OVERVIEW OF MOLECULAR MARKERS
IN AML
Nilam Virani
Molecular Diagnostics
July 28, 2011
AML Classification
FAB Classification
M0 Minimally differentiated
M1 Without maturation
M2 With maturation
M3 Acute promyelocytic leukemia
M4 Acute myelomonocytic
leukemia
M5 Acute monoblastic leukemia
M6 Acute erythroleukemia
M7 Acute megakaryoblastic
leukemia
2008 WHO Classification
Cytogenetic Abnormalities
Swerdlow et al. 2008
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
Molecular Abnormalities
Cytogenetic Risk Group
Recommended Molecular Studies
t(15;17)
t(8;21)
inv(16)/t(16;16)
KIT
Normal karyotype
Nonrecurrent abnormalities
NPM1
CEBPA
FLT3-ITD
inv(3)/t(3;)
t(6;9)
t(6;11)
Complex karyotype
KIT Mutations in CBF AMLs
• 10-20% of the CBF
AMLs have KIT
mutations
• While the cytogenetic
abnormalities that
define CBF AMLs
normally confer a good
prognosis, those with
KIT mutations do
poorly
Paschka et al., J Clin Oncol, 2006
inv(16)
t(8;21)
Prognostic Significance of Molecular
Markers
Established
Favorable
mutations
NPM1
CEBPA
Unfavorable
mutations
FLT3-ITD
Unfavorable
overexpression of
single genes
Likely
Potential
KIT
FLT3-TKD
MLL-PTD
WT1
IDH
BAALC
ERG
MN1
EVI1
HOXA9
MEIS1
FLT3 Mutations
• Member of the the class III receptor tyrosine kinase family
• Important role in proliferation, survival, and differentiation
•
•
•
•
•
of hematopoietic progenitor cells
Mutations result in constitutive activation of FLT3
30% of CN-AML have mutations in FLT3
ITDs result from duplication and tandem insertion of
small, variably sized (3-400 nt) gene fragments
TKDs are point mutations, small insertions, or deletions
that occur in activation loop in the carboxyterminal lobe,
mainly at codons 835 and 836
Identification of the ITD mutation involves a PCR-based
assay that can detect the larger PCR products indicative
of duplication
FLT3 Mutations
• Prognosis of CN-AML
• Therapeutically, there
with FLT3-ITD is
significantly inferior
compared with FLT3ITD negative CN-AML
• Prognostic relevance
of FLT3-TKD is not as
well-established, but
also appears to be
unfavorable
is accumulating
evidence that
allogeneic HSCT is an
attractive option, given
the poor outcome with
standard
chemotherapy
• Randomized phase III
trials evaluating FLT3
inhibitors are
underway
NPM1 Mutations
• Multifunctional phosphoprotein that travels between
•
•
•
•
•
nuclear compartments and the cytoplasm
Normally, predominantly located in the nucleolus
Implicated in ribosome assembly and regulation of ARF
and p53 tumor suppressor function
Mutations were first discovered by IHC because of
cytoplasmic mislocation of the mutated NPM1 protein
50-60% of CN-AML show NPM1 mutations
Detection of the mutations involves a PCR-based assay
• >40 mutations have been identified within exon 12 of the gene, but
virtually all lead to a net insertion of 4 nt
NPM1 Mutations
• Prognostic implications
• Standard induction
due to NPM1 mutations
must be made in the
context of FLT3
mutations
• Only NPM1mut/FLT3ITDneg are associated
with achievement of
complete remission and
favorable outcome
• Potential marker for
disease monitoring
chemo followed by 3-4
cycles of high-dose
cytarabine is the
recommended therapy
• Patients may not be
considered candidates
for allogeneic HSCT in
first complete remission
• May become important
marker for disease
monitoring
CEBPA Mutations
• 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
CEBPA Mutations
• Only double mutations
• Standard induction
are associated with a
favorable outcome
• A series of cases with
silencing of CEBPA
have been identified,
and associated with a
distinctly poor
prognosis
chemo followed by 3-4
cycles of high-dose
cytarabine is the
recommended therapy
• Patients may not be
considered candidates
for allogeneic HSCT in
first complete
remission
More molecular markers under
investigation
Unfavorable recurrent genetic
abnormalities
Unfavorable overexpression of
single genes
• IDH1, IDH2
• BAALC
• WT1
• ERG
• MLL-PTD
• NRAS
• KRAS
• TP53
• TET2
• ASXL2
• RUNX1
• DNMT3A
• EVI1
• MN1
References
Betz B, Hess JL: Acute Myeloid Leukemia Diagnosis in the 21st Century. Arch Pathol Lab
Med 134:1427-33, 2010.
Gorello P, et al: Quantitative assessment of minimal residual disease in acute myeloid
leukemia carrying nucleophosphomin (NPM1) gene mutations. Leukemia 20(6):11031108, 2006.
Gregory TK, et al: Molecular prognostic markers for adult acute myeloid leukemia with
normal cytogenetics. Journal of Hematology and Oncology 2:23, 2009.
Marcucci G, Haferlach T, Dohner H: Molecular Genetics of Adult Acute Myeloid
Leukemia: Prognostic and Therapeutic Implications. Journal of Clinical Oncology
29:475-86, 2011.
Schlenk RF, Dohner K, Krauter J, et al: Mutations and treatment outcome in
cytogenetically normal acute myeloid leukemia.
Swerdlow SH, Campo E, Harris NL, et al: WHO Classification of Tumours of the
Haematopoietic and Lymphoid Tissues. 4th ed. Geneva, Switzerland: WHO Press;
2008.