Transcript Leukemia –otherwise unclassifed? May be not so….

Molecular newbies in normal
karyotype AML
Dr Hang Quach
Haematology Registrar
Box Hill Hospital
AML prognostic groups
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Karyotype at diagnosis = independent
prognostication
Good prognostic group70% 5 years OS
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Poor prognostic group<20% 5 year OS
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.t(8;21)(q22;22)AML1-ETO
Inv(16)MYH11-IgH
.t(15;17)(q22;q21)PML-RARa
-5,-7,abn(3q) or complex karyotype (>5abn)
Normal cytogenetics thought to be of
intermediate prognosis: 30%-45% 5yr OS
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~50%
Normal karyotypic leukaemia –
A heterogeneous group.
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FMS tyrosine like kinase (FLT-3)
Nucleophosmin (NPM1) mutations
CAAT/enhancer binding protein alpha
(CEBPalpha)
BAALC (brain and acute leukemia,
cytoplasmic)
FLT-3 = STK1=flK2
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Class III receptor tyrosine kinase.
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Structurally related to PDGFR, c-KIT
Human FLT3 gene = on chr 13q12
Expression described on early
hematopoietic and lymphoid precursors.
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Imp role in stem cell survival + myeloid
differentiation.
FLT3 mutations
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Internal tandem duplications (FLT3 ITD)
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Point mutation in tyrosine kinase domain (FLT3-TKD)
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Found in up to 30% of AML – mostly in normal karyotypes
Unfavourable prognosis (high relapse risk, decrease DFS and OS)
7% of AML
Point mutations and small deletions mostly of codons 835 and
836
Thiede et al, Blood 2002:
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FLT3 mutation in 970pts
Both mutations were associated with similar clinical
characteristics- higher WCC, higher blasts, monocytoid diff.
More prevalent in pt with normal karyotypes
Pt with high mutant/wt ratio (>0.78) worse OS and DFS
Nucleophosmin
NPM1
NPM1 = nucleolar
phosphosprotein.
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A molecular chaperone:
transport pre-ribosomal
particles through nuclear
membrane into cytoplasm.
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Controls duplication of
centrosomes during cell cycle
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regulates tumour supressor
pathway.
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Mutated NPM1cytoplasmic
translocation dimerises with
wild type NPM 1cytoplasmic
retention of NPM1.
Falini et al 2005
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1835 paraffin embedded tumour specimen:
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591 = primary AML.
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980 with extrahematopoietic neoplasms/hematopoietic
malignancies other than “primary” AML.
Immunohistochemical detection of NPM1
localisation.
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Pt were enrolled in italian trials: GIMEMA, LAM99P,
GIMEMA/EORTC -M12.
Using anti human monoclonal Ab against NPM1.
Correlation with clinical and biologic features of
disease
Method: Cont
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Cytogenetics - G banding analysis.
RT-PCR for:
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PML-RARa, AML-ETO, CBFb-MYH11, BCRABL.
FLT3 mutations.
Correlation with cytogentics
Cytogentic data available for 493/591 pt with primary AML.
Co-expression of FLT-3
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FLT3 ITD found in 59/219 pt with normal
karyotype (26.9%)
FLT-TKD (D835) found in 13/202 (6.4%).
FLT3 ITD was twice as frequent in the group
with NPMc+ disease compared NPMcdisease, p<0.003
Independent assocciation betweeen NPMc
and FLT3 ITD on multivariate logistic
regression model.
Blood Dec 2005, 106(12): 3773-39
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Retrospective analysis:
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401 AML pt with normal karyotype.
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Age 16-81.
Entered AMLCG99 between 1999-2004.
Treatment: AMLCG study
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Double induction:
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TAD (Thioguanine, AraC, Daunorubicin)/ HAM (High dose
araC, mitoxantrone) Vs HAM/HAM.
Consolidation -->radomised to maintenance for 3 yrs Vs
AutoSCT.
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Cytogenetics: G banding analysis.
Screening for NPM1 gene mutation
using melting curve based light cycler
assay.
Also screened for:
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FLT 3-ITD, FLT3-TKD mutations
NPM1 mutation, morphology and
other biologic markers.
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212/401 pt (52.9%)
were HTZG for NPM1
mutation.
Higher expression in
monocytic
differentiation.
No sig difference with
incidence of NPM1 mut
b/w age groups.
Higher WCC in NPM1
mutation:
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Mean 61.1 x10^9/L vs
39.1x10^9/L, p<0.001.
NPM1 mutations and other gene
mutations.
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NPM1-mutated
groups showed
significantly higher
incidence of FLT3
mutations:
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FLT ITD: 40.6%vs
24.5%, p=0.001
FLT-TKD: 9.5%Vs
3.8%
Prognostic impact of NPM1 mutations.
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401pt
Median follow up=484d
CR was significantly
higher in the NPM1
mutated cases:
 70.5% Vs 54%,
p=0.003
EFS: NPM mutated
group:428d vs 336
days, p=0.012.
Median OS
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Median OS in the
NPM1 mutated
group trends
towards better
prognosis:
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1012d vs 549d,
p=0.076
Effects of additional FLT3
mutations
Median OS:
 NPM1+/FLT3-: 1183d
 NPM1-/FLT3-: 601d
 NPM1-/FLT3+: 401d
 NMP1+/FLT3+: 321d
Event free survival
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Same pattern was
found with EFS:
NPM1+/FLT3-:773d
NPM1-/FLT3-: 365d
NPM1-/FLT3+: 279d
NMP1+/FLT3+: 234d
Relapse free survival.
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RFS was significantly
better for the
NPM1+/FLT3- group
compared to all
other 3 groups, p=
0.001
Summary
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NPM1 gene mutation occurred more
frequently than any other muation in normal
karyotypic AML: ~53% in this study
Favourable impact on outcome:
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Longer EFS (med 428 vs 336d,p=0.12)
Trend to longer OS (med 1012 vs 549d, p= 0.076)
Favourable impact of NPM1 mutation is lost with
concomitant FLT3-ITD mutations.
CCAAT/enhancer binding
protein(C/EBP)
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A member of the leucine zipper transcription
factor family-gene located on 19q13.1
In human: genes recently isolated and shown
to be preferentially expressed in
myelomonocytic cells (not erythroid, T or B
lineages)
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Specifically up regulated during granulocyte
differentiation.
Regulates promoters of granulocyte specific
genes.
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C/EBP deficient mice lacked mature granulocytes
(zhang et al proc Natl Acad Sci USA, 1997)
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C/EBP mutations found in 7% of AML
Blood 2002)
Mutation resulted in a truncated C/EBP protein.
 Inhibits wild type C/EBPa DNA binding.
Frequency was highest in those with FAB subtype
M2, the majority of whom had normal cytogenetics.
In pt with t(8;21)AML1-ETO fusion protein down
regulates CEBPa expression to a level insufficient for
granulocyte differentiationAML-M2
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(Gombart eg al,
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Objective:
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assess the prognostic relevance of CEBPA mutations in young
adults with normal karyotypic AML
Search for cooperating mutations.
Diagnostic BM or PB samples from 236 pts (16-60yo) with
normal cytogenetics:
 72pts: AML HD93 (1993-1998):
 164pts: AMLHD98(1998-2002)
Treatment:
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Double induction: ICE x 2 (Ida 12mg/m2 d1,3,5; AraC100mg/m2
d1-7, Etop 100mg/m2 d1-3)
consolidation with HAM (AraC3g/m2bd d1-3, mitox 12mg/m2 d2,3)
2nd consolidation: HD93: HAM; HD98: HAM or autoSCT.
Method
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Cytogenetics – G banding analysis
FISH for other common AML-associated
aberrations.
Analysis of CEBP coding regions:
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amplified by PCR, abn products
clonedSequenced the entire coding regions.
Other mutations also assessed:
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FLT3 ITD, FLT3-TKD
MLL PTD
Results:
•36/236 (15%) pt demonstrated at least one CEBPa
mutation
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FLT3 mutations were significantly less freqent in pt
with CEBP mutations: 28% v 49%, p=0.01
None of the pts with CEBP mutation had MLL PTD.
Response to induction
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Rates of CR (standard criteria) and resistant
disease were not significantly different
in patients with or without CEBP
mutations, p=0.17
Remission duration
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Median follow up 30
months
Median duration of
remission:
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Remision duration
26 months in those
without CEBP
mutation.
Not reached for
group with CEBP
P=0.01
Multivariate analysis
Overall survival
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Overall survival
OS longer for
patients with CEBP
mutations compared
to wild type.
P=0.05
Multivariate analysis - OS
CEBPA – an independent prognostic marker
affecting remission duration and OS
Effects of additional FLT3
mutation
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Among 36 pt with CEBPA mutation,
presence of FLT 3 mutation (both ITD
and D835) did not significantly
influence OS, p=0.71
Summary - CEBP
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CEBPA mutations detected in 15% of pts
with normal karyotype AML.
CEBPA = an independent favourable
prognostic marker on multivariate analysis
(remission duration and OS)
Presence of FLT3 mutations had no -ve
impact on pt with CEBPA mutations.
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(not consistent with other studies)
BAALC (Brain and acute
leukaemia, cytoplasmic)
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Recently identified gene -chr 8q22.3
Encodes a protein of yet unknown
function.
In hematopoietic cells: BAALC
expression restricted to progenitor cells.
BAALC
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BAALC expression found in AML and CML in blast
crisis, but not in CML in chronic phase. (Tanner et al, Proc
Natl Acad, Sci USA 2001:)
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In AML with normal cytogenetics, high mRNA
expression of BAALC seem to predict poor prognosis
(baldus et al, Blod 2003)
High BAALC expression: (Baldus et al, JCO 2006)
 Predictive of resistant disease
 Higher cumulative incidence of relaspe
 Inferior overall survival.