33 Acute Myeloid Leukemia AML Flashcards
AML
– Acute Myeloid Leukemia
– the most common type of leukemia in adults
– Heterogeneous disease
– The result of somatic genetic alterations in hematopoietic progenitors
– Mutations affect normal proliferation, self-renewal and differentiation
– Inherited AML is rare
What is FAB AML classification system?
– French-American-British system for classify AML
– based on cell morphology and immunochemical characteristics
– not showing a significant prognostic difference
Inherited germline mutations link to AML
CEBPA
SRP72
DDX41
RUNX1
GATA2
Gilliland and Griffin: two-hit model for AML pathogenesis
Class 1 mutations:
– activate signal pathways to promote proliferation and survival of progenitors
– happened later in leukemogenesis
Class 2 mutations:
– affect transcription factors that impair differentiation
– occur early in leukemogenesis
– founder mutations
– stable during the disease course
– class 2 mutations do not coexist
Gilliland and Griffin Class 1 mutations
FLT3, KIT, NRAS, KRAS
Gilliland and Griffin Class 2 mutations
Gene fusions:
t(8;21) - RUNX1/RUNX1T1
inv(16) - CBFB/MYH11
Gene mutations:
NPM1, CEBPA, RUNX1
AML: t(8;21) (q22;q22) affected gene
RUNX1-RUNX1T1
AML: t(15;17) (q22;q12) affected gene
PML-RARA
AML: inv(16) (p13.1 q22) or t(16;16) (p13.1;q22) affected gene
CBFB-MYH11
AML: t(9;11) (p22;q23) affected gene
MLLT3-KMT2A (MLL)
AML: t(6;9) (p23;q34) affected gene
DEK-NUP214
AML: inv(3) (q21q26.2) or t(3;3) (q21;q26.2) affected gene
GATA2, MECOM (EVI1)
AML: t(1;22) (p13;q13) affected gene
RBM15-MKL1
AML with NPM1 mutation affected gene
NPM1
AML with biallelic CEBPA mutations
CEBPA
Which cytogenetic abnormalities can be used solely to diagnose if the patient has AML or not without considering >20% blast-count rule?
t(8;21) : RUNX1-RUNX1T1
inv(16) or t(16;16) : CBFB-MYH11
t(15;17) : PML-RARA
The first step to diagnose AML
– morphologic evaluation such as presence of Auer rods
– >20% myeloid blasts and monocytic progenitors
Which immunophenotypic analysis is often used to distinguish AML from lymphoblastic leukemia?
Flow cytometry
After established AML in patient, what are the next tests for further subclassification?
– Karyotyping
– FISH
– Sanger sequencing
what specimens can be used for AML diagnosis?
– blood
– bone marrow aspirate
What cytogenetic abnormalities are classified by WHO as “Favorable” risk category?
t(15;17) : PML-RARA
t(8;21) : RUNX1-RUNX1T1
inv(16)/t(16;16) : CBFB-MYH11
What cytogenetic abnormalities are classified by WHO as “Intermediate” risk category?
t(9;11) : MLLT3-KMT2A (MLL)
Normal cytogenetics
+8 alone
Other karyotype
What cytogenetic abnormalities are classified by WHO as “Poor” risk category?
Complex cytogenetics (>=3 abnormalities)
inv(3)/t(3;3) : GATA2, MECOM (EV11)
t(6;9) : DEK-NUP214
11q23 abnormalities other than t(9;11)
t(9;22)
-5, del(5q)
-7, del(7q)
What gene mutations are classified by WHO as “Favorable” risk category?
Normal cytogenetics without FLT3-ITD and with either NPM1 or biallelic CEBPA mutations
What gene mutations are classified by WHO as “Intermediate” risk category?
t(8;21) or inv(16)/t(16;16) with KIT mutation
What gene mutations are classified by WHO as “Poor” risk category?
Normal cytogenetics with FLT3-IDT mutation
CBF AML
Core-Binding Factor AML
What does “Favorable” risk category mean?
excellent prognosis
better response to chemotherapy
Function of CBF
– transcription factor complex
– role in hematopoiesis
Two subunits of CBF
– RUNX1(AML1, CBFA2)
– CBFB
Hematopoietic neoplasms caused by CBF disruption or abnormalities
– Myelodysplastic syndrome (MDS)
– Acute lymphoblastic leukemia (ALL)
– AML
Cytogenetic abnormalities or chromosomal abnormalities associated with CBF AML
- t(8;21) that creates a RUNX1-RUNXIT1 (AML1-ETO) gene fusion
- inv(16) or t(16;16) that results in the CBFB-MYH11 gene fusion
If the cytogenetic analysis did not detect t(8;21) or inv(16/t(16;16), CBF AML is suspected by morphology. How do you confirm the diagnosis of CBF AML?
Use FISH or RT-PCR to detect t(8;21) or inv(16)/t(16;16)
what gene mutations are frequently seen in CBF AML?
Mutations in KIT, FLT3, or RAS
APL
Acute Promyelocytic Leukemia
accounts 12% of AML
Cytogenetic abnormality and fusion gene associated with APL
t(15;17) – PML-RARA fusion gene
what does t(15;17) – PML-RARA fusion gene do to the APL progression?
– halts myeloid progenitor differentiation
– expansion of neoplastic promyelocytes
Gene: RARA
retinoic acid receptor alpha
Gene: PML
promyelocytic leukemia
t(15;17), what are the breakpoints on chr. 17?
chr. 17 intron 2 of RARA gene
t(15;17), what are the breakpoints on chr. 15?
chr. 15 intron 6 (bcr1: 55% of APL cases)
chr. 15 exon 6 (bcr2: 5% of APL cases)
chr. 15 intron 3 (bcr3: 40% of APL cases)
3 PML-RARA isoforms
1) Long: L or bcr1: 55% of APL cases
2) Variant: V or bcr2: 5% of APL cases
3) Short: S or bcr3: 40% of APL cases)
% APL cases of PML-RARA with bcr1 isoform?
55%
% APL cases of PML-RARA with bcr2 isoform?
5%
% APL cases of PML-RARA with bcr3 isoform?
40%
Which PML-RARA isoform has the least % of APL?
bcr2 isoform
A minor subset of APL cases harbor variant translocations that fuse RARA with a different partner genes other than PML. What are these partner genes for a variant RARA translocation?
BCOR, FIP1L1
NPM1, NUMA
PRKAR1A
STAT5B
ZBTB16 (PLZF)
What are the RARA alternative partner genes to cause APL?
BCOR, FIP1L1
NPM1, NUMA
PRKAR1A
STAT5B
ZBTB16 (PLZF)
what is the APL-specific treatment that must be given ASAP after the initial diagnosis?
ATRA = all-trans retinoic acid
What causes APL patient death during the first few days after diagnosis?
Coagulopathy (凝血病)
ATRA
all-trans retinoic acid
Which alternative RARA fusion genes lead to poor outcome and resistance to ATRA in APL patients?
PLZF-RARA
STAT5B-RARA
Residual disease monitoring for APL use which method?
RT-PCR of PML-RARA
consecutive negative tests are associated with remission, long-term survival and possible cure
Relapse: two consecutive RT-PCR positive results (one in bone marrow)
Which cytogenetic abnormality is very rare and predominantly occur in patient less than 3 years old AML patients?
t(1;22)
RBM15-MKL1
What is CN-AML?
50% of AML lacks chromosomal abnormalities
these are called cytogenetically normal (CN) AML.
Genes mutated in CN-AML
NPM1
FLT3-ITD
CEBPA
Genes mutated in CBF-AML
KIT
Gene mutations that are tested in guiding post-remission therapy in AML
NPM1
FLT3-ITD
CEBPA
KIT
% of NPM1 mutations in AML patients
30% in all AML patients
50% in CN-AML patients
What NPM1 stands for?
Nucleophosmin
also called nucleolar protein B23, numatrin, or NO38,
an abundant phosphoprotein
located on chr. 5q35, 12 exons
Function of NPM1
transport preribosomal particles through nuclear membrane
shuttle between the nucleus and the cytoplasm
where is mutated NPM1 localized in a cell?
cytoplasm
where is wild type NPM1 usually localized in a cell?
nucleus
NPM1 mutations
frameshift mutations within exon 12
a net 4bp insertions
loss of a nucleolar localization signal
generation of a novel nuclear export signal
Disease outcome of NPM1 without FLT3-IDT mutation in AML patients
better overall survival
event-free survival
better response to treatment
patients are not candidates for allogeneic stem cell transplantations
Why do we test NPM1 and FLT3-IDT for CN-AML patients?
NPM1 without FLT3-IDT mutation is prognostic favorable
Method to detect NPM1 mutations
PCR fragment-sizing assay:
PCR amplify exon 12–>capillary electrophoresis
4bp insertions
LOD: 2% in 20% blasts
50 variants known
Disadvantages of qPCR detection of NMP1 mutations
only designed to detect specific NMP1 mutations
qPCR is better for minimal residual disease (MRD) monitoring
FLT3
FMS-like tyrosine kinase 3
receptor tyrosine kinase
regulate proliferation of hematopoietic progenitor cells
which is the second common gene mutations in AML?
FLT3 mutations
25-30% of all AML patients
Two types of FLT3 mutations in AML
- internal tandem duplication (FLT3-ITD)
- tyrosine domain mutations (FLT3-TKD)
% of AML with FLT3-ITD mutations
20-25% of AML
% of AML with FLT3-TKD mutations
5-10%
Function of FLT3-ITD mutations and FLT3-TKD mutations
constitutive activation of the FLT3 receptor
FLT3-ITD mutations
Duplication and tandem insertion of 3 to several hundreds bp of FLT3 gene
insertion located in exon 14 (common) or 15 of FLT3 gene
in-frame mutations
gain-of-function
FLT3-ITD mutations detection method
PCR fragment-sizing assay:
PCR–> capillary electrophoresis
15% of FLT3-ITD AML patients have multiple IDT mutations
Biallelic ITD shown taller peaks on capillary electrogram than the WT control
FLT3-TKD mutations
missense mutations such as D835 (codon 835 aspartic acid)
The outcomes of AML with the following combinations:
FLT3-ITD + NPM1 mutations
FLT3-ITD + CEBPA mutations
poor
Is NGS a efficient method for FLT3-ITD testing?
No, due to large duplications
FLT3-TKD mutations detection method
PCR amplification
EcoRV digestion at D835
capillary electropherogram
WT-digested by EcoRV
FLT3-TKD D835 mutants, can not be digested
see figure
Are FLT3-ITD mutations good test markers for monitoring MRD?
No. FLT3-ITD mutations might be lost during the relapse.
CEBPA
CCAAT/enhancer-binding protein alpha
a member of the basic region leucine zipper (bZIP) family of TFs
CEBPA role in hematopoiesis
differentiation of myeloid progenitors
% of CEBPA mutations in AML patients
8-15% of all AML
commonly seen in CN-AML
CEBPA mutations in AML
Majority of cases with two mutations:
N-terminal frameshift mutation in one allele
C-terminal in-frame mutation in another allele
Only double mutated CEBPA associated with favorable AML outcomes.
CEBPA mutations function in AML
loss of function of the differentiation promoting p42 isoform
expression of pro-proliferative p30 isoform
How do the CEBPA mutations compared to FLT3 and NPM1 mutations?
CEBPA mutations are highly variable and spanned the entire coding region
CEBPA mutations detection method
fragment-sizing analysis: for insertions and deletions
Sequencing
Why NGS is not a preferred method for CEBPA mutations detection method?
High GC content of CEBPA
Sanger sequencing limitations for CEBPA mutations?
low sensitivity (~ 10%)
KIT
The KIT gene: chr. 4q11-12
a 145-kDa transmembrane glycoprotein
a member of the type III tyrosine kinase family
Binding of stem cell factor (KIT ligand) to the KIT receptor activates downstream signaling pathways important for cell proliferation, differentiation, and survival
KIT mutations
occur in ~30% of CBF-AML patients
mostly occur in exon 8 or 17
KIT Exon 8 mutations in AML
small in-frame insertions/deletions that affect codon 419
KIT Exon 17 mutations in AML
substitution at codon D816 or N822
substitution at codon D816 in CBF-AML (t8;21) associated with a higher risk of relapse
RUNX1
Runt-related TF 1
role in hematopoietic differentiation
RUNX1 mutations
10-15% of CN-AML
seen in MDS and AML with myelodysplasia-related changes
initiating events for AML
often found in the absence of fusion genes and NPM1 and CEBPA mutations
MDS
Myelodysplastic syndrome
a type of blood cancer (leukemia) in which the bone marrow produces immature blood cells
RUNX1 gene mutations in AML
substitution, insertions, deletions
throughout the coding region
IDH1 and IDH2
isocitrate dehydrogenase
are key enzymes that function at a crossroads of cellular metabolism, epigenetic regulation, redox states, and DNA repair
IDH1 and IDH2 are NADP+ dependent, share considerable sequence similarity
Where IDH1 expressed?
in the mammalian liver and moderately expressed in other tissues
It contains a C-terminal tripeptide peroxisome targeting signal 1 sequence
localizes to the cytoplasm and peroxisome of yeast and mammalian cells
Where IDH2 expressed?
IDH2 contains an N-terminal mitochondrial signal peptide
localizes to the mitochondria
It is highly expressed in mammalian heart, muscle, and activated lymphocytes
% IDH1 and IDH2 mutations in AML
15-30% of AML
common in CN-AML
IDH 1 and IDH2 mutations
substitutions
R132 of IDH1
R140 or R172 of IDH2
IDH1 and IDH2 mutations occur in a mutually exclusive fashion
Function of IDH 1 and IDH2 mutations
affect active site of IDHs
lead to high level of d-2-hydroxyglutarate (2HG)
IDH 1 and IDH2 mutations found in which cancers?
AML
glioma
DNMT3A
DNA (cytosine-5)-methyltransferase 3 alpha
function in DNA methylation
% of DNMT3A mutations in AML
20% of AML
common in CN-AML
DNMT3A mutations
affect epigenetic modification of DNA
R882 is the most common DNMT3A mutations (60%)
in-frame and frame-shift mutations
DNMT3A mutations testing methods
NGS
high resolution melting of R882 (exon23)
Sanger seq of R882 (exon23)
KMT2A (MLL)
encodes a DNA-binding protein that methylates histone H3
positively regulates expression of target genes, including multiple HOX gene
epigenetic modifier
KMT2A (MLL) mutations
epigenetic modifier
5-10% of CN-AML
prognosis: inconclusive
ASXL1
the additional sex combs like-1 gene
a member of trithorax and polycomb (ETP) family
ASXL1 function
transcriptional regulator
has chromatin-binding activity
chromatin modifier
WT1
Wilms tumor 1 gene
TF
a tumor suppressor or an oncogene
WT1 gene mutations
10-15% of CN-AML
germline with WAGR syndrome
majority mutations found are frameshift insertions and deletions on exon 7
WAGR syndrome
a disorder that affects many body systems and is named for its main features: Wilms tumor, aniridia (無虹膜), genitourinary anomalies, and intellectual disability (formerly referred to as mental retardation)
ASXL1 mutations
5-10% of CN-AML
prognosis unfavorable
frameshift or nonsense mutations in exon 12
found in AML and MDS
TET2
ten-eleven-translocation 2 gene
demethylation of DNA
epigenetic modifier
TET2 mutations
found in AML and MDS
10% of CN-AML
inactivated TET2
increased stem cells renewal and myeloproliferation
TP53
cell cycle regulator
tumor suppressor
TP53 mutations
2-5% in CN-AML
75% of TP53 mutated AMLcases have poor outcome
RAS
membrane associated signaling
regulate proliferation, differentiation and apoptosis
KRAS and NRAS mutations
~20% in all human cancers
10% of CN-AML
Overexpression of genes in AML
BAALC - a negative prognostic indicator for CN-AML
ERG
EVI1
associated with worst outcome
minimal residual disease testing (MRD) for APL, RT-PCR
PML-RARA
MRD for CBF-AML, RT-PCR
RUNX1-RUNX1T1
CBFB-MYH11
Future testing methods for AML
NGS
microRNA
