Genetic Markers in Acute Myeloid Leukemia

1. Genetic Markers
in
Acute Myeloid Leukemia (AML)
Prepared by:
Mary Suzette Angeles, RCh
Regina Marie Jastiva, RCh
Hannah Aloyon, BSc
Ferdinand F. Fatiga, RMT

2. BIOMARKERS in AML
Cytogenetic Abnormalities
• Chromosomes aberrations
• Karyotyping
Genetic Lesions
• Mutations  SNPs

4. Prognostic
Subgroup
Cytogenetic Abnormality
Favorable • t(15;17)/PML-RARA
• t(8;21)
• inv(16)/t(16;16)
Intermediate • Normal karyotype
• t(9;11)
• Gains of whole
chromosomes or loss of Y
chromosome
Unfavorable • t(6;9)
• inv(3)/t(3;3)
• Complex karyotype

5. Normal karyotypic leukemia
(A heterogeneous group)
Helps classify AML
patients to a more
Specific
Prognostic group

6. Significance of Genetic
Markers
1. Prognostic Impact
2. Detection of Minimal Residual Disease

7. Classic “Two Hits” Model in Mutation
Class I Mutation
 Confers Myeloproliferation of abnormal WBCs
 Genes coding for receptor and nonreceptor
Protein Tyrosine Kinase
• FLT3, JAK2, C-KIT, ABL1
 Genes coding for Proteins of GTPase activity
• N-RAS and K-RAS
Class II Mutation
 Involves Differentiation arrest
 Affects genes of Transcription Factors (TFs)
 Chromosome Aberration  Gene Fusion
– CBF, MLL, EVI1, TEL, RARA
 Point Mutation (CEBPA and NPM1)

8. ESTABLISHED Genetic Markers
 FMS - like Tyrosine Kinase
(FLT-3)
 Nucleophosmin 1
(NPM1)
 CAAT/enhancer binding protein alpha
(CEBPA)

9. Established Likely Potential
Favorable
mutations
NPM1
CEBPA
Unfavorable
mutations
FLT3-ITD KIT
FLT3-TKD
MLL-PTD
WT1
IDH
Unfavorable
overexpression
of single genes
BAALC
ERG
MN1
EVI1
HOXA9
MEIS1
Genetic Markers

10. FLT-3
 Human FLT3 gene is found on Chromosome 13q12
 aka CD 135
 Proto-oncogene
 Encodes for Class III Transmembrane
Tyrosine Kinase receptor (Cytokine receptor)
 Normally expressed by hematopoietic stem cells,
early Myeloid and Lymphoid precursors
 Supports proliferation and survival of
progenitor cells

12. FLT3 Mutation Categories
• Internal tandem duplications (FLT3 ITD)
– Found in up to 30% of AML – mostly in normal karyotypes
– Unfavorable 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

14. Prognosis of CN-AML with FLT3-ITD is significantly
inferior compared with FLT3-ITD negative CN-AML
Prognostic relevance of FLT3-TKD is not as
well-established, but also appears to be
unfavorable

15.  Located on Chromosome
5q35
 Multifunctional
phosphoprotein
 Molecular chaperone
• Transport pre-
ribosomal particles
through nuclear
membrane into
cytoplasm
 Controls duplication of
centrosomes during cell cycle
 Regulates tumor suppressor
pathway

16. 1) NPM1 Gene Alteration
Mutated NPM1 cytoplasmic translocation 
dimerises with wild type NPM 1 
cytoplasmic retention of NPM1
2) NPM functional Loss
mutations of NPM1 seem to simultaneously
dampen a tumor-suppressor pathway4 (p53–ARF) and
enhance an oncogenic one (MYC)

18. Survival Rates Associated with
NPM1 mutation

19. NPM1 Prognostic Impact
•Prognostic implications due to NPM1
mutations must be made in the context of
FLT3 mutations
•Only NPM1mut/FLT3-ITDneg are associated
with achievement of complete remission
and favorable outcome

22. 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.

23. • Transcription factor whose function is crucial for the
development and differentiation of granulocytes from
hematopoietic precursors
• Mutations lead to a loss of function, and thought to promote
leukemogenesis by blocking granulocyte differentiation
• 15% of CN-AML have CEBPA mutations
• Variety of mutations occur throughout the coding region, but
fall into two major types:
• N-terminal frameshift → truncation of protein
• C-terminal in-frame → impaired dimerization and DNA binding
• Identification of mutations requires DNA sequencing
• Majority of mutations are biallelic, compound heterozygous
mutations
CCAAT/enhancer binding
protein(C/EBP)

24.  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 differentiationAML-M2
CCAAT/enhancer binding
protein(C/EBP)

25. CEBPA Prognostic Impact
• Only double mutations are associated with a
favorable outcome
• A series of cases with silencing of CEBPA have
been identified, and associated with a
distinctly poor prognosis

26. 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

27. 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

28. Multivariate analysis

29. Overall survival
 OS longer for patients
with CEBP
mutations compared
to wild type.
 P= 0.05
Overall survival

30. Multivariate analysis - OS
CEBPA – an independent prognostic marker
affecting remission duration and OS

31. 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

32. 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)

33. More molecular markers under investigation
Unfavorable recurrent
genetic abnormalities
• IDH1, IDH2
• WT1
• MLL-PTD
• NRAS
• KRAS
• TP53
• TET2
• ASXL2
• RUNX1
• DNMT3A
Unfavorable overexpression
of single genes
• BAALC
• ERG
• EVI1
• MN1

34. Thank you! 