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at the heart of a world city....

Austin G Kulasekararaj

Consultant Haematologist

King’s College Hospital, London

Aplastic Anaemia Genomics: Current data and

interpretation

T cell clones

(Immune) Escape clones- PNH, +8, 13q and 6pUPD

Good Clones vs Bad clones

Aged clones vs disease clones

True vs Pseudo clonality

Clonal Flux (Fluctuant/transient)

Clonal elimination (spontaneous/therapy)-

• PNH and transplant (!)

Clonal Wars- clonal Force Awakens

Adapted and modified from Young NS et al. Blood 2006;108:2509–2519

AA associated with diseases of clonal

hematopoiesis

RBDS

VSAA

SAA

NSAA

PNH

Hypo MDS

AMLLow risk

High

risk

MDS

LGL

(clonal or polyclonal)

5q-

MDS/MPD

DC

Hypo

AML

AA/PNH

FA

PIGA STAT3

MDS/AML-type somatic mutations

RPS5, RPS11,

RPS 19

TERC,

TERT,TINF2

FANC

Trisomy 81Copy number

neutral 6p

loss of

heterozygosity2

Ch6, chromosome 6; CTL, cytotoxic T lymphocytes; HLA, human leukocyte antigen;HSC, hematopoietic stem cell; HSPC, hematopoietic stem/progenitor cell; LOH, loss of heterozygosity; TCR, T cell receptor; WT1, Wilms tumor protein

1. Sloand EM et al. Blood 2005;106:841–851; 2. Katagiri T et al. Blood 2011;118:6601–6609 3..Hosakawa K et al. Haematologica

2012;97:1845–1849

Cytogenetic clones in AA that evade

immune attack

CD8+ T cell

clonal expansion

HSC

WT

1Cytokin

es

c-myc

Survivi

n

CDK1

→→

→

8+ LOHch6

LOH

ch6

LOH

ch6

LOH

ch6

LOH

ch6

LOH

ch6

ch6

ch6

CTL

CTL

CTLCTL

CTL

TCR

TCR

TCR

HLA

HLA*

HSP

C

Recombinat

ion of 6p

arms

A*02:01

A*02:06

A*31:01

B*40:02

GPI-AP, glycosylphosphatidylinositol-anchored protein; FISH, fluorescent in situ hybridization; UPN, unique patient number 3. Hosakawa K et al. Haematologica 2012;97:1845–1849

De

l(1

3q)

ce

lls (

%)

0 5 10 15 25 40 5020 30

Months from diagnosis

Changes in the proportion of

del(13q) cells after IST in 8

patients

0

20

60

80

100

40

4535

UPN 3del(13q) FISH (Peripheral blood)

32.2%

UPN 7del(13q) FISH (Peripheral blood)

4.0%

FSC-A CD55, CD59 FSC-A CD55, CD59

SSC-A CD11b SSC-A CD11b

del(13q) FISH

0%

del(13q) FISH

12%

del(13q) FISH

0%

del(13q) FISH

12%

10

00

80

0

60

0

40

0

20

0

0

10 4

10 3

10 2

10 1

10 0

10

00

80

0

60

0

40

0

20

0

0

10 4

10 3

10 2

10 1

10 010

00 20

0

40

0

60

0

80

0

10

00

10 1

10 2

10 3

10 4

0 20

0

40

0

60

0

80

0

10

00

10 0

10 1

10 2

10 3

10 4

Del

13q3

142 DNMT3A 1.5 Non-synonymous SNV c.C5644T:p.R882C

129 DNMT3A 5 Non-synonymous SNV c.G2207A:p.R736H

130 DNMT3A 5 Non-synonymous SNV c.G2645A:p.R882H

97 DNMT3A 7 Non-synonymous SNV c.C5644T:p.R882C

93 DNMT3A 8 Stopgain SNV C2311T:p.R771X

18 DNMT3A 31 Non-synonymous SNV c.C2644T:p.R882C

2 DNMT3A 42 Non-synonymous SNV c.C1540G:p.L514V

64 DNMT3A 47 Non-synonymous SNV c.C2644T:p.R882C

21 BCOR 5 Stopgain SNV c.C526T:p.Q176X

140 BCOR 5 Frameshift deletion c.4760delC:p.P1587fs

73 BCOR 6 Frameshift insertion c.4834_4835insC:p.L1612fs

70 BCOR 14 Stopgain SNV c.T912G:p.Y304X

94 BCOR 30 Splice site Splice site c.3052-2A>G

33 BCOR 68 Stopgain SNV c.G4832A:p.W1611X

2 ERBB2 44 Non-synonymous SNV c.G922A:p.V308M

19 IKZF1 14 Non-synonymous SNV c.C640G:p.H214D

46 MPL 10 Non-synonymous SNV c.G1544T:p.W515L

10 SRSF2 20 Non-synonymous SNV c.C284T:p.P95L

5 TET2 5 Stopgain SNV c.C3100T:p.Q1034X

67 U2AF1 19 Non-synonymous SNV c.C101A:p.S34Y

Bone marrow

Skin

C>Tp.W1356

XMutant allele burden = 41%

ASXL1 (n=12) DNMT3A (n=8) BCOR (n=6)

One each of: SRSF2U2AF1TET2MPLIKZF1ERBB2

The median allele burden was 20% (range, 1.5–68%)

All heterozygous except one hemizygous BCOR

41% of mutations had

Disease durationMutations

(n=29)

Wild type

(n=121)P-value

Stressed hematopoiesis

Proliferative pressure

Depleted stem cell pool

Telomere loss Genomic instability

CR, complete response; CSA, cyclosporine A; GCSF, granulocyte-colony stimulating factor; hMDS, hypoplastic MDS; HSCT, hematopoietic stem cell transplant; NR, non response; RAEB, refractory anemia with excess blasts; RCMD, refractory cytopeniawith multilineage dysplasia; TPO, thrombopoietin; VAF, variable allele frequency Kulasekararaj AG et al. Blood 2014;124:2698–2704

Monosomy 7: Diverse origins

Age/

sex

Diagnosi

s

Initial

therapy

Disease

duration

(months)

Respons

eMC Mutation(s)

Clonal

evolutionVAF

73/M NSAA ATG + CSA 44 CR NormalASXL1

DNMT3ARCMD/-7

30

42

35/F NSAA ATG + CSA 45 NR 45,XX,-7 ASXL1 RCMD/-7 41

28/M VSAA ATG + CSA 2 NR Normal ASXL1 hMDS/-7 31

83/M NSAA CSA 62 CR 45,XY,-7 DNMT3ARCMD-

RAEB2-AML47

Road to

monosomy

7

Growth

factors

GCSF

Kostmanns

TPO mimetic

Eltrombopag

AA

HSCT

Post-

auto

Donor

leukemia

Post-

cord

Transient -7 (disappears post-ATG) Persistent -7 (MDS/AML)

Myeloid

neoplasia

Mo

no

so

my 7

(%

by F

ISH

)

Patient UPN

0

60

80

100

40

20

3 4 10 1 14 16 17 25

Control

GCSF 400 ng/mL

• Targeted sequencing of genes at diagnosis of aplastic anaemia (n=96) at King’s

• None of them had mutations with VAF >10%

• Cautious use of mutation calling in clinical practise by sequencing laboratories

Kulasekararaj et al,

Unpublished

Yoshizato et al. NEJM 2015

Lower VAF at diagnosis and its interpretation:

mutations present at diagnosis

‘Aged’ versus ‘disease’ clonesPrevalence of somatic mutations

according to age

Fre

qu

ency

Age (years)

0

0.1

0.3

0.4

0.5

0.2

Jaiswal S et al. N Engl J Med 2014;371:2488–2498

Somatic mutations that drive clonal

expansion are common in the elderly and

most frequently involve DNMT3A, TET2

or ASXL1,

and are associated with increased

overall mortality

Babushok et al. BJH 2016, Marsh et al

unpublished

Clonal eradication: spontaneous or therapy

King’s College Hospital, 2015FCC regimen

MSD, n=12, 100%

UD, n=33, 90.5%

AA

Clo

nal siz

e a

nd

fre

qu

en

cy Clinical Significance

Hypoplastic MDS or Overlap

AA/MDS (?)

Progression to MDS

?Consider early allogeneic

transplantation

Time and disease course

*BCORL1 not analysed

NA-PIGA not done for the whole cohort

VAF-variant allele frequency

CHIP-Clonal haemopoiesis of indeterminate potential

IST-immunosuppressive therapy

MDS

Overlap

AA/MDS

CHIP

Recovery of normal

haemopoiesis

IST

Age related clonal haemopoiesis

Mutational profile of AA and its evolution following IST1

Frequency of four commonly

mutated genes in AA from

two studies2,3Top four

genes

Kulasekararaj

et al.

Yoshizato et

al.

DNMT3A 8.3% 8.4%

ASXL1 8% 6.2%

BCOR/BCORL

14%* 9.3%

PIGA N/A 7.5%

Median VAF 20% 9.3%

Normal haemopoietic stem cell (HSC)

Dysplastic HSC

Dysplastic HSC with single mutation

Dysplastic HSC with multiple mutations 1. Mufti GJ et al. N Engl J Med 2015;373:1673–1676; 2. Kulasekararaj AG et al. Blood 2014;124:2698–2704; 3. Yoshizato T et al. N Engl J Med 2015;373:35–47

Acknowledgements

Tony Pagliuca

Robin Ireland

Victoria Potter

Donal McLornan

Kavita Raj

Hugues Delavallade

Rayne Institute

Joop Gaken

Alexander Smith

Jie Jiang

Azim Mohamedali

Shreyans Gandhi

Syed Mian

Nik Lea

Steve Best

Shahram Kordasti

SAAWP