Review Article

CME Article

Clonal hematopoiesis in acquired aplastic anemiaSeishi 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, in-

cluding paroxysmal nocturnal hemoglo-

binuria 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, themolecular

aspect of CH in AA has been clarified by

comprehensive detection of somatic mu-

tations thatdriveclonalevolution.Genetic

abnormalities are found in∼50%of patients

withAAand, except forPIGAmutations and

copy-neutral loss-of-heterozygosity, or

uniparental disomy (UPD) in 6p (6pUPD),

are most frequently represented by mu-

tations involving genes commonly mu-

tated in myeloid malignancies, including

DNMT3A, ASXL1, and BCOR/BCORL1.

Mutations exhibit distinct chronological

profilesandclinical impacts.BCOR/BCORL1

and PIGA mutations tend to disappear or

showstable clonesize andpredict abetter

response to IST and a significantly better

clinical outcome compared with mutations

inDNMT3A,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

ASXL1mutations, also commonly seen in

CH in the general population, indicate a

close link toCH in theagedbonemarrow, in

terms of the mechanism for selection. De-

tection and close monitoring of somatic

mutations/evolution may help with predic-

tion and diagnosis of clonal evolution of

MDS/AML and better management of pa-

tients with AA. (Blood. 2016;128(3):337-347)

Introduction

Acquired aplastic anemia (AA) is a prototype of bone marrow (BM)failure caused by immune-mediated destruction of hematopoietic stemand/or progenitor cells, where autoreactive T cells are supposed to playa central role (Figure 1).1-6 Although almost uniformly terminating in afatal outcome in a previous era, management of AA has drastically

improved since the late 1970s with the introduction of allogeneic stemcell transplantation and IST, as well as optimized supportive care.5

However, the entire picture of the disease is more complicated thanexpected for a simple immune-mediated marrow failure. Amongothers, the frequent development of late clonal diseases, including

Medscape Continuing Medical Education onlineThis activity has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council forContinuing Medical Education through the joint providership of Medscape, LLC and the American Society of Hematology.Medscape, LLC is accredited by the ACCME to provide continuing medical education for physicians.Medscape, LLC designates this Journal-based CME activity for a maximum of 1.0 AMA PRA Category 1 Credit(s)™. Physiciansshould claim only the credit commensurate with the extent of their participation in the activity.All other clinicians completing this activity will be issued a certificate of participation. To participate in this journal CME activity:(1) review the learning objectives and author disclosures; (2) study the education content; (3) take the post-test with a 75% minimumpassing score and complete the evaluation at http://www.medscape.org/journal/blood; and (4) view/print certificate. For CMEquestions, see page 464.DisclosuresLaurie Barclay, freelance writer and reviewer, Medscape, LLC, owns stock, stock options, or bonds from Pfizer. Associate EditorJean 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 areview.

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

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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paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplasticsyndromes (MDS)/acute myeloid leukemia (AML), is known as anenigmatic complication in AA, which may not instantaneously beunderstood under the category of immune-mediated disorders.7,8 Theenigma is further highlighted by the evidence suggesting the presenceof clonal hematopoiesis (CH), even in patients who do not developapparent clonal diseases.9 It should be noted that clonal evolution (ie,evolution of clones) does not necessarily mean the development ofcancer or any other diathesis, because it can be compatible with normalhomeostasis.10,11

Indeed, clonality in AA has been one of the long-standing issues inhematology, which was first implicated by Dameshek asking thequestion, What do AA, PNH, and hypoplastic leukemia have incommon?12 Since then, many authors have revisited his riddle, whichcouldnowbe rephrasedas,What is the commonunderlyingmechanismfor, and the link between, CH and the evolution of clonal disorders inAA?8 More recently, the issue has been newly addressed by usingrevolutionized sequencing technologies, unmasking a unique picture ofCH in AA frequently accompanied by somatic mutations commonlyseen inmyeloidmalignancies.13-18 Combinedwith seminal findings onCH in aged normal individuals,19-21 as well as on preleukemic clones22

in patients with hematologic malignancies,23-25 the finding on somaticmutations in AA provides new insight into the origin and themechanism of frequent CH in AA and its impact on the late develop-ment of MDS and AML.13,15,18 This review summarizes recentprogress in the current understanding of CH in AA in relation to thepathogenesis of late clonal diseases.

Late clonal diseases in AA

The early evidence suggesting a link between AA and clonality was anunduly high incidence of PNH and MDS/AML among patients withAA.8 Although their incidence substantially increases among patientswho are successfully treated by IST,9,12 the complication of clonaldiseases seems to be an intrinsic feature of AA irrespective of IST,because it had longbeennoted evenbefore thewidespread use of IST.26

PNH is a unique form of BM failure characterized by acquiredhemolytic anemia and thrombosis, which is caused by a clonalexpansion of cells derived from a hematopoietic stem cell (HSC)carrying a somaticmutation involving thePIGA gene.27PIGA-mutated

cells show defective cell surface expression of glycosylphosphatidy-linositol (GPI)-anchored proteins, such as CD55 and CD59, which isresponsible for the vulnerability to complement-mediated hemolysis.27

AA and PNH are closely related conditions: AA may develop duringthe course of PNH and vice versa (AA/PNH syndrome), both beingpredisposed toAMLandMDS.28,29Complication of clinically relevantPNH has been described in 15% to 25%of the patients withAA treatedby IST.30-35 Moreover, regardless of typical manifestations of PNH,GPI anchor–deficient, “PNH-type” cells are detectable at even higherfrequencies (up to 67%) when assessed by sensitive flow cytometryusing antibodies to CD55 or CD59 and/or fluorescent aerolysin(FLAER).36,37

Another major category of late clonal diseases includes MDS andAML. The cumulative incidence of MDS/AML increases after IST,ranging from 5% to 15% within the observation period of 5 to 11.3years.35,38-40 Because the development of MDS/AML consistentlyconveys a dismal prognosis inAApatients, it is of particular importance

antigen Class I HLA

CTL

HSC

IFNγ

TNFα

BM suppression

TNFα

IFNγIF

A B

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.

Cytopenia

ICUSAA

Dysplasia/increased RS

MDS

Excess of

blasts

Clonality (specific

cytogenetic lesions)

PNH

Figure 2. Overlaps between AA, PNH, and MDS. Although conceptually rep-

resenting 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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to predict, diagnose, and initiate due interventions to these complica-tions during the course of AA. However, this is often complicatedby difficulty in diagnosing MDS among patients with AA, whichrepresents a major challenge to clinicians and hematopathologists.

Diagnosing MDS in AA patients

MDS is defined as refractory cytopenia caused by a neoplasticproliferationof clones committed to themyeloid lineage.The clones arethought to be neoplastic, in that their growth is no longer compatiblewith normal hematopoiesis, causing ineffective production of matureblood cells, enhanced apoptosis, and even progression to AML, al-though the clinical presentation is highly variable.41,42 Thus, at leastconceptually, clonality is an essential requisite for the pathophysiologyof MDS, but for the sake of clinical/pathological diagnosis, cliniciansand pathologists usually diagnose the involvement of a neoplasticprocess primarily relying on cell morphology, in which the presence ofexcess blasts ($5%) (propensity to leukemia) and/or dysplasia (in$10% of myeloid cells) with or without increased ($15%) ringsideroblasts (deregulated myelopoiesis) empirically, but consistently,indicates that the defective hematopoiesis is caused by malignantclones, although morphologic examination may not necessarily beconsistent between observers (Figure 2).41 Alternatively, direct dem-onstration of a malignant clonal process by cytogenetics can establishthe diagnosis of MDS, even in the absence of dysplasia or increasedblast counts, but the borderline between malignant and benign lesionsis obscured: a set of abnormalities, including27/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 tosupport the diagnosis of MDS irrespective of morphology, whereas2Y, 18, and del(20q) as a sole abnormality may not, because theycan be found in aged, apparently normal individuals.41,43,44 A sub-stantial proportion of patients with persistent cytopenia lack conclusivemorphology and cytogenetics and may presumptively be classifiedas ICUS.45,46

The difficulty in diagnosing MDS among AA patients lies in thefact that both conditions often show indistinguishable phenotypes andmay even coexist in the same patients, although patients with AAare nevertheless at high risk for MDS development. Many AApatients who ever responded to IST are still cytopenic and, evenwhen a complete response is obtained, recurrence is common. InMDS, BM is typically normo- to hypercellular, but ;10% of MDSpatients show hypocellular marrow, mimicking nonsevere AA. Con-versely, AA often displays mild to moderate dysplasia, especially inthe erythroid lineage.47 Several drugs (eg, antimetabolites) and viralinfections can also produce an apparently dysplastic marrow picture.Accurate evaluation of increased blasts and the presence of myeloiddysplasia in the face of severely hypoplastic marrow is anotherdiagnostic challenge.48Lastly, clonality is commonamongAApatientsas 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 bydetecting nonrandom X-chromosome inactivation (NRXI; ie, skewingfrom the expected 1:1 ratio for random lyonization).49 van Kamp et alreported NRXI in 13 of 18 evaluable cases with AA.50 Ohashi51 andMortazavi et al52 also reported high frequencies (;40%) of NRXI,

whereas frequencies are lower in other studies (11%-20%). Thediscrepancy seems to be explained partly by small sample sizes andalso by different methods and genes/loci used for the detection ofNRXI.9,49 The interpretation of NRXI is further complicated by thepresence of extreme skewing (.3:1) in normal females, which canoccur during normal development (NRXI in neonates) but also canbe acquired during normal aging. In a large study using a highlyreliable human androgen receptor assay (HUMARA), NRXI wasseen in 37.9% of normal females aged.60 years vs 16.4% of thoseaged 28 to 32 years and 8.6% of neonates.53,54

Abnormal cytogenetics in AA

Clonality is also evidenced by cytogenetic abnormalities, which havebeen reported in 4% to 11%ofAA cases,8,18,55-58 although difficulty inobtaining sufficient numbers of metaphases from the failed marrowcould underestimate the real frequency, particularly at the time ofdiagnosis. Predominant lesions are numerical abnormalities, whichinclude those commonly seen in myeloid malignancies, such as18,27, and del(5q), whereas other lesions, including16 and115, arerarely seen in MDS/AML. Among these, MDS-specific abnormal-ities defined in theWorldHealthOrganization (WHO) criteria wouldsupport the diagnosis of MDS, even without definitive morpholog-ical evidence. Especially,27 is most frequently seen with a possibleassociation with the use of granulocyte colony-stimulating factor,and consistently predicts a poor clinical outcome in AA.35,38,40 Forother lesions, their diagnostic value for MDS/AML has not beenestablished and their presence by itself does not support the diagnosisof MDS. Del(13q) is a recurrent chromosomal abnormality assignedto MDS-specific cytogenetic lesions in the WHO criteria, althoughrecent studies suggest that del(13q)-positive MDS cases might bebetter related toAA,on the basis of their excellent response to IST.59-61

Single nucleotide polymorphism (SNP) array–based karyo-typing can complement conventional cytogenetics by enablingmetaphase-independent detection of copy number abnormalities(CNAs), and despite a compromised sensitivity for lesions carriedby,20% of total cells, it reveals more abnormalities than conventionalkaryotyping, also unmasking small cryptic lesions and copy-neutral,uniparental disomy (UPD), which otherwise escape conventionalkaryotyping.62,63 Among these most frequently observed are 6pUPDs,which are recognized on visual inspection of SNP array karyotypingin ;9% of AA cases and, with more sensitive evaluation of arraysignals, detected in an additional 4% of the cases.16,18,63

Somatic mutations in AA

Somaticmutations aremore reliable and sensitivemarkers for detectingclonal populations than NRXI or cytogenetics. The revolutionizedtechnologies of next-generation DNA sequencing (NGS), togetherwith the accumulated knowledge about themajor driver mutations inMDS/AML,42,64 have prompted investigations on somatic muta-tions inAA in relation to clonal evolution toMDS/AML. Thus, sincethe first report by Lane et al,13 there have been several NGS-basedmutation studies on AA during the past 2 years reporting varyingmutation frequencies ranging from 5.3% to 72%13-18 (Table 1). Amongthese studies, mutations were most comprehensively studied byYoshizato et al, who surveyed mutations in 106 genes commonlymutated in myeloid cancers in 439 patients using targeted-capture

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deep sequencing.18 AML/MDS-associated mutations were identi-fied in 157 (35.8%) patients.65 Mutations were more frequently foundin older patients and were dominated by age-dependent C to G tran-sitions.About one-third ofpatients hadmultiplemutations.WhenSNParray karyotyping results were combined, clonality was demonstratedin 47% of the total cohort. The mean allelic burden of mutations wassubstantially smaller in AA compared with MDS: 75% of mutationshad ,10% of allelic burden. The majority of mutations (58/79)detected after IST were shown to be already present at the time ofdiagnosis, albeit at a much lower allele frequency in most casesexamined.Mutationswere present inmultilineage compartments in6 of 6 cases, including HSCs, common myeloid progenitors, anderythroid progenitors, as well as in a minor fraction of T cells, sug-gesting that the mutations occurred at a stem cell level.

Kulasekararaj et al also surveyed somaticmutations in 150 patients,first by targeted-capture sequencing of 835 candidate genes in the dis-covery cohort (n 5 57), followed by NGS-based amplicon sequencingof detected candidates (5 genes) in an additional 93 patients.15 Exclud-ing PIGA mutations, somatic mutations were detected in 29 of 150(19.3%)patients. In accordancewithYoshizatoet al’s report,mutationswere more frequently observed in older patients and in those havinglonger (.6 months) disease durations. Allelic burdens were generallysmall, particularly in patients with shorter disease duration.

Characteristics of mutations in AA

Yoshizato et al also interrogated non-MDS/AML-related mutationsby whole exome sequencing (WES) in 52 patients, including 15 with

no detectable mutations in targeted sequencing. Four of the 15 patientswere newly found to carry 5 mutations that involved very large genes,likely representing somatic mosaicism in blood reported in ;10% ofthe humanpopulation.19Noother genes thanMDS/AML-related geneswere recurrently mutated, suggesting that the latter genes should be themajor driver targets in AA.

Mutations most frequently affected 5 genes, including PIGA,BCOR/BCORL1, DNMT3A, and ASXL1. Other MDS/AML-relatedgenes were also recurrently mutated but at lower frequencies(Figure 3).18,20,66 The highly recurrent nature of well-characterizeddriver mutations indicates that they were selected by a Darwinianmechanism, rather than stochastically achieving a clonal dominancemerely by a rapid expansion froma small number of residual stemcells(“bottleneck”).9 In several patients, some of these genes, includingPIGA, DNMT3A, ASXL1, BOCR, and RUNX1, harbor multiple,independentmutations, suggesting a strong selective pressure favoringevolution of cells having these mutations (Figure 3A).18

Whereas DNMT3A and ASXL1 are among common mutationaltargets in both AA and MDS/AML, there was a substantial overrep-resentation of PIGA and BCOR/BCORL1 mutations and underrep-resentation of mutations in TET2, TP53, RUNX1, cohesion, andsplicing factors in AA compared with MDS/AML, most likely re-flecting the difference in the mechanism of clonal selection betweenboth diseases and/or between mutations (Figure 3B). In accordancewith this, mutations exhibit different temporal behavior, age depen-dency, impacts on disease progression, overall survival, and responseto IST. DNMT3A and ASXL1 mutations tend to increase their clonesize over years, whereas BCOR/BCORL1 and PIGA mutations arelikely to show a decreasing or stable clone size. Generally, frequencyand the mean number of mutations increase with age, which is not

Table 1. Somatic mutations in AA

Study

Lane et al13 Kulasekararaj et al15 Heuser et al14 Babushok et al16Yoshizato et al18

N 5 39 N 5 150 N 5 38 N 5 22 N 5 439 (All) N 5 256 (NIH)

Ratio of male to female (M/F, n) 1.3 (22/17) 0.9 (71/79) 0.9 (18/20) 0.7 (9/13) 1.3 (244/195) 1.6 (157/99)

Median age, y (range) 34.8 (4-65.7) 44 (17-84) 30 (9-79) 14.5 (1.5-61) 40.5 (2.5-88) 29.5 (2.5-82.5)

SAA/VSAA, n (%) 29 (74) 74 (49) 27 (71) 9 (41) 344 (78) 256 (100)

Serially collected samples, n — — — — 82 62

Platform for sequencing

Targeted exons 219 genes 835 genes (n 5 57) 42 genes — 106 genes

Whole exome, n — — — 22 52 35

Mutated genes, n (%)

DNMT3A 1 (2.6) 8 (5.3) 0 (0) 0 (0%) 37 (8.4) 18 (7.0)

ASXL1 2 (5.1) 12 (8.0) 1 (2.6) 1 (4.5) 27 (6.2) 17 (6.6)

BCOR/BCORL1 0 (0) 6 (4.0) 0 (0) 1 (4.5) 41 (9.3) 28 (10.9)

PIGA ND 18 (12.0) ND 9 (40.9) 33 (7.5) 25 (9.8)

Other genes* 6 (15.1) 1 (2.6) 5 (22.7) 67 (15.3) 29 (6.6)

Any genes 9 (23.1) 29 (19.3) 2 (5.3) 16 (72.7) 157 (35.8) 90 (35.2)

Excluding PIGA alone 9 (23.1) 29 (19.3) 2 (5.3) 137 (31.2) 74 (28.9)

Clone size

Median (range) (%) ND 20 (1.5-68) ND ND 15.4 (2.4-96.4) 15.2 (2.4-76.7)

Clone size ,10%, n (%)† 7 (78) 11 (14) 0 (0) 2 (9.1) 130 (29.6) 79 (30.9)

Cytogenetic abnormalities, n (%) ND 3 (7.9) 2 (9.1) 20 (8.5) 9 (3.8)

6pUPD (1) clone, n (%) ND 1 ND 3 (13.6) 55 (13.2) 31 (13.2)

PNH clone, n (%) 4 (10)‡ 85 (57) 17 (45) 10 (46) 221 (50.3) 110 (43.0)

IST, n (%)

Yes 19 (49) 107 (71.3) ND 20 (91) 403 (92.4) 256 (100)

No 20 (51) 43 (28.7) ND 2 (9) 33 (7.6) 0 (0)

Transformation to MDS/AML, n (%) ND 17 (11.3) ND ND 47 (10.7) 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%.

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the case for the latter set of mutations. As a whole, mutations do notsignificantly affect response to IST, progression to MDS/AML,or overall survival. However, when analyzed separately, mutationsin DNMT3A, ASXL1, TP53, RUNX1, and CSMD1 (“unfavorable”mutations) are significantly associated with faster progression toMDS/AML, shorter overall survival, and poor response to ISTcompared with PIGA and BCOR/BCORL1 mutations (“favorable”mutations) (;40% vs 3% of projected progression to MDS/AMLand 60% vs;20% projectedmortality for unfavorable vs favorablemutations, respectively). Kulasekararaj et al also reported a significantassociation of mutations with faster progression to MDS (38% formutated vs 6% for unmutated cases, P, .01), particularly for patientswith longer disease durations and larger clone size, although the small

cohort size seemed to prevent the analysis of the differential effect ofmutations.15

Dynamics of clones

Yoshizato et al illustrated the dynamics of clonal evolution found inAA patients by combining WES and accurate determination of allelicburden 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 smallclone size over years. However, in other patients who progressed to

BCOR/BCORL1

PIGAASXL1RUNX1TET2TP53

SETBP1GNASU2AF1ZRSR2CSMD1RAD21SRSF2ATM

ATRXEZH2PHF6RIT1

LAMB4WT1

PEG3JAK2JAK3

Others

6pUPD/del(6p)-7

del(13q)+8

multiple mutations BCORL1 mutated

Number of cases0 20 40 60

DNMT3A

other CNA/UPD

NIH Cleveland JapanA

PIGA

24.9%

BCOR/BCORL1

19.6%DNMT3A16.9%

ASXL1

12.9%

Splicingfacotrs 4.9%

RUNX1 3.1%

cohesinTET2

2.2%

TP53EZH2 CBL

Others

9.3%

Splicing factors

25.4%

TET2

19.9%

ASXL1

8.8%

Cohesin

5.2%DNMT3A

RUNX1

4.7%TP53

2.9%EZH2 2.4%CBL 2.4%

BCOR/BCORL1 1.9%

PIGA

Others

21.1%

5.0%

2.2%

2.7%

DNMT3A

CBL

61.9%TET2

11.1%

ASXL1

9.5%

Splicingfacotrs

5.5%

TP53

5.1%

JAK2

4.8%

B

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.

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MDS/AML, the size of dominant clones invariably increasedover time.Evolution of new clones was quite common, often accompanied bycomplete sweeping of preexisting clones. Nevertheless, an increasingclone size did not necessarily herald MDS/AML progression but wascompatible with completely normal peripheral blood counts, evenwhen patients’ hematopoiesis was mostly supported by these clones.Even in the patients who eventually developed MDS/AML, thedominance of clones having typical MDS/AML-related mutationspersisted 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 originallysuggested by the studies on NRXI53,54 and has recently been investi-gated more extensively using advanced genomics. By reanalyzingSNP array data generated for .50 000 individuals who did notcarry hematologic cancer, Jacobs et al43 andLaurie et al44 demonstratedthat CNAs and UPDs were observed at increasing frequencies inperipheral blood from elderly people (;2% to 3%) compared withthose from younger people (;,0.5%) (ARCH). Some authors de-scribe the same phenomenon under the term “CHIP” (CH with inde-terminate potential), although the potential may not be totallyindeterminate. The most frequent lesions include del(20q), del(11q),del(13q), and UPDs in 4q, 11q, 13q, and 14q, which are common inmyeloid malignancies.41,67-70 Importantly, ARCH is shown to beassociated with a substantially high incidence of developing hema-tologic cancer (hazard ratio,10-35.4).19,20,43,44

Busque et al reported TET2 mutations in PNMs (not in lymphoidcells) from 10 of 182 (5.6%) elderly women (.65 years old) withNRXI, but not in 95 younger women (,60 years old) or 105 elderlywomen without NRXI.71 More comprehensive surveys of leukemia/MDS-associated mutations in the general population were con-ducted by repurposing WES data of peripheral blood DNA fromlarge population-based studies. Jaiswal et al focused on 160 candidatemutational targets reported inhematologic cancers and foundmutationsin 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 subfractionof cells. They found a total of 1918 participants having at least 1putative somatic mutation, of which 308 individuals carrying knowncancer-associated driver mutations and 195 others who had no putativedrivers but had $3 somatic mutations were considered to haveCH.19 In agreement with studies on CNAs, the frequency of CHin both studies increased with age (;1% in those,50 years old vs;10%in those.65years old) and, aswithCH inAA, itwasdominatedby age-related C to T transitions. A similar finding was reportedusing whole exome data of cancer-bearing patients with no evidenceof hematologic cancer.21 Given the limitation of WES to capturemutations with low allelic burdens, the incidence should be evenhigher when mutations are detected by deep sequencing.68 Strik-ingly, recurrent mutations were highly biased to common drivergenes implicated in myeloid malignancies and most frequentlyaffected DNMT3A, followed by TET2, ASXL1, TP53, SF3B1,JAK2, and SRSF2, which differed substantially from the case withAA (Figure 3B); in AA, PIGA and BCOR/BCORL1 were morefrequently mutated than DNMT3A and ASXL1, whereas mutationsin TET2, TP53, SF3B1, JAK2, and SRSF2 were infrequent. As isthe case with ARCH associated with CNAs, individuals carryingmutations have a higher risk for developing hematologic cancer(hazard ratio, 11.1-12.9) and all-cause mortality, compared toindividuals without CH.19,20

Mechanisms of CH in AA

CH is highly prevalent among patients with AA (;50%), in which 2types 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 specificto or overrepresented in AA (PIGA/BOCR/BCORL1 mutations and6pUPD). The 2 types of alterations show different chronologicalbehaviors, age distribution, impacts on response to IST, progression toMDS/AML, and overall survival, suggesting discrete mechanisms ofclonal selection.

Table 2. CH in AA and the general population

Aplastic anemia Normal individuals MDS

Mutations High (.50%) Low (;2%) Very high (;90%)*

Correlation to age Positive Positive Positive

Mutated ,40 y, % 20-40 ,1

Mutated .70 y, % 52 .10

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,

TP53, SF3B1

TET2, SF3B1, ASXL1, SRSF2,

DNMT3A, RUNX1, TP53

Variant allele frequency Low (generally ,10%) Low (generally ,10%) High (.30%)

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 del(20q)

del(13q) UPDs in 4q, 11q, 13q, 14q

Impact of CH on progression to

MDS/AML

;3% for BCOR, BCORL1, and PIGA

mutations

Relative risk: ;103 to ;353

;40% for DNMT3A, ASXL1, RUNX1,

TP53, and CSMD2 mutations

*By abnormal cytogenetic and somatic mutations.

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MDS/AML-related mutations in AA show a clear age dependencyand are dominated by C to T transitions,18 as also seen in ARCH,19,20

suggesting that thesemutations could have a common origin, randomlyacquired with aging (Figure 4).72 Given their tight association withMDS/AML, it might be postulated that these mutations are clonallyselected in AA and in the general population through a similar mech-anism that would operate in the development of MDS/AML. In fact,in those AA patients who developed MDS/AML, an initial clonehaving 1 or more MDS/AML-related mutations gradually expandedand eventually gave rise to MDS/AML,18 although not all pa-tients carrying those mutations progressed to MDS/AML. How-ever, despite frequent DNMT3A and ASXL1 mutations in AA andMDS/AML, relative frequencies of otherMDS/AML-relatedmutationsdiffer substantially between both conditions; for example, TET2, JAK2,and splicing factors represent common targets of mutations in ARCHbut are affected at lower frequencies inAA(Figure 3B). In contrast to thevery high rate of progression to MDS/AML among AA patients withunfavorable mutations, the absolute risk of ARCH for MDS/AMLprogression still remained modest (;0.5% to 1% per year) (Table 2).73

A possible explanation would be that in AA, BM is repopulated from asmall number of residual stem cells after IST, which could provideclones having a proliferative advantage with a higher chance of cellcycling to achieveclonal dominance (bottleneckeffects), comparedwiththe caseofARCHina normally populatedBMenvironment (Figure 4).9

Alternatively, the autoimmune environment can preferentially selectDNMT3A and ASXL1mutations over others.

Immune escape as the mechanism for CH

The other set of lesions, including 6pUPD and PIGA mutations, arehighly specific to AA, for which autoimmunity has been implicated inthemechanismsof clonal selection (“clonality as escape”) (Figure 4), asfirst proposed by Young et al.5 6pUPD represents the most frequentgenetic lesion inAA.18,63All 6pUPDscritically involve the class IHLAlocus proximally, leading touniparental expressionof the retainedHLAclass I alleles.63 Significantly, most of the patients (36/40) with 6pUPD

Aged BM

EvolutionImmune-mediateBM destruction

Selection

Rapid expansion

Escape?↑Proliferation? Loss of

selective advantage?

PIGABCORBCORL1

DNMT3AASXL1RUNX1etc

IST

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 auto-

immune insult and/or show faster cycling/less apopto-

sis than others upon BM recovery to achieve a clonal

dominance. In some cases, the dominant clones, es-

pecially those having DNMT3A, ASXL1, and other un-

favorable 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 domi-

nant clones may regress or remain stable over years.

HLA locus

1Mb

A B

CTL

TCR

ch6

6pUPD (+) cells

CTL

TCR

UPD

ch6

CTL

TCR

?

PIGA-mutated cells

C

A

CB

DRB1DQB1

DPB1

PIGAmut

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 in-

volving 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 de-

struction by CTLs. The 6pUPD-mediated mechanism

for immune escape in AA is also supported by the

presence of multiple independent 6pUPD clones affect-

ing the same parental HLA allele in some patients.63

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carry 1 or more of 4 HLA alleles (A*02:01, A*02:06, A*31:01, andB*40:02), which are significantly enriched in AA (and also in PNH74)patients compared with the general population, and are the allelesconsistently lost in UPD-positive clones.63 These observations wouldlead to a hypothesis that unknown causative antigens are presented tocytotoxic T cells via these class I HLAs, loss of which in 6pUPD-positive HSCs enables escape from cytotoxic T cell–mediated de-struction (Figure 5). A similarmechanism seems to operate in leukemicrelapse after haploidentical transplantation, inwhich relapsed leukemiccells are thought to escape alloimmunity by losing the mismatchedhaploid alleles via 6pUPD.75,76

A conspicuous correlation between AA and the PNH-type cells,togetherwith the frequent presence ofmultiplePIGA-mutated clones in10% to 30% of AA/PNH patients,15,18,77,78 indicates a strong selectiveadvantage of PIGA-mutated cells that would be sufficient for clonalexpansion of PNH-type cells within BM in AA patients,79,80 althoughthere is no direct experimental evidence supporting this hypothesis.Nevertheless, this does not necessarily exclude a possible role ofsecondary mutations in clonal selection of PIGA-mutated cells, whichhas longbeendiscussed.Variious plausible secondarydriver alterationshave been reported in occasional cases and implicated in the clonaloutgrowth ofPIGA-mutated clones, including those affectingHMGA2,NRAS, JAK2, TET2, U2AF1, and even BCR/ABL.78,81-84 However,thus far, no consistently recurrent mutational targets have been iden-tified, even with WES,78 precluding the indispensable role of thesesecondary mutations in the development of PNH.

Currently, the underlyingmolecularmechanism for immune escapeof PIGA-mutated cells in AA is poorly understood. A possiblemechanismwould include loss of expression of GPI-anchored proteinscritical for immune recognitions.79,80 PIGA-mutated clones rarelydisappear but, more often, persist, even after successful IST, which is

in agreement with the theory of natural selection in the populationgenetics;many functional variants selected against past, but not present,insults are widely observed among the human population.85 Frequentgenetic drifts displayed by PNH-type clones18,86,87 also seem to favorthe explanation.

Clinical implications of somatic mutations

In the light of these new findings onCH inAA, aswell as in the generalpopulation, an important question would be, How could we translatethem into better clinical practice and improved outcomeof patients?Anextremely elevated risk of MDS/AML progression and high mortalityrates associated with unfavorable mutations would reasonably raisea possibility of early intervention with allogeneic stem cell transplantfor AA patients with unfavorable mutations, which should be bestinvestigated in a setting of prospective trials.88 Also, the current WHOcriteria for MDS could be extended for those patients having a set ofunfavorablemutations on the basis of their impact on clinical/biologicaloutcomes (Figure 6A), which is exactly the same way the currentcytogenetic criteria have been included.

Of interest in this regard are 2 recent studies on somatic muta-tions among ICUS patients, because these patients also had persis-tent cytopenia with no definitive evidence of MDS. Kwok et alreported that a substantial fraction of ICUS patients harboredsimilar MDS/AML-related mutations showing comparable allelicburdens to those found in low-riskMDS.89 Cargo et al, on the otherhand, investigated prediagnostic samples from ICUS patients whoeventually progressed to MDS/AML and observed a high frequencyof MDS/AML-related mutations comparable to those found in

Unexplained Cytopenia

ARCH(CHIP)

CCUS

ICUS

Documented Clonality

MDS (WHO)

Aplastic Anemia

MDS (WHO)

Documented Clonality

A B

DNMT3A ASXL1

TP53 etc. PIGA BCOR/BCORL1 6pUPD etc.

Unfavorable

mutations

Clonality by

‘MDS-specific’ Cytogenetic

lesions

Clonality by

‘MDS-specific’ Cytogenetic

lesions

DNMT3A TET2 ASXL1

PRM1D TP53, etc.

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 MDS-

specific 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.

344 OGAWA BLOOD, 21 JULY 2016 x VOLUME 128, NUMBER 3

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MDS patients in their number and variant allele frequency.90 Thesefindings might also suggest a possibility that these ICUS patientswith MDS/AML-related mutations (or clonal cytopenias of un-determined significance73,89) could be better considered as havingMDS or pre-MDS (Figure 6B). Lastly, it cannot be more underscoredthat regardless of the definition of MDS, careful examination ofpatients for hematologic status/morphology, symptoms, and clinicalcourse, as well as detailed history taking, should constitute theindispensable basis for a pertinent utilization of these new findingsfor better management of AA patients and other individuals pre-sented with unexplained cytopenia.

Concluding remarks

Recent advances in high-throughput sequencing technologies haverevealed anunexpected complexity ofCH inAA,which is closely relatedto, but in several respects distinct from, CH found in the aged normalpopulation, in which immune-mediated BM destruction and subsequentrecovery in AA seem to play a critical role. CH is common in AA, inwhich clones may eventually proceed to malignant transformation insome cases, but in others, may continue to support apparently normalhematopoiesis. Mutation types can predict the behavior of clones andpatients’ clinical course to some extent, but is not totally predictable,suggesting a potential value of monitoring CH using NGS now widelyavailable. Understanding of the mechanism underlying clonal selection

of different mutations should help in the accurate diagnosis/prediction ofmalignant evolution and in better management of AA patients.

Acknowledgments

The author thanks TetsuichiYoshizato andNeal S.Young for helpfuldiscussions on CH in AA.

This work was supported by Grants-in-Aid from the Ministry ofHealth, Labor, and Welfare of Japan (Health and Labor SciencesResearch Expenses for Commission and Applied Research forInnovative Treatment of Cancer).

Authorship

Contribution: S.O. conceived this reviewarticle, analyzed the literature,wrote the manuscript, and prepared the illustrations.

Conflict-of-interest disclosure: The author declares no competingfinancial interests.

Correspondence: Seishi Ogawa, Department of Pathology andTumor Biology, Graduate School of Medicine, Kyoto University,Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan; e-mail:sogawa-tky@umin.ac.jp.

References

1. Risitano AM, Maciejewski JP, Green S, PlasilovaM, Zeng W, Young NS. In-vivo dominant immuneresponses in aplastic anaemia: molecular trackingof putatively pathogenetic T-cell clones by TCRbeta-CDR3 sequencing. Lancet. 2004;364(9431):355-364.

2. Zeng W, Maciejewski JP, Chen G, Young NS.Limited heterogeneity of T cell receptor BV usagein aplastic anemia. J Clin Invest. 2001;108(5):765-773.

3. Zeng W, Nakao S, Takamatsu H, et al.Characterization of T-cell repertoire of the bonemarrow in immune-mediated aplastic anemia:evidence for the involvement of antigen-drivenT-cell response in cyclosporine-dependentaplastic anemia. Blood. 1999;93(9):3008-3016.

4. Sloand E, Kim S, Maciejewski JP, Tisdale J,Follmann D, Young NS. Intracellular interferon-gamma in circulating and marrow T cells detectedby flow cytometry and the response toimmunosuppressive therapy in patients withaplastic anemia. Blood. 2002;100(4):1185-1191.

5. Young NS, Calado RT, Scheinberg P. Currentconcepts in the pathophysiology and treatment ofaplastic anemia. Blood. 2006;108(8):2509-2519.

6. Marsh JC, Ball SE, Cavenagh J, et al; BritishCommittee for Standards in Haematology.Guidelines for the diagnosis and management ofaplastic anaemia. Br J Haematol. 2009;147(1):43-70.

7. Marsh JC, Geary CG. Is aplastic anaemia a pre-leukaemic disorder? Br J Haematol. 1991;77(4):447-452.

8. Socie G, Rosenfeld S, Frickhofen N, Gluckman E,Tichelli A. Late clonal diseases of treated aplasticanemia. Semin Hematol. 2000;37(1):91-101.

9. Young NS. The problem of clonality in aplasticanemia: Dr Dameshek’s riddle, restated. Blood.1992;79(6):1385-1392.

10. Abkowitz JL. Clone wars–the emergence ofneoplastic blood-cell clones with aging. N Engl JMed. 2014;371(26):2523-2525.

11. Martincorena I, Campbell PJ. Somatic mutationin cancer and normal cells. Science. 2015;349(6255):1483-1489.

12. Dameshek W. Riddle: what do aplastic anemia,paroxysmal nocturnal hemoglobinuria (PNH) and“hypoplastic” leukemia have in common? Blood.1967;30(2):251-254.

13. Lane AA, Odejide O, Kopp N, et al. Low frequencyclonal mutations recoverable by deep sequencingin patients with aplastic anemia. Leukemia. 2013;27(4):968-971.

14. Heuser M, Schlarmann C, Dobbernack V, et al.Genetic characterization of acquired aplasticanemia by targeted sequencing. Haematologica.2014;99(9):e165-e167.

15. Kulasekararaj AG, Jiang J, Smith AE, et al.Somatic mutations identify a subgroup of aplasticanemia patients who progress to myelodysplasticsyndrome. Blood. 2014;124(17):2698-2704.

16. Babushok DV, Perdigones N, Perin JC, et al.Emergence of clonal hematopoiesis in themajority of patients with acquired aplastic anemia.Cancer Genet. 2015;208(4):115-128.

17. Huang J, Ge M, Lu S, et al. Mutations of ASXL1and TET2 in aplastic anemia. Haematologica.2015;100(5):e172-e175.

18. Yoshizato T, Dumitriu B, Hosokawa K, et al.Somatic mutations and clonal hematopoiesis inaplastic anemia. N Engl J Med. 2015;373(1):35-47.

19. Genovese G, Kahler AK, Handsaker RE, et al.Clonal hematopoiesis and blood-cancer riskinferred from blood DNA sequence. N Engl J Med.2014;371(26):2477-2487.

20. Jaiswal S, Fontanillas P, Flannick J, et al. Age-related clonal hematopoiesis associated with

adverse outcomes. N Engl J Med. 2014;371(26):2488-2498.

21. Xie M, Lu C, Wang J, et al. Age-related mutationsassociated with clonal hematopoietic expansionand malignancies. Nat Med. 2014;20(12):1472-1478.

22. Shlush LI, Zandi S, Mitchell A, et al; HALT Pan-Leukemia Gene Panel Consortium. Identificationof pre-leukaemic haematopoietic stem cells inacute leukaemia. Nature. 2014;506(7488):328-333.

23. Damm F, Mylonas E, Cosson A, et al. Acquiredinitiating mutations in early hematopoietic cellsof CLL patients. Cancer Discov. 2014;4(9):1088-1101.

24. Sakata-Yanagimoto M, Enami T, Yoshida K, et al.Somatic RHOA mutation in angioimmunoblasticT cell lymphoma. Nat Genet. 2014;46(2):171-175.

25. Chung SS, Kim E, Park JH, et al. Hematopoieticstem cell origin of BRAFV600E mutations in hairycell leukemia. Sci Transl Med. 2014;6(238):238ra71.

26. Najean Y, Haguenauer O; The CooperativeGroup for the Study of Aplastic and RefractoryAnemias. Long-term (5 to 20 years) evolution ofnongrafted aplastic anemias. Blood. 1990;76(11):2222-2228.

27. Hillmen P, Lewis SM, Bessler M, Luzzatto L,Dacie JV. Natural history of paroxysmal nocturnalhemoglobinuria. N Engl J Med. 1995;333(19):1253-1259.

28. de Latour RP, Mary JY, Salanoubat C, et al;French Society of Hematology; FrenchAssociation of Young Hematologists. Paroxysmalnocturnal hemoglobinuria: natural history ofdisease subcategories. Blood. 2008;112(8):3099-3106.

29. Socie G, Mary JY, de Gramont A, et al; FrenchSociety of Haematology. Paroxysmal nocturnalhaemoglobinuria: long-term follow-up and

BLOOD, 21 JULY 2016 x VOLUME 128, NUMBER 3 CLONAL HEMATOPOIESIS IN AA 345

For personal use only.on July 26, 2016. by guest www.bloodjournal.orgFrom

prognostic factors. Lancet. 1996;348(9027):573-577.

30. de Planque MM, Bacigalupo A, Wursch A, et al;Severe Aplastic Anaemia Working Party of theEuropean Cooperative Group for Bone MarrowTransplantation (EBMT). Long-term follow-up ofsevere aplastic anaemia patients treated withantithymocyte globulin. Br J Haematol. 1989;73(1):121-126.

31. Frickhofen N, Heimpel H, Kaltwasser JP,Schrezenmeier H; German Aplastic Anemia StudyGroup. Antithymocyte globulin with or withoutcyclosporin A: 11-year follow-up of a randomizedtrial comparing treatments of aplastic anemia.Blood. 2003;101(4):1236-1242.

32. Rosenfeld S, Follmann D, Nunez O, Young NS.Antithymocyte globulin and cyclosporine forsevere aplastic anemia: association betweenhematologic response and long-term outcome.JAMA. 2003;289(9):1130-1135.

33. Tichelli A, Gratwohl A, Nissen C, Speck B. Lateclonal complications in severe aplastic anemia.Leuk Lymphoma. 1994;12(3-4):167-175.

34. Tichelli A, Gratwohl A, Wursch A, Nissen C,Speck B. Late haematological complications insevere aplastic anaemia. Br J Haematol. 1988;69(3):413-418.

35. Tichelli A, Schrezenmeier H, Socie G, et al. Arandomized controlled study in patients with newlydiagnosed severe aplastic anemia receivingantithymocyte globulin (ATG), cyclosporine, withor without G-CSF: a study of the SAA WorkingParty of the European Group for Blood andMarrow Transplantation. Blood. 2011;117(17):4434-4441.

36. Brodsky RA, Mukhina GL, Li S, et al. Improveddetection and characterization of paroxysmalnocturnal hemoglobinuria using fluorescentaerolysin. Am J Clin Pathol. 2000;114(3):459-466.

37. Brodsky RA, Mukhina GL, Nelson KL, LawrenceTS, Jones RJ, Buckley JT. Resistance ofparoxysmal nocturnal hemoglobinuria cells tothe glycosylphosphatidylinositol-binding toxinaerolysin. Blood. 1999;93(5):1749-1756.

38. Li Y, Li X, Ge M, et al. Long-term follow-up ofclonal evolutions in 802 aplastic anemia patients:a single-center experience. Ann Hematol. 2011;90(5):529-537.

39. Socie G, Henry-Amar M, Bacigalupo A, et al;European Bone Marrow Transplantation-SevereAplastic Anaemia Working Party. Malignanttumors occurring after treatment of aplasticanemia. N Engl J Med. 1993;329(16):1152-1157.

40. Kojima S, Ohara A, Tsuchida M, et al; JapanChildhood Aplastic Anemia Study Group. Riskfactors for evolution of acquired aplastic anemiainto myelodysplastic syndrome and acute myeloidleukemia after immunosuppressive therapy inchildren. Blood. 2002;100(3):786-790.

41. Brunning R, Orazi A, Germing U, et al.Myelodysplastic Syndromes. Lyon, France: IARCPress; 2008.

42. Cazzola M, Della Porta MG, Malcovati L. Thegenetic basis of myelodysplasia and its clinicalrelevance. Blood. 2013;122(25):4021-4034.

43. Jacobs KB, Yeager M, Zhou W, et al. Detectableclonal mosaicism and its relationship to aging andcancer. Nat Genet. 2012;44(6):651-658.

44. Laurie CC, Laurie CA, Rice K, et al. Detectableclonal mosaicism from birth to old age and itsrelationship to cancer. Nat Genet. 2012;44(6):642-650.

45. Wimazal F, Fonatsch C, Thalhammer R, et al.Idiopathic cytopenia of undetermined significance(ICUS) versus low risk MDS: the diagnosticinterface. Leuk Res. 2007;31(11):1461-1468.

46. Valent P, Bain BJ, Bennett JM, et al. Idiopathiccytopenia of undetermined significance (ICUS)

and idiopathic dysplasia of uncertain significance(IDUS), and their distinction from low risk MDS.Leuk Res. 2012;36(1):1-5.

47. Tichelli A, Gratwohl A, Nissen C, Signer E,Stebler Gysi C, Speck B. Morphology in patientswith severe aplastic anemia treated withantilymphocyte globulin. Blood. 1992;80(2):337-345.

48. Bennett JM, Orazi A. Diagnostic criteria todistinguish hypocellular acute myeloid leukemiafrom hypocellular myelodysplastic syndromesand aplastic anemia: recommendations for astandardized approach. Haematologica. 2009;94(2):264-268.

49. Busque L, Gilliland DG. X-inactivation analysisin the 1990s: promise and potential problems.Leukemia. 1998;12(2):128-135.

50. van Kamp H, Landegent JE, Jansen RP,Willemze R, Fibbe WE. Clonal hematopoiesis inpatients with acquired aplastic anemia. Blood.1991;78(12):3209-3214.

51. Ohashi H. Clonality in refractory anemia [inJapanese]. Rinsho Ketsueki. 1993;34(3):265-268.

52. Mortazavi Y, Chopra R, Gordon-Smith EC,Rutherford TR. Clonal patterns of X-chromosomeinactivation in female patients with aplasticanaemia studies using a novel reversetranscription polymerase chain reaction method.Eur J Haematol. 2000;64(6):385-395.

53. Fey MF, Liechti-Gallati S, von Rohr A, et al.Clonality and X-inactivation patterns inhematopoietic cell populations detected by thehighly informative M27 beta DNA probe. Blood.1994;83(4):931-938.

54. Busque L, Mio R, Mattioli J, et al. NonrandomX-inactivation patterns in normal females:lyonization ratios vary with age. Blood. 1996;88(1):59-65.

55. Appelbaum FR, Barrall J, Storb R, et al. Clonalcytogenetic abnormalities in patients withotherwise typical aplastic anemia. Exp Hematol.1987;15(11):1134-1139.

56. Keung YK, Pettenati MJ, Cruz JM, Powell BL,Woodruff RD, Buss DH. Bone marrow cytogeneticabnormalities of aplastic anemia. Am J Hematol.2001;66(3):167-171.

57. Maciejewski JP, Risitano A, Sloand EM, Nunez O,Young NS. Distinct clinical outcomes forcytogenetic abnormalities evolving from aplasticanemia. Blood. 2002;99(9):3129-3135.

58. Mikhailova N, Sessarego M, Fugazza G, et al.Cytogenetic abnormalities in patients with severeaplastic anemia. Haematologica. 1996;81(5):418-422.

59. Holbro A, Jotterand M, Passweg JR, Buser A,Tichelli A, Rovo A. Comment to “Favorableoutcome of patients who have 13q deletion: asuggestion for revision of the WHO ‘MDS-U’designation” Haematologica. 2012;97(12):1845-9. Haematologica. 2013;98(4):e46-e47.

60. Hosokawa K, Katagiri T, Sugimori N, et al.Favorable outcome of patients who have 13qdeletion: a suggestion for revision of the WHO‘MDS-U’ designation. Haematologica. 2012;97(12):1845-1849.

61. Saitoh T, Saiki M, Kumagai T, Kura Y, Sawada U,Horie T. Spontaneous clinical and cytogeneticremission of aplastic anemia in a patient withdel(13q). Cancer Genet Cytogenet. 2002;136(2):126-128.

62. Afable MG II, Wlodarski M, Makishima H, et al.SNP array-based karyotyping: differences andsimilarities between aplastic anemia andhypocellular myelodysplastic syndromes. Blood.2011;117(25):6876-6884.

63. Katagiri T, Sato-Otsubo A, Kashiwase K, et al;Japan Marrow Donor Program. Frequent loss ofHLA alleles associated with copy number-neutral

6pLOH in acquired aplastic anemia. Blood. 2011;118(25):6601-6609.

64. Vogelstein B, Papadopoulos N, Velculescu VE,Zhou S, Diaz LA Jr, Kinzler KW. Cancergenome landscapes. Science. 2013;339(6127):1546-1558.

65. Alexandrov LB, Nik-Zainal S, Wedge DC, et al;Australian Pancreatic Cancer Genome Initiative;ICGC Breast Cancer Consortium; ICGC MMML-Seq Consortium; ICGC PedBrain. Signatures ofmutational processes in human cancer. Nature.2013;500(7463):415-421.

66. Haferlach T, Nagata Y, Grossmann V, et al.Landscape of genetic lesions in 944 patients withmyelodysplastic syndromes. Leukemia. 2014;28(2):241-247.

67. James C, Ugo V, Le Couedic JP, et al. A uniqueclonal JAK2 mutation leading to constitutivesignalling causes polycythaemia vera. Nature.2005;434(7037):1144-1148.

68. Kralovics R, Passamonti F, Buser AS, et al.A gain-of-function mutation of JAK2 inmyeloproliferative disorders. N Engl J Med. 2005;352(17):1779-1790.

69. Sanada M, Suzuki T, Shih LY, et al. Gain-of-function of mutated C-CBL tumour suppressor inmyeloid neoplasms. Nature. 2009;460(7257):904-908.

70. Chase A, Leung W, Tapper W, et al. Profoundparental bias associated with chromosome 14acquired uniparental disomy indicates targetingof an imprinted locus. Leukemia. 2015;29(10):2069-2074.

71. Busque L, Patel JP, Figueroa ME, et al. Recurrentsomatic TET2 mutations in normal elderlyindividuals with clonal hematopoiesis. Nat Genet.2012;44(11):1179-1181.

72. Welch JS, Ley TJ, Link DC, et al. The origin andevolution of mutations in acute myeloid leukemia.Cell. 2012;150(2):264-278.

73. Steensma DP, Bejar R, Jaiswal S, et al. Clonalhematopoiesis of indeterminate potential and itsdistinction from myelodysplastic syndromes.Blood. 2015;126(1):9-16.

74. Shichishima T, Noji H, Ikeda K, Akutsu K,Maruyama Y. The frequency of HLA class I allelesin Japanese patients with bone marrow failure.Haematologica. 2006;91(6):856-857.

75. Villalobos IB, Takahashi Y, Akatsuka Y, et al.Relapse of leukemia with loss of mismatchedHLA resulting from uniparental disomy afterhaploidentical hematopoietic stem celltransplantation. Blood. 2010;115(15):3158-3161.

76. Vago L, Perna SK, Zanussi M, et al. Loss ofmismatched HLA in leukemia after stem-celltransplantation. N Engl J Med. 2009;361(5):478-488.

77. Mortazavi Y, Merk B, McIntosh J, Marsh JC,Schrezenmeier H, Rutherford TR; BIOMED IIPathophysiology and Treatment of AplasticAnaemia Study Group. The spectrum of PIG-Agene mutations in aplastic anemia/paroxysmalnocturnal hemoglobinuria (AA/PNH): a highincidence of multiple mutations and evidenceof a mutational hot spot. Blood. 2003;101(7):2833-2841.

78. Shen W, Clemente MJ, Hosono N, et al. Deepsequencing reveals stepwise mutation acquisitionin paroxysmal nocturnal hemoglobinuria. J ClinInvest. 2014;124(10):4529-4538.

79. Rotoli B, Luzzatto L. Paroxysmal nocturnalhaemoglobinuria. Baillieres Clin Haematol. 1989;2(1):113-138.

80. Luzzatto L, Bessler M, Rotoli B. Somaticmutations in paroxysmal nocturnalhemoglobinuria: a blessing in disguise? Cell.1997;88(1):1-4.

346 OGAWA BLOOD, 21 JULY 2016 x VOLUME 128, NUMBER 3

For personal use only.on July 26, 2016. by guest www.bloodjournal.orgFrom

81. Inoue N, Izui-Sarumaru T, Murakami Y, et al.

Molecular basis of clonal expansion of

hematopoiesis in 2 patients with paroxysmal

nocturnal hemoglobinuria (PNH). Blood. 2006;

108(13):4232-4236.

82. Mortazavi Y, Tooze JA, Gordon-Smith EC,

Rutherford TR. N-RAS gene mutation in patients

with aplastic anemia and aplastic anemia/

paroxysmal nocturnal hemoglobinuria during

evolution to clonal disease. Blood. 2000;95(2):

646-650.

83. Sugimori C, Padron E, Caceres G, et al.

Paroxysmal nocturnal hemoglobinuria and

concurrent JAK2(V617F) mutation. Blood Cancer

J. 2012;2(3):e63.

84. Tominaga R, Katagiri T, Kataoka K, et al.Paroxysmal nocturnal hemoglobinuria induced bythe occurrence of BCR-ABL in a PIGA mutanthematopoietic progenitor cell. Leukemia. 2016;30(5):1208-1210.

85. Bamshad M, Wooding SP. Signatures of naturalselection in the human genome. Nat Rev Genet.2003;4(2):99-111.

86. Pu JJ, Mukhina G, Wang H, Savage WJ, BrodskyRA. Natural history of paroxysmal nocturnalhemoglobinuria clones in patients presenting asaplastic anemia. Eur J Haematol. 2011;87(1):37-45.

87. Sugimori C, Mochizuki K, Qi Z, et al.Origin and fate of blood cells deficient inglycosylphosphatidylinositol-anchored protein

among patients with bone marrow failure. Br J

Haematol. 2009;147(1):102-112.

88. Young NS, Ogawa S. Somatic mutations and

clonal hematopoiesis in aplastic anemia. N Engl J

Med. 2015;373(17):1675-1676.

89. Kwok B, Hall JM, Witte JS, et al. MDS-

associated somatic mutations and clonal

hematopoiesis are common in idiopathic

cytopenias of undetermined significance. Blood.

2015;126(21):2355-2361.

90. Cargo CA, Rowbotham N, Evans PA, et al.

Targeted sequencing identifies patients with

preclinical MDS at high risk of disease

progression. Blood. 2015;126(21):2362-2365.

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online April 27, 2016 originally publisheddoi:10.1182/blood-2016-01-636381

2016 128: 337-347  

Seishi Ogawa Clonal hematopoiesis in acquired aplastic anemia 

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