review
Advances in understanding the pathogenesis of acquired
aplastic anaemia
Lucio Luzzatto1 and Antonio M. Risitano2
1
Muhimbili University of Health and Allied Sciences, Dar-es-Salaam, Tanzania, and 2Department of Clinical Medicine and Surgery,
Federico II University, Naples, Italy
Summary
This review examines the evidence that bone marrow failure
(BMF) in aplastic anaemia (AA) is due to loss of
haematopoietic stem cells (HSCs), which, in turn, is caused
by deranged immunity and inflammation. We also consider
how the course of the disease and the response to immuno-
suppressive therapy are influenced by the nature and speci-
ficity of the pathogenic process. A somatic mutation of the
PIGA gene underlies the clonal disease paroxysmal nocturnal
haemoglobinuria (PNH): there is direct evidence that the
expansion of the PIGA mutant clone results from Darwinian
selection exerted by a glycosyl-phosphatidyl-inositol -specific
auto-immune attack. Thus, PNH patients are a unique subset
of patients with AA, in whom haematopoiesis recovers
through this escape mechanism. A similar process, although
less effective, may operate when the auto-immune attack is
against a human leucocyte antigen (HLA) molecule and an
HLA mutation has produced a clone missing that molecule.
We then discuss the significance of other mutant clones that
are frequently found in AA, presumably due to a combina-
tion of genetic drift and selection. These clones are not cau-
sative of AA, but they emerge in AA and they may be pre-
leukaemic: unlike a PIGA mutant clone, in general they are
unable to effectively reconstitute haematopoiesis.
Keywords: aplastic anaemia, PNH, MDS, clonality, clonal
expansion, clonal evolution.
Introduction
Aplastic anaemia is a rare disease and a serious disease. Paul
Ehrlich is credited with first reporting a patient with this
condition in 1888; and Vaquez and Aubertin were probably
first in coining the word aplasique when describing the bone
Correspondence: Lucio Luzzatto, Professor of Haematology,
Department of Haematology and Blood Transfusion, Muhimbili
University of Health and Allied Sciences, P.O.Box 65001,
Dar-es-Salaam, Tanzania
E-mails: lluzzatto@blood.ac.tz; lucio.luzzatto@unifi.it
First published online 5 July 2018
doi: 10.1111/bjh.15443
marrow of another patient (Marmont, 1995), although the
title of their article, published in 1904, used the term perni-
cious anaemia. In what is perhaps the first major haematol-
ogy textbook, Le Emopatie (Ferrata, 1934), it is instead stated
emphatically – and of course correctly – that AA should not
be confused with pernicious anaemia: today this terminology
seems strangely twisted, because pernicious anaemia is emi-
nently curable, whereas AA is still a pernicious threat. From
the point of view of haematopoietic function, the regenera-
tion of effete blood cells is woefully inadequate in AA: hence,
the term bone marrow failure (BMF) is often used in prac-
tice as synonymous with AA.
AA can be inherited or acquired, and there are three
major groups of inherited AA (Wegman-Ostrosky & Savage,
2017); Table I.
Among several possible causes of acquired AA, the one for
which there is most definitive evidence is a heavy dose of
radiation. Indeed, an industrial accident near Belgrade (Ser-
bia, at that time Yugoslavia) caused severe BMF in at least 6
men who worked at a nuclear facility (Champlin, 1989). This
accident was an urgent stimulus to treating BMF by bone
marrow transplantation [BMT: (Mathe et al, 1958); in the
event, the procedure was unsuccessful because the crucial
histocompatibility requirements were not yet known]. Not
surprisingly, cytotoxic agents, such as cyclophosphamide,
busulphan or fludarabine, also cause dose-dependent BMF,
and are indeed used deliberately for the purpose of bone
marrow ablation as part of ‘conditioning’ prior to BMT. In
these situations, bone marrow aplasia is clearly secondary to a
physical or chemical myelo-toxic insult, and whether we
should call it AA is a matter of semantics; but the majority
of patients diagnosed with AA have not received heavy radia-
tion or cytotoxic drugs. Idiosyncrasy to non-cytotoxic drugs,
e.g. chloramphenicol, is often quoted as a possible cause of
AA (Young, 1994); whereas among infectious causes the best
known association is with viral hepatitis (Brown et al, 1997):
however, the majority of the millions of subjects who have
suffered hepatitis do not go on to develop AA. In the major-
ity of cases of acquired AA (or primary BMF) we are unable
to identify a specific cause, and therefore it is termed idio-
pathic. For the sake of brevity in this paper, unless otherwise
indicated, AA means idiopathic acquired AA.
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British Journal of Haematology, 2018, 182, 758–776
 Review

Schwachman-Diamond syndrome

AA, aplastic anaemia; GPI, glycosyl-phosphatidyl-inositol; HSCs, haematopoietic stem cells; IFNG, interferon-c; MDS, myelodysplastic syndrome; PNH, paroxysmal nocturnal haemoglobinuria; TNF,
General concepts

PNH, AA/PNH. Idiopathic AA

Pancytopenia in the peripheral blood before aplasia is often

Dyskeratosis congenita
the first finding in patients with AA; and it is, in first

MDS in the elderly?

approximation, an indictment of the haematopoietic stem

Post-radiation AA
Post-hepatitis AA
Fanconi anaemia
Disease example

cell (HSC).

Idiopatic AA
Idiopatic AA
When allogeneic BMT was introduced as a rational treat-
ment for patients with BMF – whether primary or secondary

CHIP?*
– it was observed that sometimes the transplanted bone mar-
row was rejected, but later the patient recovered (Thomas
et al, 1972; Mathe & Schwarzenberg, 1976). This remarkable
phenomenon, autologous reconstitution or regeneration of
Defective ribosome assembly/function (ribosomoapthy)

haematopoiesis [which often entails a good prognosis, (Pic-

Other target-specific auto-immune attack on HSCs
HLA-B4002-specific auto-immune attack on HSCs

cin et al, 2010)], was promptly attributed to the ‘condition-

ing’ regimen that, aiming to prevent rejection, was strongly
Abnormal telomere attrition (telomeropathy)

GPI-specific auto immune attack on HSCs

immune-suppressive (Speck et al, 1976a, 1978). This led to

the introduction of intensive immuno-suppressive treatment
(IST),including anti-lymphocyte globulin (Speck et al, 1976b,
Loss of stem cells/loss of stemness
Damage mediated by IFNG, TNF

1978, 1981; Gluckman et al, 1982; Champlin et al, 1983),

methylprednisolone(Doney et al, 1992) and cyclosporine
Direct damage to HSCs

(Frickhofen et al, 1991, 2003; Rosenfeld et al, 1995), that are

Table I. Various modalities that can result in depletion of haematopoietic stem cells and eventually in bone marrow failure.

Impaired DNA repair

still in use today. Given that IST produces remission and,

Main mechanism

often, cure of AA in over 70% of cases (Rosenfeld et al,

2003; Teramura et al, 2007; Scheinberg et al, 2011; Tichelli
*CHIP: clonal haematopoiesis of indeterminate potential, which at the moment must not be considered a disease.

et al, 2011), we can legitimately infer that, at least in a large

majority of cases, the primary mechanism of BMF in AA is
an auto-immune process. With this in mind, we must review
three steps in pathogenesis: (i) whether and in which way
stem cells are damaged; (ii) the effectors of damage; (iii) the
Mutations in genes of the Fanconi complex

source of the damage [see also (Risitano et al, 2007)]. But

first, we briefly outline the relationship between AA and
Mutation in DKC1, TERT or TERC†

paroxysmal nocturnal haemoglobinuria (PNH), because it

Many previous cell division cycles

provides a key to understanding the pathophysiology of AA.

Severe inflammatory process

AA and PNH
Main causative factor

†Less frequent genes causing dyskeratosis congenita are not listed here.
Auto reactive T cells
Mutations in SDBS

If acquired AA is rare, PNH is even more rare and, being an

acquired haemolytic anaemia, it features in a different chap-
ter from AA in textbooks. Nevertheless, AA and PNH over-
Radiation

lap in many patients and, given the rarity of both diseases,

this cannot be a coincidence (Dameshek, 1967). PNH is
defined by the presence of a large population of blood cells
that, as a result of an inactivating somatic mutation in the
X-linked gene PIGA, are deficient in all proteins that are nor-
Inherited bone marrow failure syndromes

mally tethered to the cell membrane through a glycosyl-phos-

phatidyl-inositol (GPI) molecule: because these include the
Acquired Aplastic Anaemia (AA)

complement regulatory surface proteins CD59 and CD55,

GPI-deficient red cells are exquisitely sensitive to lysis by
activated complement (Luzzatto & Araten, 2006). Clinically,
tumour necrosis factor.

PNH is typified by the triad of severe haemolysis, thrombosis

and cytopenias with high reticulocytosis, very high serum
Patient group

lactate dehydrogenase and erythroid hyperplasia in the bone

marrow, it could be hardly more different from AA. How-
Elderly

ever, cytopenias are reminiscent of AA; and the first hint to

a relationship between PNH and AA was that the clinical

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Review
picture may shift, over time, from one to the other (or vicev-
ersa) (Dacie & Lewis, 1961; Lewis & Dacie, 1967). Later, the
development of PNH in AA patients was regarded as a ‘com-
plication’ of IST (Tichelli et al, 1994): more probably, IST
had allowed AA patients to survive and to develop PNH of
their own accord, as discussed below. Another interesting
link was the finding that red cells with high sensitivity to
complement, now known to be GPI-deficient, are not
uncommon in AA patients, as first shown by Ben-Bassat et al
(1975), and subsequently confirmed by other groups (Fores
et al, 1995; Wang et al, 2002; Scheinberg et al, 2010). In
1989 it was suggested that PNH develops when a somatic
mutation, now known to be in PIGA (Takeda et al, 1993),
co-exists with BMF: indeed, that PNH develops because
PIGA mutant stem cells can escape the damage suffered by
PIGA-normal stem cells (Rotoli & Luzzatto, 1989a,b)
(Fig 1C, Fig 2). In keeping with this notion, GPI-deficient
PIGA mutant granulocytes were found in normal subjects
(Araten et al, 1999): they may derive from a mutant HSC or
from a progenitor cell downstream of a HSC. Based on the
large dataset of an international registry (Schrezenmeier et al,
2014), about one-half of PNH patients have a previous his-
tory of AA (and this is certainly an under-estimate, because a
diagnosis of mild AA preceding PNH might have been
missed). PNH is so different from AA that it must retain its
distinct status in clinical nosography: but from the point of
view of pathogenesis, the majority of PNH patients are a
subset of AA patients (Young, 1992).
Stem cells are damaged in AA
A stem cell is defined by its ability to undergo asymmetric
division, whereby one daughter cell differentiates and the
other daughter cell is still a stem cell. The stem cell status
of an HSC depends on its niche (Zhang et al, 2003; Crane
et al, 2017), which comprises bone marrow stromal cells,
the extracellular matrix and the local levels of cytokines
(Riether et al, 2015; Crane et al, 2017). In experimental ani-
mals, where HSCs can be quantified by in vivo assay, it has
been amply demonstrated that the shortage of stem cells is
associated with AA (Scheinberg & Chen, 2013). In humans,
the closest surrogate of HSCs are the so–called long-term
culture initiating cells (LTC-IC): their numbers in the
peripheral blood and in the bone marrow of patients with
AA are drastically reduced (Fig 1B; Marsh et al, 1990;
Maciejewski et al, 1996). CD34+ cells are also decreased in
AA (Maciejewski et al, 1996; Manz et al, 1996). We infer
therefore that BMF results from stem cell failure. In princi-
ple, stem cells might be damaged in at least 3 different ways
(Fig 3). They might be (i) killed, (ii) inhibited, or (iii)
induced to undergo symmetric rather than asymmetric divi-
sion: if both daughter cells differentiate the HSC pool will
be depleted.
At the moment we don’t know which of these mecha-
nisms predominate(s). In the meantime, we must deal with
760
two ancillary issues: (1) whether HSC failure is endogenous
or exogenous; (2) whether it is reversible or irreversible.
Regarding (1), given that mutations in any of several genes
can cause inherited AA (see above and Table I), it is always
possible that what appeared to be acquired AA is in fact inher-
ited AA (Wegman-Ostrosky & Savage, 2017); or that a muta-
tion in one of the same genes that can cause inherited AA, or
even in other genes, may make HSCs more susceptible to
developing acquired AA (Calado & Young, 2009). An analogy
between BMF in dyskeratosis congenita (DC) and in acquired
AA is that significant shortening of telomeres is found in both
(Ball et al, 1998; Brummendorf et al, 2001). In DC this results
from specific mutations (Calado & Young, 2009), whereas the
cause is not known in acquired AA, but it signifies that the
stem cells have become abnormal. Although the possible role
of the bone marrow stroma in causing AA has been investi-
gated [e.g., the physiological immunomodulatory activity of
mesenchymal stem cells may be impaired in AA (Bacigalupo
et al, 2005)], patients can recover from AA following allo-
geneic BMT while retaining their own bone marrow stroma
(Spyridonidis et al, 2005); and stromal cells from AA marrows
can form a functional haematopoietic stem cell niche (Miche-
lozzi et al, 2017). Thus, there is overwhelming evidence that
the central problem is in the HSCs themselves (Fig 1A. Marsh
et al, 1991). Transcriptome analysis of CD34+ cells from AA
patients has shown increased expression of genes implicated in
apoptosis, cytokine/chemokine signalling, stress response, and
defence/immune response; whereas the expression of cell cycle
progress-enhancing genes was decreased (Zeng et al, 2004).
Regarding (2), clinical experience tells us that patients can
recover from AA without BMT, i.e., without a supply of allo-
geneic HSCs: at least in these cases, either not all HCSs were
killed, or the damage was reversible.
The effectors of damage
Antibodies have been detected in patients with AA against a
number of proteins (e.g., kinectin, moesin, post-meiotic seg-
regation 1, diazepam-binding inhibitor-related protein 1)
(Hirano et al, 2003, 2005; Feng et al, 2004; Takamatsu et al,
2007; Espinoza et al, 2009), which could be auto-antigens:
however, these auto-antibodies have not been shown to be
cytotoxic and, rather than being pathogenic, they seem more
likely to be an epiphenomenon, as in other auto-immune
diseases.
Stem cells are maintained by a precise genetic and epige-
netic programme. Retaining stemness whilst at the same time
being capable of differentiation through asymmetric divisions
is a bit like walking a tightrope: it is not surprising that the
delicate balance can be disturbed, probably in more ways
than one. Among several extracellular regulatory molecules,
interferons, which are normally produced in response to
infection, have emerged as powerful and potentially disturb-
ing. In the short term, interferons enhance the production of
neutrophils and lymphocytes, which is advantageous for an
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British Journal of Haematology, 2018, 182, 758–776
 Review

(A) (B)

Fig 1. Historic steps in understanding the pathogenesis of idiopathic acquired aplastic anaemia (AA). (A) Stem cells failure in AA (from Marsh
et al, 1991). One sees a marked decrease in progenitor cells (granulocyte-macrophage colony-forming units; CFU-GM) generated by long-term
culture-initiating cells in patients with AA. The figure also shows that normal stroma does not correct the stem cell defect of AA patients; con-
versely, stroma for AA does not affect stem cells from normal controls. (B) In vitro growth of hematopoietic progenitors in AA. Anti-interferon-c
(black bars), compared to controls (white bars) markedly increases formation of colonies (colony-forming unit cells; CFU-C) from peripheral
blood mononuclear cells from a majority of 10 patients with severe AA (Zoumbos et al, 1985a). Different columns represent bone marrow
mononuclear cells grown in culture medium containing (a) no antiserum, (b) control hyperimmune sheep antiserum, (c) control hyperimmune
sheep antiserum, (d) sheep antiserum to human a-interferon (neutralizing antibodies, (e) sheep antiserum to human a-interferon (neutralizing
antibodies). C. Close relationship between the pathophysiology of AA and paroxysmal nocturnal haemoglobinuria (PNH). Rotoli and Luzzatto
(1989a) explicitly suggested that in PNH, a disease already known to be clonal, glycosyl-phosphatidyl-inositol-deficient haematopoietic stem cells
expand and take over haematopoiesis in the bone marrow of a patient with AA. The different panels represented different stages/clinical situa-
tions: an important implication was that patients with PNH are a subset of patients with AA. AAA, acquired aplastic anaemia; AL, acute leukae-
mia. (A) reproduced with permission from Marsh J.C., Chang J., Testa N.G., Hows J.M. and Dexter T.M. In vitro assessment of marrow ‘stem
cell’ and stromal cell function in aplastic anaemia. British Journal of Haematology 78, 258-267. © 1991 John Wiley & Sons, Inc. (B) reproduced
from Zoumbos, N.C., Gascon P., Djeu J.Y. and Young N.S. (1985). Interferon is a mediator of hematopoietic suppression in aplastic anemia
in vitro and possibly in vivo. Proc Natl Acad Sci U S A 82(1): 188-192. (C) Reprinted from Bailliere’s Clin Haematol, 2 (1), Rotoli, B. and Luz-
zatto, L., Paroxysmal nocturnal haemoglobinuria, 113-138, © 1989, with permission from Elsevier.

effective response to infection. A major advance in character-
izing how haematopoiesis is disturbed in patients with AA
was the discovery that the level of interferon-c (IFNG) was
raised in their sera, and that addition of anti-IFNG markedly
increased numbers of in vitro haematopoietic colonies from
bone marrow cells of AA patients, but not from normal con-
trols (Zoumbos et al, 1985a) (Fig 1B). These results have
been extensively confirmed (Selleri et al, 1996). Of course, all
conventional in vitro colonies are generated by progenitor
cells that are downstream of HSCs, and therefore we cannot
automatically extrapolate to the latter from the former. IFNG
may act on HSCs both directly and indirectly: directly, once
bound to the IFNG receptor, IFNG modulates the STAT and
SOCS2 pathways (Krebs & Hilton, 2001), thus potentially
affecting proliferation and retention of stemness; in addition,
IFNG up-regulates FAS expression, thus making cells more
prone to apoptosis (Maciejewski et al, 1995). Very recent
data suggest that IFNG may compromise the survival of
HSCs by forming a heterodimer with thrombopoietin (TPO),
with consequent impairment of TPO-cMPL signalling
(Alvarado et al, 2017). Indirectly, IFNG acts on IFNG
receptor-expressing macrophages and stromal cells, that in
turn may affect stem cell quiescence and exert a pull towards
producing progenitor cells (Smith et al, 2016). A polymor-
phic site in the IFNG gene may increase susceptibility to
developing AA, as well as its severity (Bestach et al, 2015),
and may influence response to IST (Zeng et al, 2015). AA
develops in mice genetically modified to express constitu-
tively low levels of IFNG (Lin et al, 2014). Tumour necrosis
factor-a (TNFa could also affect HSCs (Riether et al, 2015):
TNFa-producing lymphocytes are increased in AA patients
(Schultz & Shahidi, 1994; Selleri et al, 1995), and their disap-
pearance tends to correlate with clinical response to IST
(Dufour et al, 2001).
The finding that these cytokines are increased in AA signi-
fies that an inflammatory process is on-going (see below and
Table II). Multiple threads of evidence both in mice (Schein-
berg & Chen, 2013) and in humans (Sloand et al, 2002),
may not be definitive proof that IFNG is a culprit, but they
indict it as a prime suspect.

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Fig 1. Continued.
The source of damage
Spurred by the fact that IST can overcome AA, a large body
of accumulated evidence indicates that the damage to HSCs
originates from T cells (Young et al, 2006; Risitano et al,
2007). Here we will focus on two extreme possibilities. (i)
On one hand, a variety of abnormalities in the bone marrow
can produce, as their final common pathway, a local level of
IFNG and other cytokines sufficient to cause serious damage
to a majority of HSCs. (ii) On the other hand, a small subset
of T cells, possible a ‘forbidden clone’, may recognize a
specific molecular target in HSCs, whereby IFNG may be still
relevant in an ancillary capacity. These two possibilities are
not mutually exclusive.
Evidence for deranged immunity and inflammation
Abnormalities in relative proportions of T cells subsets and
in the T cell transcriptome have been reported in patients
762
with AA (reviewed in Young et al, 2006). Indirect evidence
for involvement of CD4+ T cells was the over-representation
of certain human leucocyte antigen (HLA) alleles in AA
patients (Nakao et al, 1994). Activated CD8+ T cells are
cytotoxic for bone marrow cells (Zoumbos et al, 1985b), lar-
gely through IFNG secretion. IFNG can be produced by
CD4+ T cells, CD8+ T cells and Natural Killer T (NKT) cells,
all of which are increased in AA patients. Disorders of NKT
or NKT-like cells (large granular lymphocytic cells, LGL) can
be associated with single or multiple cytopenias. LGLs secrete
soluble Fas receptors that are elevated in the serum of AA
patients; in addition, Fas-Receptor is overexpressed on
CD34+ cells of AA patients, consistent with susceptibility to
apoptosis of HSCs (Maciejewski et al, 1995).
The combination of locally increased levels of IFNG in the
bone marrow and increased susceptibility to apoptosis could
cause sufficient damage to HSCs to produce the clinical pic-
ture of AA. T-helper cell type 1 (Th1) polarization of T cells
in AA patients is associated with TBX21 over-expression,
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British Journal of Haematology, 2018, 182, 758–776
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PIGA+ blood cell PIGA– blood cell

PIGA+ ‘crippled’ blood cell Noxious agent

Fig 2. Somatic mutation and Darwinian selection of mutant somatic cells in paroxysmal nocturnal haemoglobinuria (PNH). This cartoon pre-
dicted that a PIGA mutation, by preventing formation of glycosyl-phosphatidyl-inositol (GPI) and GPI-linked proteins, probably pre-existing,
provided a clone invulnerable to GPI-specific auto-immune attack. The 3 panels on the right depict possible outcomes. Top: PNH, with ongoing
auto-immune process; centre: PNH after demise of normal haematopoietic stem cells (HSCs); bottom: PNH with survival of some normal HSCs,
a situation that explains rare cases of spontaneous recovery from PNH. Modified from Luzzatto, L., Bessler M. and Rotoli B. Somatic mutations
in paroxysmal nocturnal hemoglobinuria: a blessing in disguise? Cell 88(1): 1-4. © 1997 John Wiley & Sons, Inc.

potentially accounting for active transcription of IFNG
(Solomou et al, 2006), as well as an increase in Th17 cells at
diagnosis: again these decline in good responders to IST
(de Latour et al, 2010). The number of type-1 cytokine
producing cells is inversely correlated with the number of
regulatory T (Treg) cells, which tend to be decreased at diag-
nosis and are eventually restored after effective IST (Solomou
et al, 2007). A broad imbalance between increased Th1, Th2
and Th17 T cells versus decreased Tregs seem characteristic
of patients with AA (Kordasti et al, 2012). A detailed analysis
of different subsets of T cells has revealed subtle changes
(Kordasti et al, 2016) in sub-populations of Treg cells,
regarded as the master regulators of the T cell network,
adding further support to the notion that AA is a
cell-mediated auto-immune disease.
Evidence for specific (clonotypic) T cell populations and
possible molecular targets
If an auto-immune attack recognizes a specific antigenic
molecule, the T cells involved must have the appropriate
T-cell receptor (TCR) for recognition of that molecule within
the patient’s major histocompatibility complex (MHC), or
other presenting molecule. In this case, the T cells involved
would probably be a very small number amongst all T cells:
they cannot be identified by analysis of conventional T cell
subsets, but only by ad hoc methodologies. The first such
methodology, spectratyping analysis, measures the size of the
TCR-b chain complementarity determining region 3 (CDR3)
of each of the 24 b chain families. By this analysis a skewed
size distribution – suggesting the presence of sizable T cell
clones (clonotypes) – was first observed in a group of
patients with PNH (Karadimitris et al, 2000), and then in a
larger series of patients with AA (Risitano et al, 2004), as
well as in other studies (Zeng et al, 1999, 2001). Subse-
quently, sequence analysis of the TCR-b chain confirmed the
existence of recurring clonotypes in patients with PNH (Gar-
giulo et al, 2007) and in children with hepatitis-associated
severe AA (Krell et al, 2013). Both AA and PNH can be asso-
ciated with LGL (Karadimitris et al, 2001; Risitano et al,
2005), a disease that consists of an expanded NKT or NKT-
like cell clone.
Recurring T cell clonotypes may recognize specific mole-
cules on HSCs. If a putative antigen were HLA-restricted, the
TCR structure recognizing it would be different, depending
on the HLA genotype of the patient, possibly explaining why

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(A) Normal haematopoiesis (B) Direct killing of HSCs

XXX Direct killing

Self- Self-
XXX
HSCs HSCs
renewal renewal

HPCs HPCs

Granulocytes, platelets, RBCs Pancytopenia

(C) Functional inhibition of HSCs (D) Depletion consequent to loss of asymmetric division

X
Inhibition of
proliferation Inhibition of
asymmetric division
Self- No Self-
HSCs HSCs
renewal renewal

HPCs HPCs

Pancytopenia Pancytopenia

Fig 3. Different modes of injury to haematopoietic stem cells (HSCs) and haematopoietic progenitor cells (HPCs). (A) Normal haematopoiesis:
HSCs undergo asymmetric division, generating HPC which eventually produce mature blood cells [granulocytes, platelets and red blood cells
(RBCs)], while the HSC pool is preserved. (B) Direct killing. HSCs may be killed by a noxious agent (e.g. cytotoxic T cells): the HSC pool is
gradually depleted, with consequent impaired production of HPCs and of mature blood cells. (C) Functional inhibition. A noxious agent (e.g.,
interferon-c) may inhibit the proliferation of HSCs: the consequences will be as in (B). D. Loss of asymmetric division. HSCs (perhaps as a result
of an epigenetic change) may undergo symmetric rather than asymmetric division: if both daughter cells differentiate, the HSC pool will be
depleted, finally resulting in pancytopenia.

the same clonotype is not found in all AA patients (Risitano
et al, 2004). Alternatively, the antigen may not be HLA-
restricted: e.g., if it were a glycolipid it would be CD1d-
restricted instead. This hypothesis has been tested in a subset
of AA patients who have PNH, by using CD1d dimer tech-
nology and human GPI, obtained ad hoc by organic synthesis
(Richichi et al, 2011), as the putative antigen. By this
approach, GPI-specific T cells have been detected in the
peripheral blood of all PNH patients: the numbers were very
small (usually less than 1% of all CD8 + cells), but signifi-
cantly above the control background (Gargiulo et al, 2013).
This finding points to a specific molecule, GPI, as the target
of the auto-immune attack in the pathogenesis of those AA
patients who have PNH. Although direct damage to GPI(+)
HSC by GPI-specific T cells has not yet been demonstrated,
these findings support the escape model outlined above,
because GPI-positive HSCs (the majority) would suffer dam-
age, and only PIGA mutant GPI-negative HSCs would be
spared (positive Darwinian selection: Table III, Fig 4). A pos-
sible objection to this mechanism is that, because GPI is
ubiquitous, GPI-specific T cells might damage cells other
than HSCs: however, only cells expressing CD1d would be
susceptible to such damage and, in addition, GPI-specific T
cells may be more abundant in the bone marrow than else-
where. Of course, GPI may not be the target in all cases: the
escape model is versatile (Risitano, 2017; Luzzatto & Notaro,
2018) and if, as a result of mutation in a gene other than
PIGA, a different target molecule were missing from a HSC,
its progeny might be able to escape damage. This seems to
be the case in a group of patients with AA who are

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British Journal of Haematology, 2018, 182, 758–776

Table II. Genes frequently observed to have somatic mutations in residual haematopoiesis in patients with aplastic anaemia.
Map Major function of Most frequent
Gene location protein encoded mutations
BCOR Xp11
4 Transcriptional co-repressor Inactivating
via histone deacetylases
ASXL1 20q11
21 Chromatin conformation Inactivating
(polycomb family)
DNMT3A 2p23
3 De novo DNA methylation Missense
PIGA Xp22
2 Biosynthesis of GPI anchor Inactivating
HLA locus 6p21
31 Antigen presentation Inactivating
GPI, glycosyl-phosphatidyl-inositol.
*Could be resistance to apoptosis, increased proliferation, other.
†Multiple severe congenital malformations thought to be related to HOX genes dysfunction.
‡Tall stature and severe cognitive impairment.
heterozygous for the HLA-B allele 4002, and who have lost
its expression through a somatic point mutation or deletion
of this allele (Zaimoku et al, 2017); and very recently autolo-
gous cytotoxic T cells that specifically recognize induced
pluripotent stem cell-derived B4002+ CD34+ cells have been
obtained from these patients (Espinoza et al, 2018). Patients
who have B4002 are relatively few and, therefore, their pre-
sumed escape mechanism cannot account for what happens
in the majority of patients with AA; but a similar mechanism
of selection may take place with respect to other HLA alleles
(Babushok et al, 2017a). At the same time, GPI-specific
CD1d-restricted T cells have been found at diagnosis in 75%
of Japanese AA patients, most of whom had GPI-negative
blood cells above the noise level, but did not have PNH
(Gargiulo et al, 2017).
Mutant clones in AA
Amongst AA patients it is clear that the subset of PNH
patients has a clonal disorder (Oni et al, 1970) that is conse-
quent upon a specific somatic mutation in the PIGA gene;
and about one-half of all patients with AA also have a PIGA
mutation, even though a mutant clone has not expanded (as
it has in PNH). Cytogenetic abnormalities (Appelbaum et al,
1987; Socie, 1996) and skewed X chromosome inactivation
patterns (van Kamp et al, 1991) in AA have been known for
a long time; but only over the last few years has massive par-
allel DNA sequencing technology made the detection of
somatic mutations possible, even if present only in a minor-
ity of cells. Patients with AA have been tested for mutations
in a set of candidate genes (Lane et al, 2013; Heuser et al,
2014; Kulasekararaj et al, 2014), or by full exome sequencing
(Babushok et al, 2015; Yoshizato et al, 2015). Reviews of this
work (Ogawa, 2016; Cooper & Young, 2017; Stanley et al,
2017) have raised several points of interest. First, about one-
third of AA patients have mutant clones, either single or
multiple. Second, although mutations have been found in
some 30 different genes, the large majority of recurrent
ª 2018 British Society for Haematology and John Wiley & Sons Ltd
British Journal of Haematology, 2018, 182, 758–776
Review
Possible mechanism of somatic Disease caused by germ-line
mutant clone persistence/expansion mutations
Uncertain* Oculo-facial-cardio-dental syndrome
Uncertain† Boring-Opitz syndrome†
Uncertain* Tatton-Brown-Rahman syndrome‡
Escape from GPI-targeted Multiple congenital abnormalities-
auto-immune attack hypotonia-seizures
Escape from auto-immune attack None
mutations are in only 5 genes: (Table II). Third, in each
individual patient the clones identified by mutant genes
make up a very variable proportion of total residual haema-
topoiesis: less than 10% in some cases, but up to 90% in
others.
Three of these genes (Table II) are concerned with chro-
matin structure and/or transcriptional regulation. BCOR has
been so-called because it is a co-repressor of BCL6 (known
to be involved in B cell development and lymphomagenesis).
In AA, BCOR, an X-linked gene, has mostly inactivating
mutations: if the only copy of BCOR is inactivated, BCL6
will be less repressed. Loss-of-function mutations of BCOR
are associated with enhanced cell proliferation and myeloid
differentiation (Cao et al, 2016). ASXL1 belongs to the poly-
comb gene family: it too has inactivating mutations causing
loss of polycomb repressive complex 2 (PRC2)-dependent
histone H3 methylation (Abdel-Wahab et al, 2012), presum-
ably through haploinsufficieny. The ASXL1 protein forms,
with BAP1 protein, a complex required for the deubiquitina-
tion of mono-ubiquitinated histone 2A (Balasubramani et al,
2015), whereby gain of function mutations found in AA will
influence chromatin structure and transcription. DNMT3A
encodes one subunit of an enzyme responsible for de novo
methylation of DNA. Here one finds mostly missense muta-
tions, with a recurrent mutation at codon 882: this has been
shown to act as dominant-negative by interfering with the
dimerization of the DNMT3A protein (Russler-Germain
et al, 2014), but other mutations may exert their effect
through haploinsufficiency (Cole et al, 2017). Each one of
these genes must have an important role in development:
indeed, germ-line mutations cause multiple complex devel-
opmental abnormalities (Table II).
Clonal haematopoiesis versus haematopoiesis
with clones
In the subset of AA patients who have PNH, haematopoiesis
is supported effectively by a PIGA mutant clone: therefore,
765
Review

Table III. Population genetics of organisms and of somatic cells

In a population of organisms In a population of somatic cells

Event/feature Consequences Examples Consequences Examples

Mutation
Any Creates a mutant Many Creates a mutant Many
individual/family cell/clone
Lethal No offspring Many No clone Many
Neutral No visible effect Many No visible effect Many
Genetic drift
neutral or not Frequency of a certain Unusually high A mutant clone can Mutant clones in AA
mutant gene increases frequency of more easily expand
when founder population inherited if original normal
small (bottleneck) polycythaemia cells scarce
due to germ-line
VHL mutations*
Selection†
With absolute growth Mutant progeny will Fixation of Clone will expand/ Leukaemogenic mutations
advantage gradually take over species-specific become dominant
genes
With conditional growth Mutant progeny will HbS gene (HBB Clone will expand in a PIGA mutant clone in the
advantage increase in a certain E7V) in certain environment presence of GPI-specific
environment malaria-endemic T cells (PNH)
area
Convergent evolution
Involving different genes Increase in frequency of Several genes related Clones with mutations in Clones with JAK2, CALR,
mutant alleles to melanogenesis in different genes yielding MPL mutations in MPNs
tropical areas similar disease
Involving the same gene Increase in frequency of Many different G6PD Clones with different Clones with different PIGA
mutant alleles mutant alleles mutations in the same mutations may co-exist in PNH
underlying G6PD gene yield similar disease
deficiency in may concurrently expand
malaria-endemic
areas

AA, aplastic anaemia; GPI, glycosyl-phosphatidyl-inositol; PNH, paroxysmal nocturnal haemoglobinuria.

*One VHL mutation (Arg200Trp mutation (C598T) has a high prevalence in the Chuvash region of Russia (Sergeyeva et al, 1997); the same VHL
mutation has a high prevalence on the island of Ischia (Italy), resulting in typical Chuvash polycythaemia (Perrotta et al, 2006).
†Unlike with genetic drift, population size is not influential.

clonal haematopoiesis describes this situation well. In other
AA patients, haematopoiesis remains inadequate in spite of
the presence of mutant clones. The phrase ‘clonal disease’
might convey the impression that the disease results from
mutant clone(s); in fact, they are prominent as a conse-
quence of the disease, the pathogenic cause of which consists
instead, as abundantly outlined above, in the demise of nor-
mal HSCs. The clones that we see are reminiscent of residual
vegetation in a de-populated landscape.
In order to outline the pathogenic significance of these
clones, currently a subject of lively debate (Cooper & Young,
2017), we resort to the principles of populations and evolu-
tionary genetics (see Box 1 for definitions) – classically devel-
oped in studying populations of organisms – to the extent
that they can apply to population of somatic cells (see
Table III). The engine of evolution consists of mutations:
these are stochastic events. The fate of a mutant depends on
whether each generation is a representative sample of the
previous generation (i.e., genetic drift, still a stochastic phe-
nomenon), and on whether the environment is selective for
or against the mutant. In this light, the clonal composition
of an aplastic marrow probably results from a mix of drift
and selection (see also Box 2, Fig 4). Drift is favoured by the
overall paucity of cells, whereby a pre-existing mutant clone
has a greater chance to expand than the same mutant clone
would have in a non-aplastic marrow. At the same time, by
the very fact that these clones are present in AA, we must
postulate they have preferentially survived at the time of
onset of the disease: i.e., they have been selected for. It does
not seem likely that selection is mediated by escape from a
target-specific attack (as is the case for PIGA mutant cells):
rather, cells with mutations of ASXL1, BCOR, DNMT3A or
other genes may be overall more resistant to attack, be it T
cell-mediated or mediated by inflammatory cytokines.

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British Journal of Haematology, 2018, 182, 758–776
 Review

(A)

Time

Normal (polyclonal) set of HSC providing normal haematopoiesis

(B) Inflammation Persistant

and myelotoxic inflammatory
cytokines milieu

IFNG
IFNG

TGF-β TGF-β

TNF-α
TNF-α

Time

Normal HSC depletion Overt bone marrow failure: AA

(polyclonal) → oligoclonal
set of HSC haematopoiesis

(C)
Protracted GPI-targeted autoimmune
T-cell attack

±IST

PIGA
mutation

Time
Normal Loss of non-mutant HSCs and immune Rescue of haematopoiesis
(polyclonal) selection for GPI-deficient mutant HSC by expansion - clonal dominance -
set of HSC of PIGA mutant HSC: PNH

Fig 4. Dynamics of haematopoiesis in different situations. (A) Normal steady haematopoiesis in a normal bone marrow. (B) AA caused by a persis-
tent inflammatory process. (C) AA with PIGA mutants. In patients with PIGA mutant HSC and GPI-specific T cells AA converts to PNH: a PIGA
mutation seems to be the only case where haematopoiesis can be fully restored in a patient with AA. Given that some HSCs without PIGA muta-
tion may still be present, this diagram also accounts for spontaneous recovery from PNH, as previously suggested (Hillmen et al, 1995) and
recently confirmed (Babushok et al, 2017b). (D) AA with HLA-deficient mutant clones. In patients with loss of an HLA allele targeted by an auto-
immune process the HLA-deficient mutant clone can support haematopoiesis until IST helps recovery. (E) AA with other mutant clones: although
for clarity these are shown only in this panel, they may coexist also in the situations depicted in panels B–D. Note that mutant clones, which can
arise in normal people (as in panel E), are not the cause of AA. The emergence of mutant clones is favoured by the overall paucity of cells in the
bone marrow of AA patients (genetic drift: see Table III), it implies that the mutations most frequently seen must have an intrinsic slight survival
or growth advantage. The mutations of BCOR, ASXL1, DNMT3A (as well as others) are not leukaemogenic per se, because they are commonly
seen in the absence of leukaemia: however, they are probably in some cases the first step in a sequence of mutations that can cause leukaemia.
For additional explanations see Box 2. AA, aplastic anaemia; HLA, human leucocyte antigen; HSCs, haematopoietic stem cells; IFNG, interferon-c;
IST, immuno-suppressive treatment; PNH, paroxysmal nocturnal haemoglobinuria; TGF-b, transforming growth factor b; TNF-a, tumour necrosis
factor a

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British Journal of Haematology, 2018, 182, 758–776
Review

(D)
Protracted HLA-targeted autoimmune
T-cell attack

Successful
IST

6p LOH
(or B*4002)
mutation

Normal Loss of non-mutant HSCs and immune Restoration of haematopoiesis

(polyclonal) privilege of HLA-deficient mutant HSC without clonal dominance of
set of HSC HLA-deficient mutant HSC

(E) Other situations with mutant clones

DNMT3A ASXL1
mutation BCOR mutation
Clonal haematopoiesis
mutation
of indetermined potential
Slightly increased
HSC ‘fitness’?

Autoimmune T-
cell attack and/or Aplastic anaemia
with mutant clone(s)
inflammation

Additional
mutations
Myeloid malignancy

T cells Cytokines

Normal
(polyclonal)
set of HSC

Fig 4. Continued.

mutated in more than one patient. This is a compelling

AA and PNH are not cancer
The question of how PNH and AA relate to leukaemia has
been recurrent in the literature since the landmark editorial
written by William Dameshek half a century ago, as a com-
mentary to three case reports on patients with PNH who
developed acute myeloid leukaemia (AML) (Dameshek,
1967). Although more similar case reports have appeared
subsequently, it is clear that they are exceptions rather than
the rule: the majority of patients with PNH have not devel-
oped AML. This is particularly significant because PNH is a
very long-term disease: we and others have followed for dec-
ades individual patients who are still alive. The proportion of
patients with PNH who develop AML is estimated to be less
than 2% (de Latour et al, 2008); it is rather higher, perhaps
around 15% (Socie et al, 2000; Scheinberg et al, 2011), for
patients with AA who do not have PNH.
These clinical facts have been corroborated by full exome
sequencing of blood cells from patients with PNH (Shen et al,
2014; Asad et al, 2017). The most consistent finding was that
PIGA is always mutated. Additional somatic mutations were
found in 1 to 6 other genes, but only two of the genes were
confirmation, in a totally unbiased analysis, of the unique
PNH-genic role of inactivating mutations of PIGA. When
serial samples were available, it emerged that when the first
mutation is in PIGA, it is sufficient to cause clonal expansion,
whereby the superimposition of further mutations does not
affect the process much. On the other hand, when the PIGA
mutation takes place in a cell belonging to a clone that has
already a mutation in another gene, it is the clone that has
both mutations that expands much more than the clone that
has the non-PIGA mutation alone. In a separate study (Yoshi-
zato et al, 2015), again a PIGA mutation was found in every
case. Thus, the two previously reported patients with an
HMGA2 mutation in addition to a PIGA mutation (Inoue
et al, 2006) remain exceptional: in almost all PNH patients a
PIGA mutation, apart from being responsible for GPI-negative
haematopoiesis, is also the key to clonal expansion.
Unlike other clones in AA, which may survive and expand
by a combination of drift and selection, a PIGA mutant clone
is able to expand by a specific selective mechanism (Luzzatto
et al, 1997; Young & Maciejewski, 2000). It is unfortunate that
an editorial (Lee & Abdel-Wahab, 2014) accompanying the

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British Journal of Haematology, 2018, 182, 758–776
 Review

Box 1. Clones and somatic mutations

1 A clone is the progeny of a single cell. Clonal population is used to designate a part of a clone: for instance, the GPI-
negative erythrocytes in a PNH patient are a clonal population (the entire PNH clone also includes other GPI-negative
blood cells and their precursors). Sometimes clonal population is used as synonym of clone.
2 A clone is of course not in itself pathological: for instance, the epithelial cells of a single intestinal crypt are a clonal pop-
ulation arising from a stem cell located at the base of the crypt.
3 A mutant clone is the progeny of a cell in which a mutation has taken place: it may or may not have a recognizable
phenotype, depending on the consequences of the mutation. Again, a mutant clone is not necessarily pathological: a
freckle or a naevus is a clone of cells with different pigmentation from surrounding skin.
4 Given that immunoglobulin and T-cell receptor rearrangements are part of the physiological development of the immune
system, there are myriads of normal B cell clones and T cell clones in our body.
5 A sub-clone is again the progeny of a single cell (see 1 above). This term is often used when a second mutation takes
place in one cell of a mutant clone.
6 Clonal haematopoiesis has been known for a long time in patients with leukaemia or PNH. Recently this term has been
used to indicate that, in contrast to normal haematopoiesis to which many stem cells contribute, in AA few clones aris-
ing from mutant HSCs may account for the production of some or even most most mature blood cells.
7 Clonal expansion indicates growth of a clone. Given that growth in the haematopoietic tissue is a normal process, the
term clonal expansion is appropriate only when the clone is growing at a higher rate than neighbouring non-mutant
cells. In AA, clonal expansion may be favoured by the vacuum created through the demise of non-mutant HSCs, but as
marrow aplasia is not corrected, the expansion is in relative, not in absolute terms.
8 The term clonal dominance is used when a clone or a sub-clone has expanded in size to the extent of over-shadowing
other cell populations in a particular tissue: for instance, a leukaemic clone will dominate or even overwhelm normal
haematopoiesis. In other cases, like in AA, dominance may be only relative (see clonal expansion above).
9 Clonal evolution* means that one or more sub-clones can develop from a clone; it is not synonymous with leukaemoge-
nesis, because many clones with two or even more mutations may not be leukaemic. On the other hand, it is true that a
mutation causing blast crisis constitutes the clonal evolution of chronic myeloid leukaemia.
10 A clonal disease is any disease caused by a mutant clone: it is not necessarily malignant (e.g., PNH). However, it is true that
a clonal disease can evolve to malignancy (e.g., MGUS to MM). Although there is clonal haematopoiesis in AA, it does not
seem appropriate or useful to call it a clonal disease, because the clones are not the cause of the bone marrow failure charac-
teristic of AA: rather, they are a consequence: in fact, not all AA patients have clones, indicating that they are not a must.

* The term clonal architecture is sometimes used to describe, in a particular condition, the set of clones, whether indepen-
dently arisen or evolved from one another, that are present, as well as their sizes. We think this phrase is not ideal and may
be misleading, because architecture conveys the notion of a deliberate plan: instead, mutations are random events and the
fate of mutant clones depends on genetic drift and selection (see Table III). If at all, clonal composition or clonal landscape
may be better.

paper by Shen et al (2014) gave the impression that PNH is
‘strikingly similar to other myeloid neoplasms’. Not only this
is not the case, but we can also visualize why. In Table II, PIGA
stands out because it has a fundamentally different function
from some of the others listed as examples of genes mutated in
AA. Mutant clones of BCOR, ASXL1 and DNMT3A, with their
altered chromatin arrangement, may evolve to malignant
sub-clones through additional mutations; in contrast, there is
no biological basis for regarding a PIGA mutant clone as
pre-leukaemic.
We can probably also explain why the development of
AML is so much rarer in PNH patients compared to other
AA patients. Given that mutations are stochastic events,
leukaemogenesis is not a clockwise process (see also Box 2),
and it may not happen; but it is a stepwise process, and the
probability of further mutations is proportional to the size of
pre-leukaemic clones (Luzzatto & Pandolfi, 2015). In AA
patients, sizable mutant clones of BCOR, ASXL1 and
DNMT3A may persist even after IST: hence, following addi-
tional mutations, some 15% of AA patients eventually
develop AML. However, in PNH, most haematopoiesis has
been taken over by the PIGA mutant clone: indeed, a single
PIGA mutant HSC may, on its own, sustain haematopoiesis
even for decades (Nishimura Ji et al, 2002). Independent
(non-PIGA mutant) clones, if any, or PIGA-mutant sub-
clones that have other mutations, if any, are on average
smaller: hence the much lower probability of AML.
AA and myelodysplastic syndrome
Myelodysplastic syndrome (MDS) is not within our remit; but
it has been noticeable for a long time that AA and ‘hypoplastic

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British Journal of Haematology, 2018, 182, 758–776
Review
Box 2. Clones and their fate in aplastic anaemia
1 Random mutations probably occur just as frequently
in AA patients as in normal people (Rondelli et al,
2013).
2 Mutant clones found in AA patients almost certainly
pre-existed AA, as they can be found also in normal
persons (Genovese et al, 2014; Jaiswal et al, 2014; Xie
et al, 2014).
3 Survival advantage may be absolute (as for BCR-
ABL1 mutant cells in chronic myeloid leukaemia), or
it may be relative to a particular situation, e.g. an
inflammatory environment or an auto-immune
attack.
4 The epigenetic profile of mutant cells may be critical
to survival, e.g. by making cells less prone to apopto-
sis: this may be why several of the most common
mutations in clones found in AA (see Table II) are in
genes related to chromatin structure.
5 Growth advantage does not necessarily follow survival
advantage: in AA, when IST is successful and normal
haematopoiesis is restored, mutant clones may retain
the percentage of space they had acquired, but expand
very little further or not at all (Yoshizato et al, 2015).
6 The phrase ‘clonal haematopoiesis of indeterminate
potential (CHIP)’ was coined (Steensma et al, 2015)
following the finding that in normal subjects, particu-
larly the elderly (Genovese et al, 2014; Jaiswal et al,
2014; Xie et al, 2014), somatic mutations may be pre-
sent that are identical to some found in MDS and
leukaemia.
7 In most cases, leukaemogenesis is a multi-step pro-
cess, and a mutation of ASXL1 (or BCOR or
DNMT3A) may be one step. Thus, if n steps are
required for a particular type of leukaemia to develop,
a cell with one of these mutations will now only need
n-1 steps.
8 Multi-step versus clockwise. Since mutations are
stochastic events, the n steps do not take place in a
clockwise manner: rather, each event happens by
chance. Therefore, an elderly person or a patient with
AA who has a very small ASXL1 mutant clone is at an
increased risk of leukaemia (compared to a person
who does not have that small clone); however, that
person does not have leukaemia, and may not develop
leukaemia, because the next mutation(s) may not take
place within the person’s lifetime.
MDS’ overlap: molecular analysis has only reinforced the rela-
tionship, as clones with the very same somatic mutations may
be found in patients who had been diagnosed with one or the
other condition. The nature and number of these somatic
mutations are predictive of transformation to AML (Malcovati
770
et al, 2017). In addition, there is wide overlap in marrow mor-
phology between MDS and PNH (Araten et al, 2001).
That there is BMF in MDS is not a new concept (Galton,
1989): indeed, when patients with MDS were treated as
though they had AML, the outcome was grim (Tricot &
Boogaerts, 1986; Fenaux et al, 1988; Loffler et al, 1992; Ber-
nasconi et al, 1998), because normal haematopoiesis failed to
regenerate, suggesting that there were few, if any, viable nor-
mal HSCs. It is also known that, in the age distribution of
AA there are two peaks: one in young people, the other
around 65 years old (Montane et al, 2008), within the typical
age group of MDS. The differential diagnosis between AA
and hypoplastic MDS is hard, because they are probably the
same thing (Barrett et al, 2000). If the bone marrow is exam-
ined at an early stage the diagnosis will be AA; if it is exam-
ined when some of the clones arising in AA have already
established themselves, the diagnosis will be MDS. The signif-
icant beneficial results of IST in a subset of patients with
MDS (Saunthararajah et al, 2002, 2003; Sloand & Barrett,
2010) supports this notion.
At the same time, in elderly people there are at least two
hypothetical but plausible mechanisms other than auto-
immune attack that could cause BMF. One of them is telom-
ere attrition (Calado & Young, 2008; Alter et al, 2015): the
distribution of telomere length at birth is wide, but all of us
lose telomere length as we age. In some people, by 70 years
of age, the telomeres may be sufficiently worn out as to com-
promise ‘normal’ HSCs, whereas some mutant clones might
express telomerase activity or alternative lengthening of
telomeres (ALT) mechanisms to enable their survival.
Another possibility is loss of stemness: at every cell division
there is a risk that both daughter cells will differentiate,
entailing the loss of one HSC (see Fig 3). A mutant clone
might provide abnormal stem cells that are more capable of
retaining stemness. These hypothetical mechanisms might
explain why the mutational landscape in MDS is wider than
in AA: there may be a greater variety of selective mechanisms
at play.
Concluding remarks
Our understanding of inherited syndromes leading to bone
marrow failure has made great strides with the identification
of a discrete set of genes, the mutation of which underlie
these syndromes (see Table I). Similarly, it is clear that heavy
radiation (which may be accidental) or high doses of cyto-
toxic agents (such as those used for deliberate pre-transplant
haematopoietic ablation) cause acquired bone marrow aplasia
by eliminating HSCs.
In contrast, unravelling the pathogenesis of acquired ‘idio-
pathic’ AA has proven harder, because it is a subtler process.
The beneficial and often curative effect of intensive IST
remains, to date, the most compelling evidence that AA is, in
most cases, a disease of deranged immunity: but by now this
evidence is further corroborated by a multitude of studies
ª 2018 British Society for Haematology and John Wiley & Sons Ltd
British Journal of Haematology, 2018, 182, 758–776
 Review

demonstrating in this condition consistent changes in the
levels of certain cytokines, the relative proportions of T cell
subsets and their function, as assessed by analysis of their tran-
scriptome. It is conceivable that, in some cases, such changes
might amount to an inflammatory state sufficiently powerful
to damage HSCs as innocent bystanders. However, the efficacy
of an agent targeting T cells specifically, such as alemtuzumab
(Risitano et al, 2010; Scheinberg et al, 2012), makes one won-
der whether this may be true in the majority of cases.
PNH, being even more rare than AA, has seemed for a
long time to be an accessory to the crime. There is now
overwhelming evidence that, in fact, PNH is a subset of AA;
and because of the unique molecular lesion – a PIGA muta-
tion – these patients have provided us with a special insight
into pathogenesis. In PNH, normal haematopoiesis is largely
and sometimes completely replaced by clonal (phenotypically
GPI-negative) haematopoiesis, driven by a single PIGA-
mutant stem cell (Nishimura Ji et al, 2002): meaning that it
must have been spared by whatever has injured the other
HSCs. Given that there is no difference between PIGA-
mutant and non-mutant HSC in all the essential biological
characteristics of HSCs, including stem cell renewal and
potential for tri-lineage differentiation, it is not easy to imag-
ine a damage effector mechanism that is sufficiently sophisti-
cated to discriminate between the two, if not by immune
recognition. Thus, at least for PNH, at the time the ‘escape’
model was formulated nearly three decades ago, the notion
of a non-specific inflammatory process had to be rejected:
the target of the immune attack had to be specific, although
at that time we had no clue as to whether it was GPI or any
of the GPI-linked proteins. Now we know that PNH has
GPI-specific T cells that recognize GPI in the CD1d groove
(Gargiulo et al, 2007), whereby it appears that GPI is the real
target. With the expansion of the GPI-negative clone, AA is
cured and the diagnosis becomes PNH: the escape model is
fully vindicated (Fig 2).
But what about the other patients with AA? In some cases,
the demise of HSCs may be caused by a non-targeted inflam-
matory process. But in a substantial fraction of cases the tar-
get of auto-reactive T cells may be still GPI, except there is
no PIGA-mutant HSC, and therefore AA does not get cured
by PNH. In some cases, an escape process similar to that of
PNH may apply, as in patients with loss or mutation of
HLA-B4002, where the target of an auto-immune attack
might be a peptide from that allele: this would be MHC-
restricted rather than CD1d-restricted. In these cases, the cell
population with the mutant HLA-B4002 allele tends to
decrease upon IST, meaning that, unlike in PNH, recovery is
not mediated by expansion of that mutant clone, but by
residual non-mutant HSCs.
It is always tempting to conclude by considering therapeu-
tic implications, and we will not resist this temptation. IST
(Scheinberg et al, 2011), recently supplemented by the use of
eltrombopag (Townsley et al, 2017), as well as allogeneic
BMT (Marsh et al, 2011) have a high rate of success in AA:
both procedures subjugate or annihilate immune cells, and
the latter provides healthy HSCs in addition. However, these
therapeutic strategies seem rather blunt instruments: a more
targeted intervention would seem preferable. If and when the
pathogenic agent is a specific population of T cells, this pop-
ulation could be isolated, and a specific antiserum produced:
for instance, against GPI-specific T cells or even against their
TCR. Such an antiserum can be expected to have far fewer
side effects than antithymocyte globulin or BMT, and would
introduce a new type of precision medicine.
Acknowledgements
We are very grateful to Dr Rosario Notaro and Dr Regis Pef-
fault de Latour for reviewing the manuscript and for
thoughtful suggestions. This paper is dedicated to the mem-
ory of Bruno ROTOLI: a great haematologist, an invaluable
colleague and collaborator of some 30 years to one of us, an
exceptionally inspiring mentor to one of us, and a dear
friend for us both.
Author contribution
LL and AMR have written the paper together and agreed on
the final text.

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