Molecular newbies in normal karyotype AML

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Molecular newbies in normal karyotype AML

• Dr Hang Quach
• Haematology Registrar
• Box Hill Hospital

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AML prognostic groups

• Karyotype at diagnosis independent
  prognostication
• Good prognostic group?70 5 years OS
• .t(821)(q2222)?AML1-ETO
• Inv(16)?MYH11-IgH
• .t(1517)(q22q21)?PML-RARa
• Poor prognostic group?lt20 5 year OS
• -5,-7,abn(3q) or complex karyotype (gt5abn)
• Normal cytogenetics ?thought to be of
  intermediate prognosis 30-45 5yr OS
• 50

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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)

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FLT-3 STK1flK2

• 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.

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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 (gt0.78) ?worse OS
  and DFS

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

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Falini et al 2005

• 1835 paraffin embedded tumour specimen
• 591 primary AML.
• Pt were enrolled in italian trials GIMEMA,
  LAM99P, GIMEMA/EORTC -M12.
• 980 with extrahematopoietic neoplasms/hematopoieti
  c 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

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Method Cont

• Cytogenetics - G banding analysis.
• RT-PCR for
• PML-RARa, AML-ETO, CBFb-MYH11, BCR-ABL.
• FLT3 mutations.

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Correlation with cytogentics Cytogentic data
available for 493/591 pt with primary AML.
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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, plt0.003
• Independent assocciation betweeen NPMc and FLT3
  ITD on multivariate logistic regression model.

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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 --gtradomised 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
• FLT 3-ITD, FLT3-TKD mutations

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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 x109/L vs 39.1x109/L, plt0.001.

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NPM1 mutations and other gene mutations.

• NPM1-mutated groups showed significantly higher
  incidence of FLT3 mutations
• FLT ITD 40.6vs 24.5, p0.001
• FLT-TKD 9.5Vs 3.8

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Prognostic impact of NPM1 mutations.

• 401pt
• Median follow up484d
• CR was significantly higher in the NPM1 mutated
  cases
• 70.5 Vs 54, p0.003
• EFS NPM mutated group428d vs 336 days, p0.012.

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Median OS

• Median OS in the NPM1 mutated group trends
  towards better prognosis
• 1012d vs 549d, p0.076

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Effects of additional FLT3 mutations

• Median OS
• NPM1/FLT3- 1183d
• NPM1-/FLT3- 601d
• NPM1-/FLT3 401d
• NMP1/FLT3 321d

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Event free survival

• Same pattern was found with EFS
• NPM1/FLT3-773d
• NPM1-/FLT3- 365d
• NPM1-/FLT3 279d
• NMP1/FLT3 234d

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Relapse free survival.

• RFS was significantly better for the NPM1/FLT3-
  group compared to all other 3 groups, p 0.001

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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,p0.12)
• Trend to longer OS (med 1012 vs 549d, p 0.076)
• Favourable impact of NPM1 mutation is lost with
  concomitant FLT3-ITD mutations.

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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.

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• 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(821)?AML1-ETO fusion protein down
  regulates CEBPa expression to a level
  insufficient for granulocyte differentiation?AML-M
  2

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• 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.

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Method

• Cytogenetics G banding analysis
• FISH for other common AML-associated aberrations.
• Analysis of CEBP? coding regions
• amplified by PCR, abn products cloned?Sequenced
  the entire coding regions.
• Other mutations also assessed
• FLT3 ITD, FLT3-TKD
• MLL PTD

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Results

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

• FLT3 mutations were significantly less freqent in
  pt with CEBP? mutations 28 v 49, p0.01
• None of the pts with CEBP? mutation had MLL PTD.

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Response to induction

• Rates of CR (standard criteria) and resistant
  disease were not significantly different in
  patients with or without CEBP? mutations, p0.17

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Remission duration

• Median follow up 30 months
• Median duration of remission
• 26 months in those without CEBP? mutation.
• Not reached for group with CEBP?
• P0.01

Remision duration
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Multivariate analysis
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Overall survival

• OS longer for patients with CEBP? mutations
  compared to wild type.
• P0.05

Overall survival
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Multivariate analysis - OS

• CEBPA an independent prognostic marker
  affecting remission duration and OS

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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, p0.71

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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)

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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.

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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.