses were
observed across all lineages with the majority of responders
having increased white blood counts, and achieving platelet
and RBC transfusion independence. The study was continued
by Desmond et al., in an expanded cohort of 43 patients [113].
Forty percent of patients responded at a median of
3–4 months. Of the five patients who discontinued therapy
at a median of 28.5 months after achieving robust responses
to eltrombopag, all maintained stable counts at a median of
13 months off the medication. Eight (including six nonrespon-
ders) patients had evidence of clonal evolution (16%), and five
of them had a chromosome 7 abnormality. However, none of
them had clinical evolution to MDS/AML.
Based on its promising activity in the relapse setting, the
drug has been explored in the frontline setting in conjunction
with IST (i.e. ATG + Cys). Kadia et al. conducted a phase II
single-arm study serially enrolling 31 untreated AA patients,
the first 17 receiving IST alone while the next 14 patients were
treated with IST + E-PAG [114]. The latter group had patients
who were comparatively older in age and had had signifi-
cantly lower blood counts. Despite these unfavorable charac-
teristics, there was a trend toward improved overall response
rates in patients receiving IST + E-PAG (79% vs. 59%, P = .28). A
higher WBC count and ANC, and the presence of a PNH clone,
were associated with response and response was associated
with an improved overall survival (12-month OS: 100% vs. 58%
P = .01). The regimen is well tolerated with manageable grade
3/4 toxicities. This was followed closely by another study by
Townsley et al. similarly evaluating the IST + E-PAG in a phase
2 single-center trial [115]. A total of 88 patients, divided into
three cohorts based on differing dosing schema, were enrolled
in this study. Of all evaluable patients, ORRs at 3 and 6 months
were 80% and 85%, respectively. Interestingly, cytogenetic
abnormalities had occurred in seven patients. The investiga-
tors concluded that clonal evolution to MDS occurred at a
similar frequency compared to their historic experience with
standard IST. The role of eltrombopag alone or in conjunction
with other ISTs will be further established in future and
ongoing clinical trials [NCT01328587, NCT01703169,
NCT01891994, NCT01623167, NCT02099747, NCT02773225,
and NCT02404025].
3.2.3 Other ATG combinations
Efforts at improving immunosuppression by combining agents
like mycophenolate mofetil or CY to ATG and cyclosporine
have been abandoned after the combination regimens proved
to be toxic while not producing higher response rates
[116,117]. Although early studies showed equivalent response
rates with either modified high-dose CY plus cyclosporine or
ATG and cyclosporine [118], more recent data suggest the
combination of cyclosporine and CY may prove toxic to justify
this regimen as a viable treatment alternative. In fact, a rando-
mized control trial comparing high-dose CY + Cys with
ATG + Cys was terminated prematurely due to higher infec-
tions and early deaths in the former group [117]. Toxicities are
considerable even with moderate dose CY, and hence, its use
is not currently justified with other less toxic alternatives
available [119].
Frickhofen et al. reported mature data of their initial study
cohort sample in an 11-year follow-up update [60,108]. As was
expected, response rates were higher (70% vs. 41%, with or
without cyclosporine; P = .015), median time to response was
shorter (median, 60 vs. 82 days; P = .019) and the failure-free
survival (39% vs. 24% at 11 years; P = .04) was longer in the
cyclosporine group. However, overall survival and relapse
rates were not statistically different between the groups. In
spite of this, the ATG + Cys + steroid regimen continues to
enjoy favor because of lower time at risk from pancytopenic
complications effects and decreased need for salvage.
Outcomes with ATG + Cys have improved considerably, as
reflected in the survival trends from the EBMT database, over
the past 3 decades (1973–80 vs. 2000–07, 49% vs. 72%, respec-
tively) [120]. This is largely attributed to improved care sup-
portive and better informed second-line salvage treatment.
3.2.4. Rabbit ATG
Unfortunately, 10–20% fail initial treatment, 30–50% even-
tually relapse in the long term, and a smaller percentage
may undergo a disease evolution to other disorders [61,121].
Relapse does not impact overall survival and patients do
respond to a second round albeit at lower rates in the refrac-
tory setting [121]. Responses to second IST, incorporating
either hATG or rabbit ATG (rATG), may vary from 22–64%
after failure to first IST with hATG treatment [107,121].
Among the various ATG preparations, horse ATG has usually
been the first choice due to its larger supply and well-docu-
mented efficacy as first-line therapy. Scheinberg et al. prospec-
tively evaluated outcomes comparing horse ATG and rabbit
ATG in conventional regimens [65]. Outcomes were superior in
the hATG regimen group both in terms in hematologic rates
and overall survival [65]. This was followed by another study,
by Marsh et al., which similarly demonstrated an inferior effi-
cacy of rATG, with the best response rates being 60% and
67%, 2-year overall survival of 68% and 86% (P = .009), and
transplant-free survival of 52% and 76% (P = .002), for rATG
and hATG, respectively [66]. Hence, rATG is generally reserved
for second-line therapy unless hATG is not tolerated or
unavailable.
Rabbit ATG has been extensively studied in the second-
line setting after failure or relapse after first-line ISTs with
hATG. Its utility, in the relapsed/refractory setting after failure
to hATG, has been well documented with responses ranging
from 22% to 77% in various investigational studies [107,121].
Means et al. first distinguished that immunoglobulin from a
different source (rATG in their study) was able to effect
responses in patients who failed initial ATG treatment [122].
The role of rATG as an effective second-line treatment of AA
was further established by Di Bona et al. [123]. Patients with
SAA who had failed frontline hATG, CSA, methylpredniso-
lone, and G-CSF treatment were treated with rATG, CSA,
and G-CSF. Of the 30 evaluable patients, 23 patients (77%)
became transfusion independent, with 9 (30%) and 14 (47%)
patients achieving CR and PR, respectively. Importantly, no
immediate or late reactions were observed. Scheinberg et al.
retrospectively reviewed outcomes in 43 relapsed/refractory

patients who were treated with rATG/CSA; 22 were refractory
and 21 had relapsed after treatment. Overall response rates
were far higher in relapsed patients (66%) than in refractory
patients (27%) in refractory patients. As might be expected,
there was a trend toward improved overall survival in
responders compared with nonresponders (1000 day: 90%
vs. 65%, P = .18) [124]. Given the roughly equivalent
responses to either ATG preparation as second-line therapy,
immunoglobulin from a different animal source may be pre-
ferred to decrease the risk of acute, potentially life-threaten-
ing hypersensitivity reactions. Outcome data with other
preparations of ATG such as porcine ATG, although limited
to a single institutional experience, demonstrate comparable
efficacy to rATG and hATG and may offer suitable cost-effec-
tive alternative in the future [125].
Rabbit-ATG, in conjunction with other immunosuppres-
sants, was also shown to be effective as frontline therapy. Its
role in the frontline setting was first evaluated by Stein et al. in
57 MAA/SAA patients, 49 of whom had no prior treatment
therapy with hATG. Treatment regimen in the study consisted
of r-ATG+prednisone ± cyclosporine ± androgens. Of the 57
patients, responses occurred in 16 (28%), the majority of them
(46%) occurring in patients who received rATG and cyclospor-
ine [126]. Similarly, another study recently updated by Kadia
et al. [64] reported data suggesting rATG to be an effective
alternative to hATG. In the updated study, 14 of 22 evaluable
patients had a response (6 CR, 8 PR) within a median of
3 months. However, comparison studies have consistently
reported hATG/CSA to be superior to rATG/CSA across the
AA severity spectrum, both in terms of response rates and
long-term survival [65,127,128]. Based on the available evi-
dence, rATG is best reserved for second-line therapy after
failure to hATG but may be considered as a first-line option
if hATG is not tolerated or available.
3.2.5. Alemtuzumab
Alemtuzumab is a humanized monoclonal antibody that binds
CD52, an antigen expressed on B- and T-lymphocytes. Antibody
binding triggers a potent lympholytic effect via antibody and
complement mediated cytotoxicity. Single-agent alemtuzumab
has a modest activity and appears to be an inferior option in
the treatment-naïve AA setting [129,130]. However, alemtuzu-
mab + cyclosporine combination has demonstrated activity as a
first-line approach with results from different groups reporting
response rates ranging from 37% to 58% [129,131]. The efficacy
of alemtuzumab monotherapy in the R/R setting is more
encouraging with comparable response rates to
rATG + cyclosporine (33% vs. 37%). Hematologic toxicities
such as severe neutropenia, infusion reactions, and infectious
risks including reactivation of latent cytomegalovirus infection
stress the need for vigilance and preventative antimicrobial
strategies while on therapy [130]. Alemtuzumab is an effective
salvage option in R/R patients not eligible for transplant.
4. Conclusions and future directions
The role of transplant in AA continues to expand with
improvements in supportive care, improved matching of
EXPERT REVIEW OF HEMATOLOGY 11
unrelated transplants, and optimization of pretransplant con-
ditioning/posttransplant GVHD prophylaxis regimens. The elig-
ibility for transplant shouldn’t be solely based on age, and the
role of allogeneic transplant in the frontline setting needs to
be further clarified in the older patients (above 40 years). With
evidence showing improved survival when transplanted at the
earliest in the disease course, the rationale of choosing IST
over unrelated donor transplant as the initial frontline therapy
and the time frame of determining failure to first-line IST
before proceeding to transplant as salvage needs to be clearly
defined. Although the potential risk of growth factor and
immunosuppressive therapies in promoting clonal evolution
has not been substantiated, the risk of its development
remains a pertinent concern especially with a prolonged sur-
vival expected in AA.
The development of novel therapies may prove valuable
especially in patients with severe aplastic anemia who fail ISTs
and are ineligible for transplant. Decades of observational and
investigational study have led to a vastly improved under-
standing of AA pathogenesis and helped inform and develop
novel treatment strategies. As a generation of new therapies
emerges, fascinating insights into underlying disease pro-
cesses may provide exciting opportunities in which to explore
new therapeutic avenues.
5. Expert commentary
Bone marrow failure in aplastic anemia leads to profound
pancytopenia, transfusion dependence, and the clinical seque-
lae resulting from cytopenias. About 15% of patients may
transform to a clonal myeloid malignancy such as MDS or
AML. A more durable treatment of aplastic anemia is achieved
through allogeneic SCT in selected individuals or through
combination immunosuppressive therapy in those who are
not candidates for SCT. Importantly, pronounced deficits in
LTC-IC numbers continue to exist irrespective of treatment
with BMT or SCT, suggestive of persistent suppressive
mechanism with potential implications on disease
relapse [132].
It is through the fortuitous discovery of the response to
immunosuppresion that researchers began to understand
the underlying pathophysiology of idiopathic AA. The
clinic–pathologic manifestations of AA appear to be the
result of an auto-directed, T-lymphocyte-mediated immune
destruction of HSCs. Responses to immunosuppressive ther-
apy with horse ATG in patients with AA were observed in
approximately 30% of cases. Initial attempts to improve
immunosuppression with the combination of horse ATG
and cyclosporine led to durable responses in over two-thirds
of patients and continue to be a mainstay of therapy.
Further attempts at intensifying immunosuppression with
the use of the more potent rabbit ATG or with the addition
of more immunosuppressive drugs have not improved
response rates. In patients who are refractory to frontline
therapy or have relapsed after initial response, different
immunosuppressive therapies such as alemtuzumab and
the small-molecule thrombopoietin-mimetic, eltrombopag,
have demonstrated promising activity. Other studies have
12 P. C. BODDU AND T. M. KADIA
provided more information into a different side of the dis-
ease. Recurrent cytogenetic abnormalities and, more
recently, recurrent somatic mutations point to an underlying
clonal hematopoietic disorder that shares similarities to
other myeloid malignancies such as MDS or AML. Studies
on telomere length reveal ongoing telomere attrition in the
hematopoietic cells from patients with AA, which may result
from replication in the face of immune attack and result in
genomic instability. Several of these abnormalities may carry
prognostic significance, predict for response to immunosup-
pressive therapy, and help select patients who may be
better candidates for early allogeneic SCT.
6. Five-year view
As we look ahead to state-of-the-art management of aplastic
anemia, it is critical to continue collaborative research in this
rare disease and build upon the current research and clinical
experience. Allogeneic SCT has been a curative approach for
selected patients with AA. However, its use in the frontline is
limited to younger patients with matched-related siblings.
With advances in supportive care and reduced intensity con-
ditioning, efforts to broaden the eligible age for SCT should be
explored. Similarly, recent experience using MUD transplants
for newly diagnosed AA may prompt broader use of alterna-
tive donor SCTs in this setting. With the widespread applica-
tion of next-generation sequencing, there is an intense
interest in defining the clonal heterogeneity and clonal evolu-
tion among patients with aplastic anemia. There are already
indications that these recurrent abnormalities have prognostic
significance and predictive value when considering immuno-
suppressive therapy. Going forward, wider validation of these
genetic abnormalities will allow them to be incorporated into
a more informative risk stratification system that does not rely
on clinical factors alone. Deciphering the significance of these
genetic abnormalities may stimulate the development of more
specific therapies among defined subsets. Telomere biology in
congenital and acquired bone marrow failure states has
opened a whole area of basic and clinical research that will
have important implications for diagnostics, prognostication,
and treatment approaches over the next several years.
Integrating these advances and having the appropriate
resources to manage these patients will require expertise,
education, and development of key centers of excellence to
treat this disease.
Key issues
● Attempts at intensification beyond ATG, steroids and
cyclosporine have not been fruitful with the standard ATG,
Cyclosporine, steroid combination strategy still favored as
the current frontline immunosuppressive therapy of choice.
● Expansion of the role of hematopoietic stem cell transplant
in domains of elderly AA and alternative donor transplant
should be addressed in future investigational studies.
● Among the more recently investigated agents, alemtuzu-
mab and eltrombopag have been shown to demonstrate
effectiveness and offer valuable salvage options in HSCT
ineligible relapsed/refractory SAA patients.
● There is evidence to suggest multiple simultaneously and
sequentially operating pathophysiological mechanisms
including immune mediated damage, a role for telomere
attrition, and the less clear role of stromal/adipocyte inhibi-
tion – thus providing opportunities for targeting through
novel strategies, many of which are still in the early infancy
of development.
● Current hematological parameter based risk stratification
criteria, such as the Camitta criteria, lack firm clinical
grounds and are slowly falling out of favour in the modern
era of AA treatment
● Integration of cytogenetic/molecular markers, and more
recent concepts on telomere attrition to blood count data
may help develop more rational prognostic risk stratifica-
tion criteria informing prognosis and choice of treatment in
future protocol studies.
Funding
This manuscript was supported by the University of Texas MD Anderson
Cancer Center Support Grant (P30 CA16672) and award P01 CA049639 (Dr.
Richard Champlin) from the National Cancer Institute (NCI).
Declaration of interest
The authors have no relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial conflict with
the subject matter or materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or royalties.
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