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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 81
Copy 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+ LOH
ch6
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 <10% clone
Mutations detected were missense (n=12), nonsense (n=12), frameshift (n=6), non-frameshift deletion (n=1) and splice-site changes (n=1)
Kulasekararaj AG et al. Blood 2014;124:2698–2704
Frequency and incidence of mutationsUPN Gene
Mutant allele
burden (%)Variant class
Nucleotide and
protein change
70 ASXL1 2 Stopgain SNV c.G2026T:p.E676X
81 ASXL1 3 Stopgain SNV c.T2324G:p.L775X
88 ASXL1 7 Frameshift deletion c.2126delC:p.A709fs
2 ASXL1 30 Frameshift insertion c.1927_1928insG:p.G643fs
16 ASXL1 30 Frameshift insertion c.2469_2470insAG:p.L823fs
107 ASXL1 30 Stopgain SNV c.T2468G:p.L823X
40 ASXL1 31 Non-frameshift deletion c.2894_2896del:p.965_966del
69 ASXL1 34 Stopgain SNV c.2077T:p.R693X
79 ASXL1 36 Stopgain SNV c.G2026T:p.E676X
66 ASXL1 37 Frameshift deletion c.2433delT:p.N811fs
6 ASXL1 38 Stopgain SNV c.C2242T:p.Q748X
29 ASXL1 41 Stopgain SNV c.G4068A:p.W1356X
Disease durationMutations
(n=29)
Wild type
(n=121)P-value
<6 months (n=63) 9 54
Transformation into MDS 3 3 <0.03
Median mutant allele burden
<10%7 NA
>6 months (n=87) 20 67
Transformation into MDS 8 3 <0.0002
Median mutant allele burden
<10%4 NA
Somatic mutation found in 29/150 (19%)
In the presence of a somatic mutation:
1. Risk of MDS = 11/29 (38%) compared with 6/121 (6%); P<0.001
2. If disease duration >6 months, risk of MDS is 40% compared with 4%
Do they predict risk of evolution to MDS?
Median follow-up 46 months (range, 1–360)
Kulasekararaj AG et al. Blood 2014;124:2698–2704
What is the significance of somatic mutations in
AA
(excluding PIGA mutations)?
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 studies2,3
Top 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
