Genetic Markers in

Acute Myeloid Leukemia (AML) Prepared by:

Mary Suzette Angeles, RCh

Regina Marie Jastiva, RChHannah Aloyon, BSc

Ferdinand F. Fatiga, RMT

BIOMARKERS in AML

Cytogenetic Abnormalities• Chromosomes aberrations• Karyotyping

Genetic Lesions• Mutations SNPs

CYTOGENETIC ABNORMALITIES

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

Normal karyotypic leukemia

(A heterogeneous group)

Helps classify AML patients

to a more Specific

Prognostic group

Genetic Markers

Significance of Genetic Markers

1. Prognostic Impact 2. Detection of Minimal Residual Disease

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)

ESTABLISHED Genetic Markers

FMS - like Tyrosine Kinase (FLT-3)

Nucleophosmin 1(NPM1)

CAAT/enhancer binding protein alpha (CEBPA)

Established

Likely Potential

Favorable mutations

NPM1CEBPA

Unfavorable mutations

FLT3-ITD KITFLT3-TKD

MLL-PTDWT1IDH

Unfavorable overexpression of single genes

BAALCERGMN1EVI1

HOXA9MEIS1

Genetic Markers

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

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

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

NPM1 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

NPM11) 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 simultaneouslydampen a tumor-suppressor pathway4 (p53–ARF) and enhance an oncogenic one (MYC)

Survival Rates Associated with NPM1 mutation

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

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.

• 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)

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

CCAAT/enhancer binding protein(C/EBP)

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

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

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)

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

Thank you!