Molecular newbies in normal karyotype AML

Dr Hang QuachHaematology Registrar

Box Hill Hospital

AML prognostic groups Karyotype at diagnosis = independent

prognostication Good prognostic group70% 5 years OS

.t(8;21)(q22;22)AML1-ETO Inv(16)MYH11-IgH .t(15;17)(q22;q21)PML-RARa

Poor prognostic group<20% 5 year OS -5,-7,abn(3q) or complex karyotype (>5abn)

Normal cytogenetics thought to be of intermediate prognosis: 30%-45% 5yr OS ~50%

Normal karyotypic leukaemia – A heterogeneous group.

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 Class III receptor tyrosine kinase.

Structurally related to PDGFR, c-KIT Human FLT3 gene = on chr 13q12 Expression described on early

hematopoietic and lymphoid precursors. Imp role in stem cell survival +

myeloid differentiation.

FLT3 mutations Internal tandem duplications (FLT3 ITD)

Found in up to 30% of AML – mostly in normal karyotypes Unfavourable prognosis (high relapse risk, decrease DFS and OS)

Point mutation in tyrosine kinase domain (FLT3-TKD)

7% of AML Point mutations and small deletions mostly of codons 835

and 836 Thiede et al, Blood 2002: 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

NucleophosminNPM1

NPM1 = nucleolar phosphosprotein.

A molecular chaperone: transport pre-ribosomal particles through nuclear membrane into cytoplasm.

Controls duplication of centrosomes during cell cycle

regulates tumour supressor pathway.

Mutated NPM1cytoplasmic translocation dimerises with wild type NPM 1cytoplasmic retention of NPM1.

1835 paraffin embedded tumour specimen: 591 = primary AML.

Pt were enrolled in italian trials: GIMEMA, LAM99P, GIMEMA/EORTC -M12.

980 with extrahematopoietic neoplasms/hematopoietic malignancies other than “primary” AML.

Immunohistochemical detection of NPM1 localisation. Using anti human monoclonal Ab against NPM1.

Correlation with clinical and biologic features of disease

Falini et al 2005

Method: Cont

Cytogenetics - G banding analysis. RT-PCR for:

PML-RARa, AML-ETO, CBFb-MYH11, BCR-ABL.

FLT3 mutations.

Correlation with cytogentics Cytogentic data available for 493/591 pt with primary AML.

Co-expression of FLT-3 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 NPMc- disease, p<0.003

Independent assocciation betweeen NPMc and FLT3 ITD on multivariate logistic regression model.

Blood Dec 2005, 106(12): 3773-39

Retrospective analysis: 401 AML pt with normal karyotype.

Age 16-81. Entered AMLCG99 between 1999-2004.

Treatment: AMLCG study Double induction:

TAD (Thioguanine, AraC, Daunorubicin)/ HAM (High dose araC, mitoxantrone) Vs HAM/HAM.

Consolidation -->radomised to maintenance for 3 yrs Vs AutoSCT.

Cytogenetics: G banding analysis. Screening for NPM1 gene mutation

using melting curve based light cycler assay.

Also screened for: FLT 3-ITD, FLT3-TKD mutations

NPM1 mutation, morphology and other biologic markers.

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:

Mean 61.1 x10^9/L vs 39.1x10^9/L, p<0.001.

NPM1 mutations and other gene mutations.

NPM1-mutated groups showed significantly higher incidence of FLT3 mutations: FLT ITD: 40.6%vs

24.5%, p=0.001 FLT-TKD: 9.5%Vs

3.8%

Prognostic impact of NPM1 mutations.

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

Median OS in the NPM1 mutated group trends towards better prognosis: 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

Same pattern was found with EFS:

NPM1+/FLT3-:773d NPM1-/FLT3-: 365d NPM1-/FLT3+: 279d NMP1+/FLT3+: 234d

Relapse free survival. RFS was

significantly better for the NPM1+/FLT3- group compared to all other 3 groups, p= 0.001

Summary NPM1 gene mutation occurred more

frequently than any other muation in normal karyotypic AML: ~53% in this study

Favourable impact on outcome: 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) 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) Specifically up regulated during granulocyte

differentiation. Regulates promoters of granulocyte specific

genes.

C/EBP deficient mice lacked mature granulocytes (zhang et al proc Natl Acad Sci USA, 1997)

C/EBP mutations found in 7% of AML (Gombart eg al, 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 differentiationAML-M2

Objective: 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: 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 Cytogenetics – G banding analysis FISH for other common AML-associated

aberrations. Analysis of CEBP coding regions:

amplified by PCR, abn products clonedSequenced the entire coding regions.

Other mutations also assessed: FLT3 ITD, FLT3-TKD MLL PTD

Results:

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.

•36/236 (15%) pt demonstrated at least one CEBPa mutation

Response to induction

Rates of CR (standard criteria) and resistant disease were not significantly different in patients with or without CEBP mutations, p=0.17

Remission duration Median follow up

30 months Median duration

of remission: 26 months in

those without CEBP mutation.

Not reached for group with CEBP

P=0.01

Remision duration

Multivariate analysis

Overall survival OS longer for

patients with CEBP mutations compared to wild type.

P=0.05

Overall survival

Multivariate analysis - OS

CEBPA – an independent prognostic marker affecting remission duration and OS

Effects of additional FLT3 mutation

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 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. (not consistent with other studies)

BAALC (Brain and acute leukaemia, cytoplasmic)

Recently identified gene -chr 8q22.3

Encodes a protein of yet unknown function.

In hematopoietic cells: BAALC expression restricted to progenitor cells.

BAALC 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:)

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.