| |

Received: 4 April 2018    Revised: 23 July 2018    Accepted: 24 July 2018

DOI: 10.1111/ejh.13153

REVIEW ARTICLE

Aplastic anemia: Etiology, molecular pathogenesis, and

emerging concepts

Rory M. Shallis1  | Rami Ahmad1 | Amer M. Zeidan1,2

1
Division of Hematology/Medical
Oncology, Department of Medicine, Yale
University School of Medicine, New Haven,
Connecticut
2
Cancer Outcomes, Public Policy, and
Effectiveness Research (COPPER)
Center, Yale University, New Haven,
Connecticut
Correspondence: Amer M. Zeidan, Section
of Hematology, Department of Internal
Medicine, Yale University, 333 Cedar Street,
PO Box 208028, New Haven, CT (amer.
zeidan@yale.edu).
Abstract
Aplastic anemia (AA) is rare disorder of bone marrow failure which if severe and not
appropriately treated is highly fatal. AA is characterized by morphologic marrow fea‐
tures, namely hypocellularity, and resultant peripheral cytopenias. The molecular
pathogenesis of AA is not fully understood, and a uniform process may not be the
culprit across all cases. An antigen‐driven and likely autoimmune dysregulated T‐cell
homeostasis is implicated in the hematopoietic stem cell injury which ultimately
founds the pathologic features of the disease. Defective telomerase function and re‐
pair may also play a role in some cases as evidenced by recurring mutations in related
telomerase complex genes such as TERT and TERC. In addition, recurring mutations in
BCOR/BCORL, PIGA, DNMT3A, and ASXL1 as well as cytogenetic abnormalities,
namely monosomy 7, trisomy 8, and uniparental disomy of the 6p arm seem to be in‐
timately related to AA pathogenesis. The increased incidence of late clonal disease
has also provided clues to accurately describe plausible predispositions to the devel‐
opment of AA. The emergence of newer genomic sequencing and other techniques is
incrementally improving the understanding of the pathogenic mechanisms of AA, the
detection of the disease, and ultimately offers the potential to improve patient out‐
comes. In this comprehensive review, we discuss the current understanding of the
immunobiology, molecular pathogenesis, and future directions of such for AA.

KEYWORDS
aplastic anemia, cytogenetic, molecular, pathogenesis, review

1 |  I NTRO D U C TI O N dedicated treatment is not considered. Multiple etiologies for

Bone marrow failure is a characteristic finding in a number of
inherited syndromes such as dyskeratosis congenita, Fanconi
anemia, Diamond‐Blackfan anemia, and Shwachman‐Diamond
syndrome; however, acquired etiologies of marrow aplasia may
present similarly.1,2 Originally described by Dr. Paul Ehrlich in
1888 and further defined by 1904 by Dr. Anatole Chauffard,
aplastic anemia (AA) is a nonmalignant hematologic disorder
characterized by an injured, markedly hypocellular, and thus inef‐
fective bone marrow. 3 The resultant pancytopenia presents diag‐
nostic challenges and confers an extreme risk of mortality when
AA have been proposed which in due course lead to an immune‐
mediated destruction of hematopoietic stem cells (HSCs) resid‐
ing in the bone marrow, but the bulk of cases remain idiopathic.
Allogeneic stem cell transplantation (alloSCT) offers much of the
limited prospect of cure for patients (who do not respond to im‐
munosuppressive therapy), but unfortunately a significant subset
of patients are not suitable candidates. Immunosuppressive ther‐
apies otherwise continue to be the mainstay for patients with AA
not proceeding to alloSCT, but the increasing understanding of
the pathophysiology of HSC injury is expected to incrementally
broaden therapeutic options.

Eur J Haematol. 2018;1–10. wileyonlinelibrary.com/journal/ejh   © 2018 John Wiley & Sons A/S. |  1
Published by John Wiley & Sons Ltd
 |
2       SHALLIS et al.
2 |  E TI O LO G Y A N D A NTI G E N I C
E X P OS U R E
Aplastic anemia is rare disease with an incidence of approximately
two to three cases per million per year based on dedicated studies,
but may be threefold higher in Asian populations. It is a disease of
the young and typically that of the first three decades of life with
a median age of about 20 years old, though a second peak occurs
around age 60.4-7 Geographic differences in incidence implicate
environmental and host‐related factors and ultimately corroborate
prior study into possible causative factors. Etiologic association with
occupational exposure to chemical haptens such as benzene and
pesticides, specifically the organochlorines and organophosphates
has been demonstrated, although these are exceedingly rare causes
of AA when examining cases worldwide.7-9 In Asian populations spe‐
cifically, exposure to not only similar pesticides, but also animal fer‐
tilizer and animals themselves have been linked to AA.10,11
Medication exposures with subsequent idiosyncratic reactions
are archetypal (yet still rare) causes of AA‐related marrow fail‐
ure. Chloramphenicol is a classic example which is still without a
known, culprit mechanism, although the myelosuppressive effect
is attributed to mitochondrial injury. The etiology of resultant AA
has been attributed to accumulation of drug metabolites toxic to
12,13
HSCs. Ocular chloramphenicol and the antiepileptics carbamaz‐
epine and valproic acid have been implicated as a cause of AA, al‐
though these are likewise exceedingly infrequent.14,15 A handful of
other supposed offending medications are limited to case reports
only.
Aplastic anemia has also been associated with pregnancy, which
interestingly enough was also the purported cause for the devel‐
opment of AA of the case first described by Dr. Paul Ehrlich.3,16,17
Infection with human immunodeficiency virus (HIV) is associated
with the development of AA, although this association is limited to
case reports and small case series which may be a result of a de‐
creasing incidence of HIV infection.18,19 Seronegative hepatitis has
been implicated in approximately 5% of AA cases. 20,21 Association
with eosinophilic fasciitis is limited to less than two dozen cases
reported in the literature, although many patients have concomi‐
tant autoimmune disorder and/or possible environmental exposure
which could otherwise be implicated. 22,23 Clear infectious etiologies
beyond hepatitis and HIV have yet to be established and be con‐
founded by the fact that undiagnosed postinfectious aplasia can be
clinically mistaken for AA.
3 |  TH E PATH O G E N I C I M M U N O B I O LO G Y
The injury to hematopoiesis observed in AA has primarily been
shown to be facilitated by an antigen‐driven immune response,
which may be related to the suspected etiologic factors discussed
previously. The broad and heterogeneous nature of these associated
etiologic factors may call into question whether a single mechanism
is responsible for the resultant profound cytopenias with which AA
manifests. In fact, several pathophysiologic pathways have been
proposed to explain the pathologic nature of the disease, but the
greatest proportion of cases is hypothesized to be a result of uni‐
form T cell‐mediated autoimmunity and marrow destruction lead‐
ing to defective and nearly absent hematopoiesis. Strong laboratory
data suggest that suspected yet unidentified antigenic exposure
leads to a polyclonal expansion of CD4+ T cells dysregulation at the
genetic level and ultimately an overproduction of proinflammatory
cytokines such as interferon (IFN)‐γ as well as tumor necrosis factor
(TNF)‐α. Oligoclonal expansion of a dysregulated cytotoxic CD8+ T‐
cell population has also been demonstrated in ex vivo bone marrow
models of AA patients. 24-27 Increases in T helper 17 (Th17) cells, the
effector cells which produce the proinflammatory cytokine inter‐
leukin‐17 (IL‐17), are also found in the peripheral blood and bone
marrow of AA patients. 28 The degree of Th17 cell elevation in one
study was also found to correlate with the amount of IFN‐γ‐produc‐
ing cells and overall disease activity; Th17 cell populations also nega‐
tively correlated with Treg populations. 28 The concept of aberrant,
disordered T‐cell populations initiating the pathology of AA is also
supported by the finding that that T cell‐directed immunosuppres‐
sive therapy (IST) such as the combination of antithymocyte globulin
and cyclosporine is able to induce response in up to 80% of patients
with severe aplastic anemia. 29
AA patient marrows are also noted to have both marked quan‐
titative and qualitative deficits of regulatory T‐cells (Tregs), which
normally suppress autoreactivity of other T‐cell populations to nor‐
mal tissue including the bone marrow environment and HSCs.30,31
CD8+CD57+ cells, a subset of T‐cells known as effector memory
cells which activate as a result of antigen affinity and stimulation,
are shown to be expanded in the peripheral blood of AA patients,
with a majority demonstrating features of oligoclonality.32 Given the
role of CD8+CD57+ effector memory cells in immune surveillance,
their expansion may precipitate the abnormal oligoclonal expansion
seen in AA. A recent study of Treg population deep phenotyping
using mass cytometric analyses reproducibly defined two Treg sub‐
populations (deemed Treg A and Treg B) with specific phenotypes,
gene expression signature, and function.33 Dominance of a Treg B
subpopulation, defined in part by higher expression of specifically
human leukocyte antigen (HLA)‐DR, FOXP3, CD95, and CCR4, lower
expression of CD45RA, and expression of the interleukin‐2 (IL‐2)/
STAT5 pathway were found in AA patient with response to IST.33
Earlier studies of HLA expression in AA have also supported their
role in provoking the aberrant T‐cell homeostasis based on the find‐
ing of a higher frequency of both HLA‐DR2 and HLA‐DR15 (a split
of HLA‐DR2) presence in AA patients.34,35 The overrepresentation
of these specific HLA molecules in AA patient may effectively aug‐
ment the otherwise physiologic and constitutive expression of HLA
class II molecules on antigen‐presenting cells to interact with CD4+
T‐cells and ultimately present a HSC‐derived antigen resulting in
subsequent HSC immune‐mediated destruction.34 In addition, cells
expressing the HLA‐DR2 antigen (after IFN‐γ priming) have been
previously shown to augment the release of TNF‐α, a known partic‐
ipant in the apparatus of this destruction.35 Together, the increasing
SHALLIS et al.
F I G U R E 1   Immunopathogenic mechanism of hematopoietic stem or progenitor cell apoptosis in the aplastic anemia bone marrow
data on the understanding of Treg and Th17 subsets or subpopula‐
tions in the AA marrow or peripheral blood at diagnosis may soon
solidify a reliable immune signature which predicts response to stan‐
dard IST and as a result guide front‐line treatment decisions.
Despite eventual antigen clearance which under physiologic
circumstances would dictate abatement of the appropriate, proin‐
flammatory immune response, the AA marrow milieu appears to be
one of clonal persistence and continued aberrant immune response
leading to the observed pathology. Increasing understanding of the
culprit apoptotic mechanisms observed in AA has aided the identi‐
fication of the precise population of marrow cells affected by such
T‐cell dysregulation. Fas (first apoptosis signal) receptor (FasR), a
chief element in apoptotic signaling, has specifically been shown
to be overexpressed in CD34+ progenitor cells and lymphocytes in
marrow specimens of AA patients. Such data provide evidence that
the ensuing marrow failure is a consequence of the inflammatory
cytokine‐induced and Fas‐mediated apoptosis as well as that CD34+
progenitor cells appear to be the predominant population whose an‐
nihilation leads to HSC deficiency.36,37 Normal FasR expression in
such cells in patients with AA in remission further supports a Fas‐
38
based mechanism of apoptosis in AA. (Figure 1).
A genetic and molecular basis for why such clonal T‐cell ex‐
pansion occurs and becomes pathogenic is being progressively
|
      3
illuminated. Increased incidence of proinflammatory cytokine poly‐
morphisms, specifically in IFN‐γ, TNF‐α, and interleukin‐6, is repeat‐
edly found in cases of AA. HSC FasR expression is likely upregulated
by this cytokine overproduction. Hypersecreting genotypes of the
multifunctional and regulatory transforming growth factor beta 1
(TGFβ1) have also been demonstrated and taken together this may
suggest a quicker and more robust immune response to antigenic
exposure and myelosuppression.39 MicroRNAs (miRNAs) including
miR‐532‐5p, MiR‐4651, and miR‐126‐5p, which modulate down‐
stream proinflammatory pathway components such as Toll‐like re‐
ceptors and TNF‐α, are uniquely and commonly identified in AA.40,41
miR‐126‐5p is also found to be decreased in patients with AA re‐
sponding to IST, suggesting miRNA interference of cytokine and
growth factor‐related proinflammatory and immune signaling.41
4 | TE LO M E R E H O M EOS TA S I S
PATH O LO G Y
Inherent HSC biological flaws are also suspected to play a role in the
pathogenesis of AA. Defective homeostasis of telomeres (the repeti‐
tive sequences of DNA which cap linear chromosomes and facilitate
cell proliferation) has also been implicated in a significant subset
 |
4       SHALLIS et al.
of AA patients and is an emerging category of disease in general.
Telomere shortening (occurring if suppressor or other protective
genes are inactivated or by physiologic attrition) leads to cell pro‐
liferative arrest and eventual apoptosis.42 To combat the attrition,
germ line cells utilize telomerase reverse transcriptase (TERT), telom‐
erase RNA component (TERC), and the stabilizing protein dyskerin
(DKC1) to assemble the telomerase complex and maintain telomere
length, which is naturally and expectantly lengthy in HSCs.42 Human
lymphocytes are the primary somatic cells which can upregulate
TERT with resultant telomerase activity and provide a transient pro‐
liferative advantage, although this does not avert eventual replica‐
tive senescence.43,44
Approximately 35% of AA patients are found to have periph‐
eral granulocyte and mononuclear cell telomere length shortening,
pointing toward a progenitor cell defect.45,46 Critical loss of HSC
telomere length in about 9% of acquired AA cases has been associ‐
ated with mutations in TERC, TERT, and DKC1 which are known to be
the cause of inherited or constitutional aplastic anemia such as dys‐
keratosis congenita.47,48 Rare mutations in other telomerase genes
such as TERF1 and TERF2 have been identified as possible driver mu‐
tations in AA.49 Biallelic mutations in regulator of telomere elonga‐
tion helicase 1 (RTEL1), which encodes a protein critical to telomere
homeostasis, is a known cause of congenital bone marrow failure,
but has been identified in 1%‐2% of AA cases; most RTEL1 variants,
however, are felt not to be pathogenic in the latter condition.50
The degree of telomere length shortening or erosion has been
shown to correlate with the severity of AA, risk of relapse, overall sur‐
vival, and risk of clonal evolution (via acquisition of new cytogenetic
aberrancy) to conditions such as myelodysplastic syndrome (MDS),
51
which will be discussed in more detail later. Telomere length short‐
ening in cases of AA is likely insufficient to lead to AA and ultimately
calls into question the distinction between acquired and inherited
bone marrow failure syndromes such as AA given it frequently oc‐
curs in adult patients without a clear family history of such.
Sex hormones have been shown to exert significant effects on
telomere homeostasis. Previous in vitro evidence has shown that
HSC exposure to several types of androgens induced telomerase
activity and this correlated with higher levels of TERT mRNA.52 This
effect was also seen in peripheral blood lymphocytes heterozygous
for loss‐of‐function TERT mutations which at baseline were associ‐
ated with reduced telomerase, but with the addition of androgen
52
were restored to normal. Subsequent murine models corroborated
this observation of androgen‐induced telomere lengthening with
eventual improved hematopoiesis.53 This was ultimately confirmed
in human studies using danazol, a strong androgen receptor agonist
which increased telomerase activity and correlated with upregu‐
lation of TERT.54 However, a recent retrospective study found no
difference in the telomere attrition rates of androgen‐treated or un‐
treated patients with dyskeratosis congenita irrespective of the type
of androgen therapy used or leukocyte subset evaluated; such data
imply a mechanism of clinical response other than androgen‐related
effects on telomere homeostasis.55 It remains to be seen whether
or not abrogation of telomere length shortening seen in AA (and the
foreseeable chromosomal instability) with androgen therapy averts
the molecular pathogenesis of clonal evolution.
5 | TH E M YS TE R I O U S RO LE O F
TH RO M B O P O I E TI N
Increasing data support the role of thrombopoietin (TPO) and TPO
signaling in hematopoiesis specifically related to HSC homeostasis,
proliferation, and survival. Knockout murine models lacking the c‐
mpl gene (which encodes the TPO receptor which itself is also ex‐
pressed on HSCs) show significant deficiencies in HSCs and thus
suggest that TPO is integral to HSC homeostasis.56,57 Higher levels
of plasma TPO are observed in AA patients at diagnosis suggesting
a physiologic response to the diminished hematopoiesis and throm‐
bopoiesis. In addition, TPO levels decrease after response to IST.58
IFN‐γ, the principal proinflammatory cytokine responsible for the
HSC destruction in AA, is shown to decrease the interaction of TPO
and c‐MPL via direct TPO‐IFN‐γ heterodimerization and likely rep‐
resent the key mechanism by which inflammatory cytokines directly
disrupt hematopoiesis in AA.59
Given the role of TPO in normal hematopoiesis and the re‐
cently enlightened TPO‐related mechanism of pathology in AA, the
use of TPO mimetics is garnering an increasing clinical role in AA.
Eltrombopag, a nonpeptide TPO mimetic and c‐MPL agonist, has
previously shown response of 44% (including trilineage responses)
in patients with AA refractory to IST.60,61 Subsequent investigations
noted to that eltrombopag interaction with c‐MPL is not affected
by direct TPO‐IFN‐γ heterodimerization and eventually abrogates
the myelopoietic failure induced by such.59 Recently, eltrombopag
was combined with standard IST at varying doses and schedules in
newly‐diagnosed AA patients; induction of trilineage hematopoiesis
as well as overall response and complete response rates of up to
94% and 58%, respectively, was observed.62 Similarly, the TPO mi‐
metic romiplostim has been shown to stimulate primitive HSC, aug‐
ment their differentiation into later progenitor cells, and ultimately
demonstrate efficacy in patients with AA refractory to IST.63-65
Despite prior theoretical concerns, romiplostim therapy in AA has
not been shown to increase the subsequent development of cytoge‐
netic abnormalities or clonal evolution.64,65
TPO agonism likely promotes proliferation of the remaining,
though distinctly reduced, HSC population. The HSC compartment
has been shown to be markedly reduced (in the order of approx‐
imately 10% of normal) even in patients who have responded to
IST with appropriate count recovery and more robust responses to
IST have been observed in patients with less severe AA (and likely
a larger and functional HSC compartment).66,67 These findings sug‐
gest that a certain threshold for count recovery is required and is
hypothesized to be the result of a possible protective mechanism of
HSC to prevent total exhaustion of the HSC pool, but may also be
the result of recurrently‐observed telomere length shortening in AA
patients.60,66 TPO mimetics do not appear to abolish the pathogenic
autoimmunity of AA, and TPO biology to date has not been shown
SHALLIS et al.
to have any direct effect on cytotoxic or regulatory T‐cell expansion
or perturbation. However, this impressive clinical activity might ad‐
vocate for the incorporation of TPO mimetics such as eltrombopag
into front‐line therapies for AA.
6 | C Y TO G E N E TI C A B N O R M A LITI E S A N D
TH E I M M U N E M EC H A N I S M
Cytogenetic aberrations commonly‐observed in other myeloid malig‐
nancies are recurrent in AA such as monosomy 7 and trisomy 8, oc‐
curring in up to 13% and 7% of AA cases, respectively.68,69 Data from
Sloand et  al70 have previously shown that bone marrow specimens
of patients with MDS (13% of which had progressed from AA) and
evidence of trisomy 8 clonal disease showed a marked expansion of
CD8+ T‐cell populations. Specific T‐cell receptor subfamilies, namely
Vβ, demonstrated significant suppression of trisomy 8 myeloid cells.70
The same group also showed that trisomy 8 marrow HSCs are able to
escape destruction by CD8+ T‐cells via overexpression of prosurvival
proteins cyclin D1 and survivin. Nontrisomy 8 HSCs are unable to
mount this protection and are preferentially suppressed. No data to
date support a similar mechanism for monosomy 7 AA, but a study
by Dimitriu et  al71 demonstrated that progressive telomere loss is
noted before the detection of and thus may lead to eventual mono‐
somy 7 in patient who develop MDS progressed from AA. These data
further support the relationship between cytogenetic predisposition
and the downstream immunobiologic HSC destruction. Less com‐
monly detected cytogenetic alterations in AA include del(13q), tri‐
somy 6, and trisomy 15 which occur in <5% of cases.72,73 The clinical
relevance of cytogenetic abnormalities in AA is being gradually clari‐
fied. The identification of monosomy 7 in AA patients is associated
with an increased risk of progression to MDS or AML, while trisomy
8 and del(13q) have been associated with more favorable response
to IST and improved clinical outcome, specifically improved progres‐
sion‐free survival (PFS) and overall survival (OS).72-76
TA B L E 1   Recurring cytogenetic
Abnormality
abnormalities in acquired aplastic anemia
6pUPD
Monosomy 7/del(7q)
Trisomy 8
Del(13q)
Trisomy 6
Trisomy 15
6pUPD, uniparental disomy of the 6p arm; AML, acute myeloid leukemia; IST, immunosuppressive
therapy; MDS, myelodysplastic syndrome.
a
Referenced studies had variable analyses either performed at diagnosis of aplastic anemia or later
in course of disease (without progression to MDS) as well as differences in analyses of peripheral
blood vs bone marrow specimens.
|
      5
Karyotyping a hypocellular bone marrow specimen, however,
can be problematic and underestimate an accurate percentage of
cytogenic aberrancy in AA. The development of higher fidelity SNP
array karyotyping has revealed more subtle abnormalities below the
level of detection with conventional karyotyping and has elucidated
other means of CH in AA. Because of SNP array profiling, unipa‐
rental disomy (UPD) in 6p (6pUPD), which escaped conventional
karyotyping, can now be identified in up to 13% of AA patients.72,77
The most commonly involved region by acquired CN‐LOH in AA is
the short arm of chromosome 6 at the site of the MHC locus, oc‐
curring in about half of AA patients with acquired 6pUPD (Table 1).
6pUPD may develop via the immune‐escape of such HLA‐deficient
HSCs.78 In a recent study combining targeted deep sequencing of
class I HLA and SNP array in AA patients, 17% (11/66) of patients
were found to have somatic HLA loss and those who inherited the
targeted HLA alleles, regardless of the mutation status, had a more
severe disease course with more frequent clonal complications, and
increased frequency of developing secondary MDS.79 Given these
findings, AA patients with the acquisition of HSC HLA deficiencies
such as 6pUPD or other somatic mutations may have class I HLA
loss‐of‐function which could allow immune‐escape from cytotoxic T‐
cell‐mediated destruction, as opposed to the typical HLA‐facilitated
presentation of previously mentioned, plausible AA‐related antigens
to CD8+ T‐cells. Such findings also strengthen the assertion that
understanding the pathogenesis of clonal evolution in AA may help
explicate its autoimmune nature.
7 | S O M ATI C M U TATI O N S
The advent of next‐generation sequencing (NGS) has allowed the
identification of somatic mutations as a more dominant cause of
clonality in AA.72,78 PIGA, a gene essential for the synthesis of sur‐
face glycosylphosphatidylinositol (GPI)‐linked anchoring proteins, is
detected in 60% of patients with evidence of a paroxysmal nocturnal
Incidencea, % Prognostic impact Reference(s)
75,85
13 Favorable response to IST
68,73,92
2.0‐13.3 Higher risk of progression
to MDS or AML
68,71,92,96
1.3‐6.7 Favorable response to IST,
lower risk of progression
to MDS or AML
75,85
0.4‐1.8 Favorable response to IST,
possibly better survival
71
2.4 Unknown
71
2.4 Unknown
 |
6       SHALLIS et al.

TA B L E 2   Frequently mutated genes in

Mutated gene Incidence, % Prognostic impact Reference(s)
acquired aplastic anemia
68,85
BCOR or BCORL 4.0‐9.3 Favorable response to IST, improved
PFS and OS
68,85
DNMT3A 5.3‐8.4 Poor response to IST, inferior PFS
and OS
68,85
ASXL1 6.2‐8.0 Poor response to IST, inferior PFS
and OS
85
PIGA 7.5‐40 Favorable response to IST, improved
PFS and OS
46
TERT 5.6 Unknown
47
TERC 3.3 Unknown
85
JAK2 and JAK3 1.8 Poor response to IST, inferior PFS
85
RUNX1 1.5 Poor response to IST, inferior PFS
and OS
85
TP53 1.5 Poor response to IST, inferior OS
85
CSMD1 1.0 Poor response to IST, inferior OS
68,85
TET2 0.6−0.7 Unknown
68
SRSF2 0.6 Unknown
68
U2AF1 0.6 Unknown
68
ERBB2 0.6 Unknown
68
MPL 0.6 Unknown
85
TERT 0.5 Unknown
48
TERF1 or TERF2 N/A Unknown

IST, immunosuppressive therapy; OS, overall survival; PFS, progression‐free survival.

hemoglobinuria (PNH) clone.80-82 Between 7.5% and 40% of AA pa‐
tients are found to have mutations in PIGA, but PNH clones (though
typically small) are found in up to 68% of patients at time of AA di‐
agnosis and up to 19% will eventually develop overt PNH, another
clonal bone marrow failure disorder.72,78,83,84 GPI‐deficient HSCs
may in fact have some protective role in escape from the T‐cell‐me‐
diated destruction which otherwise induces apoptosis of normal
HSCs in the stem cell compartment. HSCs with these mutations may
have survival advantages by being less immunogenic as evidenced
by more frequent PIGA mutations and may have a higher chance for
recruitment especially when the pool of HSCs is depleted.85-87
Other than PIGA mutations, the most frequently detected somatic
mutations in AA are in that in BCOR, BCORL1, and the MDS‐related
somatic mutations in DNMT3A and ASXL1, which in one study cumu‐
latively accounted for 77% of all somatic mutation‐positive patients.
About a third of AA patients have multiple, independent mutations,
including that of the same gene, which supports a stout mechanism
by which such clones are selected for expansion and evolution.72,87
Median variant allelic frequency (VAF) seems to range from 9% to
20% based on two strong studies detecting such, and three‐quarters
of all detected somatic mutations have a VAF of <10%.69,72 Although
less common, recurring mutations in JAK2/JAK3, RUNX1, TP53,
CSMD1, and TET2 have been recognized.72 Rare (in <3% of acquired
AA cases collectively) mutations in SRSF2, U2AF1, ERBB2, and MPL
were identified in one of the more robust studies using NGS and
array‐based karyotyping of AA patients to date (Table 2).72 Yoshizato
et  al72 also demonstrated a dominance of cytosine to thymine tran‐
sition at CpG dinucleotides and a predilection for nonsense, frame‐
shift, and splice‐site alterations. Interestingly, the simple presence
of an acquired somatic mutation (other than that of telomere main‐
tenance genes such as TERT and TERC) has been associated with
shorter telomere length by a mechanism hypothesized to be the re‐
sult of hematopoietic stress.69
As with cytogenic abnormalities, outcomes in AA patients seem
to be influenced by the presence of specific somatic mutations.
Mutations in PIGA, BCOR, and BCORL1 are associated with better
response to IST as well as improved PFS and OS, while AA patients
with mutated DNMT3A, ASXL1, JAK2/JAK3, or RUNX1 fare worse
with regard to IST response and survival.69,72,88 AA patients with
mutations in DNMT3A or ASXL1 are shown to exhibit a 40% chance
of developing MDS, which is far more significant than the observed
10% incidence of such in AA patients in general.69,72,89
Primary or founder clones in AA appear to be consistently small
at diagnosis, but clone sizes have been shown to be dynamic and
fluctuate without evidence of progression to MDS or AML or even
worsened phenotypic disease. In some cases, initially detectable
mutations (specifically those clones with BCOR/BCORL or PIGA mu‐
tations) can even subsequently evade measurement during the dis‐
ease course.72 The acquisition of new secondary or subclones (which
can be a product of the original or dominant HSC clone) has also
shown to be variable, ranging from no appreciable clinical impact to
(in the case of clones with mutated ASXL1 or DNMT3A) a substantial
SHALLIS et al.
increase in clone size and even an occult metric preceding progres‐
sion to malignant myelopoiesis. ASXL1 and DNMT3A and to a lesser
degree RUNX1 and U2AF1 clone sizes tend to increase through the
course of disease.72 Because of a lack of consistent predictability
and definitive understanding of clonal selection, most AA experts
recommend not basing therapeutic decision‐making on the presence
or size of clonal disease, although specific somatic mutations may
raise suspicion.90
8 | E V I D E N C E I N C LO N A L E VO LU TI O N
A N D QU E S TI O N S FRO M C LO N A L
H E M ATO P O I E S I S
Although AA is considered to be a nonmalignant disease with poten‐
tial cure using IST or alloSCT, it can be complicated by the develop‐
ment of late clonal disease. This manifestation may provide insight
as to further molecular or other biological pathogenic mechanisms
of AA development. AA patients have a higher incidence of AML
and MDS, namely 7% and 10%, respectively, at 11‐year follow‐up
after receiving IST.91 The increased prevalence of late clonal compli‐
cations like MDS and paroxysmal nocturnal hemoglobinuria (PNH) in
AA suggests a link between clonal hematopoiesis (CH) and AA.85 CH
(characterized by either a karyotypic abnormality or somatic muta‐
tion) can be detected in approximately a quarter of adult AA cases
(and up to 70% in cohorts including children).72,78 Interestingly, T‐
cell‐directed IST itself has been associated with an increased risk of
clonal evolution.80
Clonal disease is detected at time of AA diagnosis in a sub‐
stantial proportion of patients without morphologic evidence of
MDS.72,73,78,92 A more challenging task remains the distinction be‐
tween AA and an uncommon form of MDS known as hypoplastic
MDS (hMDS). hMDS comprises approximately 15% of MDS cases
and is characterized by bone marrow cellularity <30% in patients
aged <70 years or <20% cellularity in those older than age 70 years.93
The impossibility of morphologically distinguishing AA from hMDS
has led many experts to identify this phenomenon as an overlap syn‐
drome. A retrospective study of hMDS patients revealed that 62%
of patients had normal cytogenetics and only 21% had mutations
in either ASXL1, DNMT3A, or BCOR; the overwhelming majority of
hMDS patients with a somatic mutation had a prior history of AA.93
Because of these inconsistencies, the presence of CH does not aid
in the distinction between AA and hMDS. In fact, the identification
of several cytogenetic aberrations (for instance, monosomy 7, which
is identified in up to 13% of AA cases at diagnosis) by conventional
karyotyping is diagnostic of MDS even in the absence of morphologic
dysplasia as per the World Health Organization (WHO) classification
of myeloid neoplasms.68,94,95 The risk of CH (as well as aneuploidy or
copy‐neutral loss of heterozygosity) increases with age alone with up
to 10% of patients aged 70 years or old as per evidence reanalyzing
single nucleotide polymorphism (SNP) array data generated for indi‐
viduals with no history of hematological cancer.96,97 Such CH (quan‐
tified by least two metaphases or VAF of at least 2% for karyotypic
|
      7
abnormalities or somatic mutations, respectively) without dysplasia
or a cytopenia is defined as clonal hematopoiesis of indeterminate
potential (CHIP) and such an entity raises more questions than con‐
fidence in a pathogenic diagnosis.86,87 In addition, acquired genetic
mutations (including those recurring in myeloid malignancy such as
MDS [ASXL1, DNMT3A, etc]) may arise out the hematopoietic stress
induced by AA and become a substrate for clonal selection.
Recent efforts have led to promising metrics to distinguish AA
from MDS irrespective of CH. Levels of circulating S100A8, a Toll‐
like receptor ligand which exerts its proinflammatory effect via
tumor necrosis factor receptor‐associated factor (TRAF) and nuclear
factor‐κB (NF‐κB)‐related transcription of TNF‐α and IL‐1b, were
shown in a recent study to be elevated in patients with MDS, but not
those with AA (or healthy controls).98 Such a distinction may provide
a sensitive diagnostic tool to differentiate the two entities whose
treatment varies in most circumstances. The presence of CH (even
in cases with a large VAF or somatic mutation associated with patho‐
genic significance in MDS) in a AA patient does not alter therapeutic
decision‐making in current practice (such as for whom IST would be
preferred), but this may in fact change with improved understanding
of AA molecular pathogenesis.
9 | FU T U R E D I R EC TI O N S
There has been noticeable progress in dissecting the underlying and
complex pathophysiology of AA over the past years. This has been
in no small part due to the advancement of deep gene‐sequencing
techniques and SNP arrays for detecting genetic abnormalities along
with the application of multicolor flow cytometry and fluorescently
conjugated monoclonal antibodies for the assessment of protein ex‐
pression and exploring distinct subpopulation of T‐cells. Single cell
RNA (scRNA) sequencing, which is a relatively new technique, allows
direct analysis of the transcriptomes of individual cells, comparison
to a larger number of heterogeneous cells and thus will provide fur‐
ther characteristics of gene expression.99 Such a method may allow
new insight into AA pathophysiology, specifically the underlying
recurring mutations and genetic instability as it related to clonal
evolution. Ultimately this technique can potentially yield innova‐
tive experimental approaches in the future to explore gene‐targeted
therapy.100
One of the more significant challenges to the study of AA mo‐
lecular pathophysiology has been the paucity of progenitor cell
attainment to perform various biotechniques. The use of induced
pluripotent stem cell (iPSC), which can be derived into hematopoi‐
etic cell lineages, has recently provided some promise in overcom‐
ing this barrier and represents a new perspective in the study of
AA biology and the pathological genotype. Combining the use of
iPSC with the rapid improvement of RNA splicing and gene edit‐
ing techniques such as CRISPER/Cas9 may help with the induction
of mutations and discover genetic or epigenetic events related to
previously identified phenomena such as telomere shortening.
This would ultimately provide a suitable animal model to study AA
 |
8       SHALLIS et al.
in vitro as well as a therapeutic window by means of correcting
genetic mutations via gene editing. 101,102
Eventually, the above
should produce a more precise ability to distinguish founder mu‐
tations among other acquired subcloncal populations and those
that arise with age. Such may afford the ability to predict clini‐
cal outcomes of AA patients and thus allow a more appropriate
algorithm for therapeutic decisions including allogeneic stem cell
transplantation.
10 | CO N C LU S I O N
The pathogenesis of AA is directly related to the destruction of he‐
matopoietic stem cells. A myriad of environmental insults may drive
the antigen‐driven and autoimmune means of this destruction. Strong
data support the hypothesis that a disturbance of T‐cell homeostasis
is the link between these two assertions. Defective telomerase func‐
tion, acquired cytogenetic aberrations, and recurring somatic muta‐
tions have been implicated as playing a significant role in aiding the
autoimmunity leading the acquired bone marrow failure of AA. Our
current understanding of the biological and molecular mechanisms
for the development of AA has been deepened by the advent SNP
arrays and NGS, which permitted the discovery of offending genomic
abnormalities. Future development of newer technologies and tech‐
niques may soon reveal a definitive genetic origin of the tolerance
for the inappropriate and persistent immune response seen in AA.
C O N FL I C T O F I N T E R E S T
All authors report no relevant disclosures.
AU T H O R C O N T R I B U T I O N S
All authors wrote and approved the final manuscript.
ORCID
Rory M. Shallis  http://orcid.org/0000-0002-8542-2944
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How to cite this article: Shallis RM, Ahmad R, Zeidan AM.
Aplastic anemia: Etiology, molecular pathogenesis, and
emerging concepts. Eur J Haematol. 2018;00:1–10. https://
doi.org/10.1111/ejh.13153