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Review Article
CME Article
Clonal hematopoiesis in acquired aplastic anemia
Seishi Ogawa
Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
Clonal hematopoiesis (CH) in aplastic
anemia (AA) has been closely linked to
the evolution of late clonal disorders, including paroxysmal nocturnal hemoglobinuria and myelodysplastic syndromes
(MDS)/acute myeloid leukemia (AML),
which are common complications after
successful immunosuppressive therapy
(IST). With the advent of high-throughput
sequencing of recent years, the molecular
aspect of CH in AA has been clarified by
comprehensive detection of somatic mutations that drive clonal evolution. Genetic
abnormalities are found in ∼50% of patients
with AA and, except for PIGA mutations and
copy-neutral loss-of-heterozygosity, or
uniparental disomy (UPD) in 6p (6pUPD),
are most frequently represented by mutations involving genes commonly mutated in myeloid malignancies, including
DNMT3A, ASXL1, and BCOR/BCORL1.
Mutations exhibit distinct chronological
profiles and clinical impacts. BCOR/BCORL1
and PIGA mutations tend to disappear or
show stable clone size and predict a better
response to IST and a significantly better
clinical outcome compared with mutations
in DNMT3A, ASXL1, and other genes, which
are likely to increase their clone size, are
associated with a faster progression to
MDS/AML, and predict an unfavorable
survival. High frequency of 6pUPD and
overrepresentation of PIGA and BCOR/
BCORL1 mutations are unique to AA,
suggesting the role of autoimmunity in
clonal selection. By contrast, DNMT3A and
ASXL1 mutations, also commonly seen in
CH in the general population, indicate a
close link to CH in the aged bone marrow, in
terms of the mechanism for selection. Detection and close monitoring of somatic
mutations/evolution may help with prediction and diagnosis of clonal evolution of
MDS/AML and better management of patients with AA. (Blood. 2016;128(3):337-347)
Medscape Continuing Medical Education online
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Disclosures
Laurie Barclay, freelance writer and reviewer, Medscape, LLC, owns stock, stock options, or bonds from Pfizer. Associate Editor
Jean Soulier and the author declare no competing financial interests.
Learning objectives
1. Distinguish clonal hematopoiesis (CH) and somatic mutations driving clonal evolution in aplastic anemia (AA), based on a
review.
2. Determine the prognostic significance of somatic mutations driving clonal evolution in AA.
3. Identify somatic mutations unique to AA vs those also commonly seen in CH in the general population.
Release date: July 21, 2016; Expiration date: July 21, 2017
Introduction
Acquired aplastic anemia (AA) is a prototype of bone marrow (BM)
failure caused by immune-mediated destruction of hematopoietic stem
and/or progenitor cells, where autoreactive T cells are supposed to play
a central role (Figure 1).1-6 Although almost uniformly terminating in a
fatal outcome in a previous era, management of AA has drastically
improved since the late 1970s with the introduction of allogeneic stem
cell transplantation and IST, as well as optimized supportive care.5
However, the entire picture of the disease is more complicated than
expected for a simple immune-mediated marrow failure. Among
others, the frequent development of late clonal diseases, including
Submitted January 29, 2016; accepted April 20, 2016. Prepublished online as
Blood First Edition paper, April 27, 2016; DOI 10.1182/blood-2016-01-636381.
© 2016 by The American Society of Hematology
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337
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BLOOD, 21 JULY 2016 x VOLUME 128, NUMBER 3
OGAWA
A
B
CTL
antigen
Class I HLA
IFNγ
TNFα
TNFα
HSC
IF
IFNγ
BM suppression
Figure 1. Immune-mediated mechanism of AA and clonal evolution. (A) AA is thought to be initiated by recognition and destruction of HSCs by CTLs, which recognize
some unknown antigen present on HSCs via their HLA class I molecule. Supporting this hypothesis is a limited usage of T-cell receptor Vb subsets at diagnosis, suggestive of
the presence of oligoclonal expansion of CD81CD282 T cells, which diminish or disappear with successful IST.1-3 Overproduction of inflammatory cytokines, including
interferon g (IFNg) and tumor necrosis factor a (TNFa), from aberrantly activated immune cells and stromal microenvironments is also suggested to make a significant
contribution to BM failure, in which the role of FAS-mediated apoptosis has been implicated.4 (B) During and/or after immune-mediated BM destruction, a rapid expansion of
residual cells (which escaped destruction) occurs, whereby cells carrying mutations achieve clonal dominance and may progress to malignant proliferation. CTL, cytotoxic
T cell; HSC, hematopoietic stem cell; IST, immunosuppressive therapy.
paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplastic
syndromes (MDS)/acute myeloid leukemia (AML), is known as an
enigmatic complication in AA, which may not instantaneously be
understood under the category of immune-mediated disorders.7,8 The
enigma is further highlighted by the evidence suggesting the presence
of clonal hematopoiesis (CH), even in patients who do not develop
apparent clonal diseases.9 It should be noted that clonal evolution (ie,
evolution of clones) does not necessarily mean the development of
cancer or any other diathesis, because it can be compatible with normal
homeostasis.10,11
Indeed, clonality in AA has been one of the long-standing issues in
hematology, which was first implicated by Dameshek asking the
question, What do AA, PNH, and hypoplastic leukemia have in
common?12 Since then, many authors have revisited his riddle, which
could now be rephrased as, What is the common underlying mechanism
for, and the link between, CH and the evolution of clonal disorders in
AA?8 More recently, the issue has been newly addressed by using
revolutionized sequencing technologies, unmasking a unique picture of
CH in AA frequently accompanied by somatic mutations commonly
seen in myeloid malignancies.13-18 Combined with seminal findings on
CH in aged normal individuals,19-21 as well as on preleukemic clones22
in patients with hematologic malignancies,23-25 the finding on somatic
mutations in AA provides new insight into the origin and the
mechanism of frequent CH in AA and its impact on the late development of MDS and AML.13,15,18 This review summarizes recent
progress in the current understanding of CH in AA in relation to the
pathogenesis of late clonal diseases.
cells show defective cell surface expression of glycosylphosphatidylinositol (GPI)-anchored proteins, such as CD55 and CD59, which is
responsible for the vulnerability to complement-mediated hemolysis.27
AA and PNH are closely related conditions: AA may develop during
the course of PNH and vice versa (AA/PNH syndrome), both being
predisposed to AML and MDS.28,29 Complication of clinically relevant
PNH has been described in 15% to 25% of the patients with AA treated
by IST.30-35 Moreover, regardless of typical manifestations of PNH,
GPI anchor–deficient, “PNH-type” cells are detectable at even higher
frequencies (up to 67%) when assessed by sensitive flow cytometry
using antibodies to CD55 or CD59 and/or fluorescent aerolysin
(FLAER).36,37
Another major category of late clonal diseases includes MDS and
AML. The cumulative incidence of MDS/AML increases after IST,
ranging from 5% to 15% within the observation period of 5 to 11.3
years.35,38-40 Because the development of MDS/AML consistently
conveys a dismal prognosis in AA patients, it is of particular importance
Cytopenia
PNH
Dysplasia
/increased RS
MDS
Late clonal diseases in AA
The early evidence suggesting a link between AA and clonality was an
unduly high incidence of PNH and MDS/AML among patients with
AA.8 Although their incidence substantially increases among patients
who are successfully treated by IST,9,12 the complication of clonal
diseases seems to be an intrinsic feature of AA irrespective of IST,
because it had long been noted even before the widespread use of IST.26
PNH is a unique form of BM failure characterized by acquired
hemolytic anemia and thrombosis, which is caused by a clonal
expansion of cells derived from a hematopoietic stem cell (HSC)
carrying a somatic mutation involving the PIGA gene.27 PIGA-mutated
AA
Clonality
(specific
cytogenetic
lesions)
Excess
of
blasts
ICUS
Figure 2. Overlaps between AA, PNH, and MDS. Although conceptually representing discrete disease entities, AA, PNH, and MDS frequently coexist or show
mutual transitions within a same patient, apart from the ambiguity in actual diagnosis
due to the lack of conclusive evidence or misdiagnosis because of resembling clinical
presentations. Immune-mediated BM destruction can occur at the same time of
emergence of PNH or evolution of malignant clones, causing a diagnostic overlap
between these different disease concepts. Increased ring sideroblasts (RS) are
rarely seen in AA. ICUS, idiopathic cytopenia of undetermined significance.
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BLOOD, 21 JULY 2016 x VOLUME 128, NUMBER 3
to predict, diagnose, and initiate due interventions to these complications during the course of AA. However, this is often complicated
by difficulty in diagnosing MDS among patients with AA, which
represents a major challenge to clinicians and hematopathologists.
Diagnosing MDS in AA patients
MDS is defined as refractory cytopenia caused by a neoplastic
proliferation of clones committed to the myeloid lineage. The clones are
thought to be neoplastic, in that their growth is no longer compatible
with normal hematopoiesis, causing ineffective production of mature
blood cells, enhanced apoptosis, and even progression to AML, although the clinical presentation is highly variable.41,42 Thus, at least
conceptually, clonality is an essential requisite for the pathophysiology
of MDS, but for the sake of clinical/pathological diagnosis, clinicians
and pathologists usually diagnose the involvement of a neoplastic
process primarily relying on cell morphology, in which the presence of
excess blasts ($5%) (propensity to leukemia) and/or dysplasia (in
$10% of myeloid cells) with or without increased ($15%) ring
sideroblasts (deregulated myelopoiesis) empirically, but consistently,
indicates that the defective hematopoiesis is caused by malignant
clones, although morphologic examination may not necessarily be
consistent between observers (Figure 2).41 Alternatively, direct demonstration of a malignant clonal process by cytogenetics can establish
the diagnosis of MDS, even in the absence of dysplasia or increased
blast counts, but the borderline between malignant and benign lesions
is obscured: a set of abnormalities, including 27/del(7q), 25/del(5q),
del(13q), del(11q), del(12p)/t(12p), del(17p)/t(17p)/i(17q), idic(X)(q13),
and other balanced and unbalanced translocations, is proposed to
support the diagnosis of MDS irrespective of morphology, whereas
2Y, 18, and del(20q) as a sole abnormality may not, because they
can be found in aged, apparently normal individuals.41,43,44 A substantial proportion of patients with persistent cytopenia lack conclusive
morphology and cytogenetics and may presumptively be classified
as ICUS.45,46
The difficulty in diagnosing MDS among AA patients lies in the
fact that both conditions often show indistinguishable phenotypes and
may even coexist in the same patients, although patients with AA
are nevertheless at high risk for MDS development. Many AA
patients who ever responded to IST are still cytopenic and, even
when a complete response is obtained, recurrence is common. In
MDS, BM is typically normo- to hypercellular, but ;10% of MDS
patients show hypocellular marrow, mimicking nonsevere AA. Conversely, AA often displays mild to moderate dysplasia, especially in
the erythroid lineage.47 Several drugs (eg, antimetabolites) and viral
infections can also produce an apparently dysplastic marrow picture.
Accurate evaluation of increased blasts and the presence of myeloid
dysplasia in the face of severely hypoplastic marrow is another
diagnostic challenge.48 Lastly, clonality is common among AA patients
as seen below, further obscuring the border between AA and MDS.
Skewed X-chromosome inactivation
In early studies, clonality in AA and other conditions was evaluated by
detecting nonrandom X-chromosome inactivation (NRXI; ie, skewing
from the expected 1:1 ratio for random lyonization).49 van Kamp et al
reported NRXI in 13 of 18 evaluable cases with AA.50 Ohashi51 and
Mortazavi et al52 also reported high frequencies (;40%) of NRXI,
CLONAL HEMATOPOIESIS IN AA
339
whereas frequencies are lower in other studies (11%-20%). The
discrepancy seems to be explained partly by small sample sizes and
also by different methods and genes/loci used for the detection of
NRXI.9,49 The interpretation of NRXI is further complicated by the
presence of extreme skewing (.3:1) in normal females, which can
occur during normal development (NRXI in neonates) but also can
be acquired during normal aging. In a large study using a highly
reliable human androgen receptor assay (HUMARA), NRXI was
seen in 37.9% of normal females aged .60 years vs 16.4% of those
aged 28 to 32 years and 8.6% of neonates.53,54
Abnormal cytogenetics in AA
Clonality is also evidenced by cytogenetic abnormalities, which have
been reported in 4% to 11% of AA cases,8,18,55-58 although difficulty in
obtaining sufficient numbers of metaphases from the failed marrow
could underestimate the real frequency, particularly at the time of
diagnosis. Predominant lesions are numerical abnormalities, which
include those commonly seen in myeloid malignancies, such as 18,
27, and del(5q), whereas other lesions, including 16 and 115, are
rarely seen in MDS/AML. Among these, MDS-specific abnormalities defined in the World Health Organization (WHO) criteria would
support the diagnosis of MDS, even without definitive morphological evidence. Especially, 27 is most frequently seen with a possible
association with the use of granulocyte colony-stimulating factor,
and consistently predicts a poor clinical outcome in AA.35,38,40 For
other lesions, their diagnostic value for MDS/AML has not been
established and their presence by itself does not support the diagnosis
of MDS. Del(13q) is a recurrent chromosomal abnormality assigned
to MDS-specific cytogenetic lesions in the WHO criteria, although
recent studies suggest that del(13q)-positive MDS cases might be
better related to AA, on the basis of their excellent response to IST.59-61
Single nucleotide polymorphism (SNP) array–based karyotyping can complement conventional cytogenetics by enabling
metaphase-independent detection of copy number abnormalities
(CNAs), and despite a compromised sensitivity for lesions carried
by ,20% of total cells, it reveals more abnormalities than conventional
karyotyping, also unmasking small cryptic lesions and copy-neutral,
uniparental disomy (UPD), which otherwise escape conventional
karyotyping.62,63 Among these most frequently observed are 6pUPDs,
which are recognized on visual inspection of SNP array karyotyping
in ;9% of AA cases and, with more sensitive evaluation of array
signals, detected in an additional 4% of the cases.16,18,63
Somatic mutations in AA
Somatic mutations are more reliable and sensitive markers for detecting
clonal populations than NRXI or cytogenetics. The revolutionized
technologies of next-generation DNA sequencing (NGS), together
with the accumulated knowledge about the major driver mutations in
MDS/AML,42,64 have prompted investigations on somatic mutations in AA in relation to clonal evolution to MDS/AML. Thus, since
the first report by Lane et al,13 there have been several NGS-based
mutation studies on AA during the past 2 years reporting varying
mutation frequencies ranging from 5.3% to 72%13-18 (Table 1). Among
these studies, mutations were most comprehensively studied by
Yoshizato et al, who surveyed mutations in 106 genes commonly
mutated in myeloid cancers in 439 patients using targeted-capture
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Table 1. Somatic mutations in AA
Study
13
Lane et al
N 5 39
Ratio of male to female (M/F, n)
Median age, y (range)
SAA/VSAA, n (%)
15
Kulasekararaj et al
N 5 150
14
Heuser et al
N 5 38
N 5 439 (All)
1.3 (244/195)
1.3 (22/17)
0.9 (71/79)
0.9 (18/20)
0.7 (9/13)
34.8 (4-65.7)
44 (17-84)
30 (9-79)
14.5 (1.5-61)
74 (49)
N 5 256 (NIH)
1.6 (157/99)
40.5 (2.5-88)
29.5 (2.5-82.5)
344 (78)
256 (100)
27 (71)
9 (41)
—
—
—
—
Targeted exons
219 genes
835 genes (n 5 57)
42 genes
—
Whole exome, n
—
—
—
22
0 (0)
0 (0%)
37 (8.4)
1 (2.6)
1 (4.5)
27 (6.2)
17 (6.6)
0 (0)
1 (4.5)
41 (9.3)
28 (10.9)
Serially collected samples, n
29 (74)
Yoshizato et al18
Babushok et al16
N 5 22
82
62
Platform for sequencing
106 genes
52
35
Mutated genes, n (%)
DNMT3A
1 (2.6)
8 (5.3)
ASXL1
2 (5.1)
12 (8.0)
BCOR/BCORL1
0 (0)
PIGA
ND
Other genes*
6 (15.1)
Any genes
9 (23.1)
Excluding PIGA alone
9 (23.1)
6 (4.0)
18 (12.0)
18 (7.0)
ND
9 (40.9)
33 (7.5)
25 (9.8)
1 (2.6)
5 (22.7)
67 (15.3)
29 (6.6)
29 (19.3)
2 (5.3)
16 (72.7)
157 (35.8)
90 (35.2)
29 (19.3)
2 (5.3)
137 (31.2)
74 (28.9)
Clone size
Median (range) (%)
Clone size ,10%, n (%)†
ND
7 (78)
Cytogenetic abnormalities, n (%)
ND
6pUPD (1) clone, n (%)
ND
PNH clone, n (%)
4 (10)‡
20 (1.5-68)
11 (14)
1
85 (57)
ND
ND
0 (0)
2 (9.1)
15.4 (2.4-96.4)
130 (29.6)
15.2 (2.4-76.7)
3 (7.9)
2 (9.1)
20 (8.5)
9 (3.8)
ND
3 (13.6)
55 (13.2)
31 (13.2)
79 (30.9)
17 (45)
10 (46)
221 (50.3)
110 (43.0)
256 (100)
IST, n (%)
Yes
19 (49)
107 (71.3)
ND
20 (91)
403 (92.4)
No
20 (51)
43 (28.7)
ND
2 (9)
33 (7.6)
17 (11.3)
ND
ND
Transformation to MDS/AML, n (%)
ND
47 (10.7)
0 (0)
36 (14.1)
F, female; M, male; ND, not determined; NIH, National Institutes of Health; SAA, severe AA; VSAA, very severe AA; —, no corresponding samples.
*Excluding DNMT3A, ASXL1, BCOR/BCORL1, and PIGA.
†Maximum clone size in mutation-positive cases.
‡Variant allele frequency .5%.
deep sequencing.18 AML/MDS-associated mutations were identified in 157 (35.8%) patients.65 Mutations were more frequently found
in older patients and were dominated by age-dependent C to G transitions. About one-third of patients had multiple mutations. When SNP
array karyotyping results were combined, clonality was demonstrated
in 47% of the total cohort. The mean allelic burden of mutations was
substantially smaller in AA compared with MDS: 75% of mutations
had ,10% of allelic burden. The majority of mutations (58/79)
detected after IST were shown to be already present at the time of
diagnosis, albeit at a much lower allele frequency in most cases
examined. Mutations were present in multilineage compartments in
6 of 6 cases, including HSCs, common myeloid progenitors, and
erythroid progenitors, as well as in a minor fraction of T cells, suggesting that the mutations occurred at a stem cell level.
Kulasekararaj et al also surveyed somatic mutations in 150 patients,
first by targeted-capture sequencing of 835 candidate genes in the discovery cohort (n 5 57), followed by NGS-based amplicon sequencing
of detected candidates (5 genes) in an additional 93 patients.15 Excluding PIGA mutations, somatic mutations were detected in 29 of 150
(19.3%) patients. In accordance with Yoshizato et al’s report, mutations
were more frequently observed in older patients and in those having
longer (.6 months) disease durations. Allelic burdens were generally
small, particularly in patients with shorter disease duration.
Characteristics of mutations in AA
Yoshizato et al also interrogated non-MDS/AML-related mutations
by whole exome sequencing (WES) in 52 patients, including 15 with
no detectable mutations in targeted sequencing. Four of the 15 patients
were newly found to carry 5 mutations that involved very large genes,
likely representing somatic mosaicism in blood reported in ;10% of
the human population.19 No other genes than MDS/AML-related genes
were recurrently mutated, suggesting that the latter genes should be the
major driver targets in AA.
Mutations most frequently affected 5 genes, including PIGA,
BCOR/BCORL1, DNMT3A, and ASXL1. Other MDS/AML-related
genes were also recurrently mutated but at lower frequencies
(Figure 3).18,20,66 The highly recurrent nature of well-characterized
driver mutations indicates that they were selected by a Darwinian
mechanism, rather than stochastically achieving a clonal dominance
merely by a rapid expansion from a small number of residual stem cells
(“bottleneck”).9 In several patients, some of these genes, including
PIGA, DNMT3A, ASXL1, BOCR, and RUNX1, harbor multiple,
independent mutations, suggesting a strong selective pressure favoring
evolution of cells having these mutations (Figure 3A).18
Whereas DNMT3A and ASXL1 are among common mutational
targets in both AA and MDS/AML, there was a substantial overrepresentation of PIGA and BCOR/BCORL1 mutations and underrepresentation of mutations in TET2, TP53, RUNX1, cohesion, and
splicing factors in AA compared with MDS/AML, most likely reflecting the difference in the mechanism of clonal selection between
both diseases and/or between mutations (Figure 3B). In accordance
with this, mutations exhibit different temporal behavior, age dependency, impacts on disease progression, overall survival, and response
to IST. DNMT3A and ASXL1 mutations tend to increase their clone
size over years, whereas BCOR/BCORL1 and PIGA mutations are
likely to show a decreasing or stable clone size. Generally, frequency
and the mean number of mutations increase with age, which is not
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CLONAL HEMATOPOIESIS IN AA
A
NIH
Cleveland
341
Japan
BCOR/BCORL1
DNMT3A
PIGA
ASXL1
RUNX1
TET2
TP53
SETBP1
GNAS
U2AF1
ZRSR2
CSMD1
RAD21
SRSF2
ATM
ATRX
EZH2
PHF6
RIT1
LAMB4
WT1
PEG3
JAK2
JAK3
Others
6pUPD/del(6p)
-7
del(13q)
+8
other CNA/UPD
multiple mutations
BCORL1 mutated
0
20 40 60
Number of cases
B
CBL
Others
EZH2 CBL
TP53
TET2
2.2%
cohesin
2.2%
2.7%
RUNX1
3.1%
Splicing
4.9%
facotrs
JAK2
Others
TP53
9.3%
PIGA
24.9%
ASXL1
BCOR/
BCORL1
12.9%
DNMT3A
16.9%
21.1%
Splicing factors
PIGA
BCOR/
1.9%
BCORL1
CBL 2.4%
2.4%
EZH2
25.4%
ASXL1
9.5%
2.9%
TP53
19.6%
TET2
4.7%
RUNX1
4.8%
Splicing 5.1%
facotrs
5.5%
19.9%
DNMT3A
61.9%
TET2
11.1%
5.0%
5.2%
DNMT3A
ASXL1
8.8%
Cohesin
AA
MDS
ARCH
(CHIP)
Figure 3. Genetic alterations in AA. (A) Somatic mutations and other genetic lesions in AA. Mutations and CNAs indicated on the left are shown for individual patients
(shown horizontally). Frequency of each genetic lesion is presented on the right. (B) Frequency of mutations in AA, MDS, and ARCH, according to the reports from Yoshizato
et al,18 Haferlach et al,66 and Jaiswal et al,20 respectively, for which the denominators are the total numbers of mutations in each report. ARCH, age-related CH; CHIP, CH with
indeterminate potential; NIH, National Institutes of Health.
the case for the latter set of mutations. As a whole, mutations do not
significantly affect response to IST, progression to MDS/AML,
or overall survival. However, when analyzed separately, mutations
in DNMT3A, ASXL1, TP53, RUNX1, and CSMD1 (“unfavorable”
mutations) are significantly associated with faster progression to
MDS/AML, shorter overall survival, and poor response to IST
compared with PIGA and BCOR/BCORL1 mutations (“favorable”
mutations) (;40% vs 3% of projected progression to MDS/AML
and 60% vs ;20% projected mortality for unfavorable vs favorable
mutations, respectively). Kulasekararaj et al also reported a significant
association of mutations with faster progression to MDS (38% for
mutated vs 6% for unmutated cases, P , .01), particularly for patients
with longer disease durations and larger clone size, although the small
cohort size seemed to prevent the analysis of the differential effect of
mutations.15
Dynamics of clones
Yoshizato et al illustrated the dynamics of clonal evolution found in
AA patients by combining WES and accurate determination of allelic
burden of detected mutations in 28 patients at multiple time points
(2-14; median, 5) along their clinical course over a median of 5 years
(range, 1-12 years).18 In the majority of cases, clones kept their small
clone size over years. However, in other patients who progressed to
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Table 2. CH in AA and the general population
Aplastic anemia
Mutations
Normal individuals
High (.50%)
MDS
Low (;2%)
Very high (;90%)*
Positive
Correlation to age
Positive
Positive
Mutated ,40 y, %
20-40
,1
.10
Mutated .70 y, %
52
Signature
Age-related C to T transitions
Age-related C to T transitions
Age-related C to T transitions
Commonly mutated genes
BCOR/BCORL1, PIGA, DNMT3A, ASXL1
DNMT3A, TET2, ASXL1, JAK2,
TET2, SF3B1, ASXL1, SRSF2,
Variant allele frequency
Low (generally ,10%)
Low (generally ,10%)
Copy number variations, %
;20
,2
;50
Cytogenetics SNP array karyotyping
UPD in 6p
del(20q)
25/del(5q)
27/del(7q)
del(13q)
27/del(7q)
16
del(11q)
18
18
17pLOH
17pLOH
115
UPD in 4q, 9p, 11q, 14q
TP53, SF3B1
del(13q)
Impact of CH on progression to
MDS/AML
;3% for BCOR, BCORL1, and PIGA
DNMT3A, RUNX1, TP53
High (.30%)
del(20q)
UPDs in 4q, 11q, 13q, 14q
Relative risk: ;103 to ;353
mutations
;40% for DNMT3A, ASXL1, RUNX1,
TP53, and CSMD2 mutations
*By abnormal cytogenetic and somatic mutations.
MDS/AML, the size of dominant clones invariably increased over time.
Evolution of new clones was quite common, often accompanied by
complete sweeping of preexisting clones. Nevertheless, an increasing
clone size did not necessarily herald MDS/AML progression but was
compatible with completely normal peripheral blood counts, even
when patients’ hematopoiesis was mostly supported by these clones.
Even in the patients who eventually developed MDS/AML, the
dominance of clones having typical MDS/AML-related mutations
persisted for years without the diagnosis of MDS/AML.
Age-related CH in the general population
Notably, CH is also found in the general population. It was originally
suggested by the studies on NRXI53,54 and has recently been investigated more extensively using advanced genomics. By reanalyzing
SNP array data generated for .50 000 individuals who did not
carry hematologic cancer, Jacobs et al43 and Laurie et al44 demonstrated
that CNAs and UPDs were observed at increasing frequencies in
peripheral blood from elderly people (;2% to 3%) compared with
those from younger people (;,0.5%) (ARCH). Some authors describe the same phenomenon under the term “CHIP” (CH with indeterminate potential), although the potential may not be totally
indeterminate. The most frequent lesions include del(20q), del(11q),
del(13q), and UPDs in 4q, 11q, 13q, and 14q, which are common in
myeloid malignancies.41,67-70 Importantly, ARCH is shown to be
associated with a substantially high incidence of developing hematologic cancer (hazard ratio,10-35.4).19,20,43,44
Busque et al reported TET2 mutations in PNMs (not in lymphoid
cells) from 10 of 182 (5.6%) elderly women (.65 years old) with
NRXI, but not in 95 younger women (,60 years old) or 105 elderly
women without NRXI.71 More comprehensive surveys of leukemia/
MDS-associated mutations in the general population were conducted by repurposing WES data of peripheral blood DNA from
large population-based studies. Jaiswal et al focused on 160 candidate
mutational targets reported in hematologic cancers and found mutations
in 73 genes in 746 of 17 182 participants.20 Genovese et al, by contrast,
analyzed somatic mutations in 12 380 participants in an unbiased
fashion, by evaluating those variants present only in a subfraction
of cells. They found a total of 1918 participants having at least 1
putative somatic mutation, of which 308 individuals carrying known
cancer-associated driver mutations and 195 others who had no putative
drivers but had $3 somatic mutations were considered to have
CH.19 In agreement with studies on CNAs, the frequency of CH
in both studies increased with age (;1% in those ,50 years old vs
;10% in those .65 years old) and, as with CH in AA, it was dominated
by age-related C to T transitions. A similar finding was reported
using whole exome data of cancer-bearing patients with no evidence
of hematologic cancer.21 Given the limitation of WES to capture
mutations with low allelic burdens, the incidence should be even
higher when mutations are detected by deep sequencing.68 Strikingly, recurrent mutations were highly biased to common driver
genes implicated in myeloid malignancies and most frequently
affected DNMT3A, followed by TET2, ASXL1, TP53, SF3B1,
JAK2, and SRSF2, which differed substantially from the case with
AA (Figure 3B); in AA, PIGA and BCOR/BCORL1 were more
frequently mutated than DNMT3A and ASXL1, whereas mutations
in TET2, TP53, SF3B1, JAK2, and SRSF2 were infrequent. As is
the case with ARCH associated with CNAs, individuals carrying
mutations have a higher risk for developing hematologic cancer
(hazard ratio, 11.1-12.9) and all-cause mortality, compared to
individuals without CH.19,20
Mechanisms of CH in AA
CH is highly prevalent among patients with AA (;50%), in which 2
types of genetic alterations seem to be involved in the clonal selection,
although both are not necessarily mutually exclusive (Table 2): (1)
mutations and CNAs commonly seen in MDS/AML (eg, 27,
DNMT3A, ASXL1, and other mutations) and (2) those highly specific
to or overrepresented in AA (PIGA/BOCR/BCORL1 mutations and
6pUPD). The 2 types of alterations show different chronological
behaviors, age distribution, impacts on response to IST, progression to
MDS/AML, and overall survival, suggesting discrete mechanisms of
clonal selection.
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BLOOD, 21 JULY 2016 x VOLUME 128, NUMBER 3
Figure 4. A possible model for clonal evolution in
AA. Upon immune-mediated destruction of BM, some
clones are thought to be resistant to the inciting autoimmune insult and/or show faster cycling/less apoptosis than others upon BM recovery to achieve a clonal
dominance. In some cases, the dominant clones, especially those having DNMT3A, ASXL1, and other unfavorable mutations, increase their clone size, giving
rise to more selectively dominant clones therein. In
other cases, typically those carrying PIGA mutations
(and possibly BCOR/BCOR mutations), initially dominant clones may regress or remain stable over years.
CLONAL HEMATOPOIESIS IN AA
Evolution
Rapid
expansion
Immune-mediate
BM destruction
Aged BM
DNMT3A
ASXL1
RUNX1
etc
IST
PIGA
BCOR
BCORL1
Selection
Escape?
↑Proliferation?
MDS/AML-related mutations in AA show a clear age dependency
and are dominated by C to T transitions,18 as also seen in ARCH,19,20
suggesting that these mutations could have a common origin, randomly
acquired with aging (Figure 4).72 Given their tight association with
MDS/AML, it might be postulated that these mutations are clonally
selected in AA and in the general population through a similar mechanism that would operate in the development of MDS/AML. In fact,
in those AA patients who developed MDS/AML, an initial clone
having 1 or more MDS/AML-related mutations gradually expanded
and eventually gave rise to MDS/AML,18 although not all patients carrying those mutations progressed to MDS/AML. However, despite frequent DNMT3A and ASXL1 mutations in AA and
MDS/AML, relative frequencies of other MDS/AML-related mutations
differ substantially between both conditions; for example, TET2, JAK2,
and splicing factors represent common targets of mutations in ARCH
but are affected at lower frequencies in AA (Figure 3B). In contrast to the
very high rate of progression to MDS/AML among AA patients with
unfavorable mutations, the absolute risk of ARCH for MDS/AML
progression still remained modest (;0.5% to 1% per year) (Table 2).73
A
343
Loss of
selective
advantage?
A possible explanation would be that in AA, BM is repopulated from a
small number of residual stem cells after IST, which could provide
clones having a proliferative advantage with a higher chance of cell
cycling to achieve clonal dominance (bottleneck effects), compared with
the case of ARCH in a normally populated BM environment (Figure 4).9
Alternatively, the autoimmune environment can preferentially select
DNMT3A and ASXL1 mutations over others.
Immune escape as the mechanism for CH
The other set of lesions, including 6pUPD and PIGA mutations, are
highly specific to AA, for which autoimmunity has been implicated in
the mechanisms of clonal selection (“clonality as escape”) (Figure 4), as
first proposed by Young et al.5 6pUPD represents the most frequent
genetic lesion in AA.18,63 All 6pUPDs critically involve the class I HLA
locus proximally, leading to uniparental expression of the retained HLA
class I alleles.63 Significantly, most of the patients (36/40) with 6pUPD
B
HLA locus
A
1Mb
C
B
DRB1
DQB1
DPB1
C
CTL
CTL
CTL
TCR
TCR
TCR
ch6
ch6
UPD
6pUPD (+) cells
?
PIGA
mut
PIGA-mutated cells
Figure 5. Putative mechanism of immune escape
of 6pUPD-positive clones. (A) A schematic diagram of
6pUPD, which is thought to result from a recombination
between 2 homologous chromosomes, invariably involving 6pter distally. (B) Breakpoint mapping of 6pUPD
in different patients, showing a prominent breakpoint
cluster within the HLA class I region. Breakpoints in
critical cases are shown by arrows. (C) 6pUPD-positive
(1) HSCs permanently lose the relevant HLA class I
molecule required for the presentation of the putative
antigen and are thereby thought to escape destruction by CTLs. The 6pUPD-mediated mechanism
for immune escape in AA is also supported by the
presence of multiple independent 6pUPD clones affecting the same parental HLA allele in some patients.63
From www.bloodjournal.org by guest on July 26, 2016. For personal use only.
344
BLOOD, 21 JULY 2016 x VOLUME 128, NUMBER 3
OGAWA
A
Aplastic Anemia
B
Unexplained Cytopenia
MDS (WHO)
S
MDS (WHO)
U
IC
Clonality
by
‘MDS-specific’
Cytogenetic
lesions
Documented
Clonality
DNMT3A
Unfavorable
ASXL1
TP53 etc. mutations
PIGA
BCOR/BCORL1
6pUPD etc.
Clonality
by
‘MDS-specific’
Cytogenetic
lesions
DNMT3A
TET2
ASXL1
PRM1D
TP53, etc.
CCUS
ARCH
(CHIP)
Documented
Clonality
Figure 6. Clonality and MDS diagnosis in AA and other refractory cytopenia. (A) Clonality (orange) is common (.50%) in AA when assessed by gene mutations and
other genetic lesions, such as cytogenetic abnormalities, using advanced genomics. However, the clonal evolution does not necessarily mean the development of diseases,
especially malignant ones, such as MDS. According to the current World Health Organization (WHO) criteria, patients with AA are diagnosed as having MDS when cytopenia
is present and clonality is evidenced by MDS-specific cytogenetic abnormalities, such as monosomy 7, even in the absence of dysplasia or increased blast counts (pink circle).
Their clone size is not relevant as long as they are found at $2 metaphases. On the other hand, not all clonality is associated with MDS diagnosis (and vice versa). For
example, isolated abnormalities of trisomy 8, del(20q), and –Y, are not considered to be sufficient evidence of MDS diagnosis without morphological evidence or increased
blast counts, because these isolated lesions may be found in normal individuals and do not likely to correlate with typical MDS pictures. Similarly, without other evidence of
MDS, somatic mutations, including those affecting common targets of myeloid malignancies, are not thought to be evidence of malignant clonal evolution by themselves. In
fact, in many AA cases, somatic mutations or other clonality can be compatible with normal or almost normal blood counts. By contrast, patients with unfavorable mutations
(blue circle) are more likely to show poor prognosis and to satisfy the WHO criteria for MDS/AML during their clinical course than patients with other mutations. (B) A parallel
relationship is also found in unexplained cytopenia in the general population. According to the WHO criteria, the clonality (cytogenetic) criteria of MDS are reserved for MDSspecific lesions, which suffice for the diagnosis of MDS, even without evidence of dysplasia or increased blast counts. Those patients with unexplained refractory cytopenia
having no evidence of MDS (between red and blue circles) are designated as ICUS. Some ICUS patients show evidence of clonal evolution (CCUS), and recent reports have
suggested some similarity between CCUS and MDS (see main text), for which further confirmation is needed. CCUS, clonal ICUS.
carry 1 or more of 4 HLA alleles (A*02:01, A*02:06, A*31:01, and
B*40:02), which are significantly enriched in AA (and also in PNH74)
patients compared with the general population, and are the alleles
consistently lost in UPD-positive clones.63 These observations would
lead to a hypothesis that unknown causative antigens are presented to
cytotoxic T cells via these class I HLAs, loss of which in 6pUPDpositive HSCs enables escape from cytotoxic T cell–mediated destruction (Figure 5). A similar mechanism seems to operate in leukemic
relapse after haploidentical transplantation, in which relapsed leukemic
cells are thought to escape alloimmunity by losing the mismatched
haploid alleles via 6pUPD.75,76
A conspicuous correlation between AA and the PNH-type cells,
together with the frequent presence of multiple PIGA-mutated clones in
10% to 30% of AA/PNH patients,15,18,77,78 indicates a strong selective
advantage of PIGA-mutated cells that would be sufficient for clonal
expansion of PNH-type cells within BM in AA patients,79,80 although
there is no direct experimental evidence supporting this hypothesis.
Nevertheless, this does not necessarily exclude a possible role of
secondary mutations in clonal selection of PIGA-mutated cells, which
has long been discussed. Variious plausible secondary driver alterations
have been reported in occasional cases and implicated in the clonal
outgrowth of PIGA-mutated clones, including those affecting HMGA2,
NRAS, JAK2, TET2, U2AF1, and even BCR/ABL.78,81-84 However,
thus far, no consistently recurrent mutational targets have been identified, even with WES,78 precluding the indispensable role of these
secondary mutations in the development of PNH.
Currently, the underlying molecular mechanism for immune escape
of PIGA-mutated cells in AA is poorly understood. A possible
mechanism would include loss of expression of GPI-anchored proteins
critical for immune recognitions.79,80 PIGA-mutated clones rarely
disappear but, more often, persist, even after successful IST, which is
in agreement with the theory of natural selection in the population
genetics; many functional variants selected against past, but not present,
insults are widely observed among the human population.85 Frequent
genetic drifts displayed by PNH-type clones18,86,87 also seem to favor
the explanation.
Clinical implications of somatic mutations
In the light of these new findings on CH in AA, as well as in the general
population, an important question would be, How could we translate
them into better clinical practice and improved outcome of patients? An
extremely elevated risk of MDS/AML progression and high mortality
rates associated with unfavorable mutations would reasonably raise
a possibility of early intervention with allogeneic stem cell transplant
for AA patients with unfavorable mutations, which should be best
investigated in a setting of prospective trials.88 Also, the current WHO
criteria for MDS could be extended for those patients having a set of
unfavorable mutations on the basis of their impact on clinical/biological
outcomes (Figure 6A), which is exactly the same way the current
cytogenetic criteria have been included.
Of interest in this regard are 2 recent studies on somatic mutations among ICUS patients, because these patients also had persistent cytopenia with no definitive evidence of MDS. Kwok et al
reported that a substantial fraction of ICUS patients harbored
similar MDS/AML-related mutations showing comparable allelic
burdens to those found in low-risk MDS.89 Cargo et al, on the other
hand, investigated prediagnostic samples from ICUS patients who
eventually progressed to MDS/AML and observed a high frequency
of MDS/AML-related mutations comparable to those found in
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BLOOD, 21 JULY 2016 x VOLUME 128, NUMBER 3
CLONAL HEMATOPOIESIS IN AA
MDS patients in their number and variant allele frequency.90 These
findings might also suggest a possibility that these ICUS patients
with MDS/AML-related mutations (or clonal cytopenias of undetermined significance73,89) could be better considered as having
MDS or pre-MDS (Figure 6B). Lastly, it cannot be more underscored
that regardless of the definition of MDS, careful examination of
patients for hematologic status/morphology, symptoms, and clinical
course, as well as detailed history taking, should constitute the
indispensable basis for a pertinent utilization of these new findings
for better management of AA patients and other individuals presented with unexplained cytopenia.
345
of different mutations should help in the accurate diagnosis/prediction of
malignant evolution and in better management of AA patients.
Acknowledgments
The author thanks Tetsuichi Yoshizato and Neal S. Young for helpful
discussions on CH in AA.
This work was supported by Grants-in-Aid from the Ministry of
Health, Labor, and Welfare of Japan (Health and Labor Sciences
Research Expenses for Commission and Applied Research for
Innovative Treatment of Cancer).
Concluding remarks
Recent advances in high-throughput sequencing technologies have
revealed an unexpected complexity of CH in AA, which is closely related
to, but in several respects distinct from, CH found in the aged normal
population, in which immune-mediated BM destruction and subsequent
recovery in AA seem to play a critical role. CH is common in AA, in
which clones may eventually proceed to malignant transformation in
some cases, but in others, may continue to support apparently normal
hematopoiesis. Mutation types can predict the behavior of clones and
patients’ clinical course to some extent, but is not totally predictable,
suggesting a potential value of monitoring CH using NGS now widely
available. Understanding of the mechanism underlying clonal selection
Authorship
Contribution: S.O. conceived this review article, analyzed the literature,
wrote the manuscript, and prepared the illustrations.
Conflict-of-interest disclosure: The author declares no competing
financial interests.
Correspondence: Seishi Ogawa, Department of Pathology and
Tumor Biology, Graduate School of Medicine, Kyoto University,
Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan; e-mail:
[email protected].
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2016 128: 337-347
doi:10.1182/blood-2016-01-636381 originally published
online April 27, 2016
Clonal hematopoiesis in acquired aplastic anemia
Seishi Ogawa
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