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Acute Myeloid Leukaemia—
phenotype and genotype
Barbara J. Bain
Department of Haematology
St Mary’s Hospital, London
The phenotypic diagnosis
• A case of leukaemia was first clearly
described in 1845 by John Hughes Bennett
and another, 6 weeks later, by Rudolf
Virchow
• These were phenotypic diagnoses at
autopsy, the blood being examined
microscopically
• A few years later Virchow was the first to
use the term “Leukhemia”
Things moved on
The next one and a half centuries saw
advances in the phenotypic diagnosis of
leukaemia based on
• Better stains and microscopes
• Examination of the blood and, later, the
bone marrow during life
Things moved on further
Further advances improved the phenotypic
diagnosis. These were
• Cytochemical stains
• Immunophenotyping
• Integration and codification of phenotypic
diagnoses
And further still
The FAB classifications from the last quarter
of the 20th century can be seen as the
culmination of phenotypic diagnosis
• The FAB group defined, and categorized
and gave haematologists and, later,
cytogeneticists throughout the world a
common language
• Phenotypic diagnosis had become quite
sophisticated
But meanwhile —
Cytogenetic techniques were improving and
with the introduction of chromosome
banding there was a quantum leap in what
could be recognized
• The incorporation of cytogenetic
information into leukaemia classification
started, e.g. MIC Group 1986.
And furthermore —
Molecular genetics was invented
Now we have
• FISH, SKY, spectral karyotyping, CGH
• PCR and RT-PCR
• Microarray analysis
What does this mean for
leukaemia diagnosis and
classification?
• Can we move from a MIC to a MIC-M
approach
• Should we do so?
The WHO classification
The WHO classification has taken an
approach which is partly based on
cytogenetic and molecular genetic
analysis
• First, antecedent factors are considered
• Secondly, genetic features are considered
• Thirdly, phenotype is used to categorize the
remaining patients
The WHO classification
• When appropriate, patients are first
assigned to the category of therapy-related
AML, regardless of whether they have
recurring cytogenetic abnormalities
• These cases are further categorized as
– Alkylating agent-related
– Topoisomerase II-interactive drug-related
– Other
WHO and therapy-related
AML
This is, in part, a genetic classification
• Alkylating agent-related AML is
characterized by abnormalities of
chromosomes 5 and 7, inv(3), t(3;3), t(6;9),
t(8;16) and complex rearrangements
WHO and therapy-related
AML
Topoisomerase II-inhibitor-related AML is
characterized by balanced translocations
with 21q22 (AML1) and 11q23 (MLL)
breakpoints including t(3;21), t(8;21),
t(4;11), t(9;11) and t(11;19)(q23;p13.1) [but
not t(11;19)(q23;p13.3)]
WHO and therapy-related
AML
• Topoisomerase II-inhibitor-related AML is
associated not only with t(8;21)(q22;q22)
but also with other translocations usually
found in de novo AML, e.g.
t(15;17)(q22;q12) and inv(16)(p13q22)
Where does therapy-related
AML with recurrent cytogenetic
abnormality belong?
It may be important to classify such cases as
therapy-related in order to accurately judge
the magnitude of the problem of therapyinduced AML (and ALL)
These cases might also be prognostically
worse than de novo cases
Where does therapy-related
AML with recurrent cytogenetic
abnormality belong?
However—t-AML and de novo AML with the
same cytogenetic abnormality have a lot of
features in common and maybe the
prognosis is not so very different
Therapy-related AML with
t(8;21)(q22;q22)
Slovak et al. (2002) reported a median
survival of < 3 years and a 5 year survival
of < 30%
However the treatment was not standardized
and in the larger group of AML patients
with a 21q22 bp a quarter of patients were
untreated or undertreated
Slovak et al. (2002) Genes Chromosomes Cancer, 33, 379.
Therapy-related AML with
t(15;17)(q22;q21)
Andersen et al. (2002) reported, for
intensively treated patients, that there was:
• a 69% CR rate
• a median survival of 29 months
• a 5-year survival of 45%
Andersen et al. (2002) Genes Chromosomes Cancer, 33,
395.
Therapy-related AML with
t(15;17)(q22;q21)
n
CR rate
34
97%
‘secondary’
642
93%
de novo
4-yr EFS 4-yr OS
65%
85%
68%
78%
‘Secondary’ = 15 surgery, 17 RT, 10 Chemo, 10 Chemo + RT
Pulsoni et al. (2002) Blood, 100, 1972
Therapy-related AML with
inv(16)(p13q22)
Andersen et al (2002) reported , with
intensive treatment:
• 85% CR
• 29 months median survival
• 45% 5-year survival
• Andersen et al. (2002) Genes Chromosomes Cancer, 33,
395.
What should we do?
• We should follow the WHO classification but
should note the presence of relevant cytogenetic
abnormalities
• We should recognize that topoisomerase IIinhibitor-related secondary AML is quite a
different matter from t-AML related to alkylating
agents
WHO classification AML —
with recurrent cytogenetic
abnormalities
Advantages
• Certain cytogenetic/molecular genetic
categories are recognized
• AML, e.g. with t(8;21) and inv(16) with
less than 30% blasts is recognized
WHO classification AML —
with recurrent cytogenetic
abnormalities
Disadvantages
• Acute promyelocytic leukaemia with
classical and variant translocations are
lumped together
• All cases of AML with all 11q23 breakpoint
and MLL rearrangement are lumped
together
Acute promyelocytic leukaemia
and “variants” (i)
Subtype
M3/M3v/t(15;17)
M3-like/t(11;17)
(q23;q21)
M3-like/t(11;17)
(q13;q21)
M3-like/t(1;17)
Gene
ATRA
response
PML-RARA Yes
PLZF-RARA No
PML
distrib
Abnormal
Normal
NuMA-RARA Probably
Normal
NPM-RARA
Normal
Probably
Acute promyelocytic leukaemia
and “variants” (ii)
RARA and the normal cell
RAR binds to RXR. In the absence of RA, RAR-RXR
binds to RARE in the promoter of target genes, interacts
with co-repressors, attracts HDACs, leading to chromatin
condensation and repression of transcription
In the presence of RA, RAR-RXR is converted from a
repressor to an activator since it now binds to co-activators
and fails to bind to co-repressors
Acute promyelocytic leukaemia
and “variants” (iii)
RARA and the promyelocytic leukaemia cell
• PML-RAR binds more strongly to co-repressors and does
not dissociate in the presence of physiological levels of RA
• However, pharmacological levels of ATRA correct the
situation
• HDAC inhibitors should enhance the action of ATRA and
appear to do so
Jones and Saha (2002) Br J Haematol, 118, 714
Acute promyelocytic leukaemia
and “variants” (iv)
RARA and the leukaemia cell of M3-like/t(15;17)/PLZFRARA AML
• PLZF is a transfer factor that binds to co-repressors
• PLZF-RAR therefore binds to corepressors through two
binding sites —dependent on RARA and independent of it
• Pharmacological doses of ATRA don’t break the second
bond and therefore cases are refractory to this therapy
• However HDAC inhibitors should enhance and appear to
do so
Acute promyelocytic leukaemia
and “variants” (v)
Conclusion
• Not all acute leukaemias involving the RARA gene
represent the same disease
– Some respond to ATRA and some do not
– Some respond to arsenic trioxide and some do not
– A molecular classification can help us to predict or
understand response to therapy
Acute myeloid leukaemia with
11q23 breakpoints and MLL
rearrangement
Is this one disease or many?
The results of the European 11q23 workshop and
other published data suggest that this is many
diseases
Acute myeloid leukaemia with
11q23/MLL rearrangement
t(4;11)
t(6;11)
t(9;11)
t(10;11)
t(11;19)/MLL-ELL
t(11;19)/MLL-ENL
n
183
30
125
20
21
32
<1y
34 %
7%
17 %
32 %
14 %
41 %
ALL
95 %
10 %
7%
20 %
nil
66 %
Acute myeloid leukaemia with
11q23/MLL rearrangement
t(4;11)
t(6;11)
t(9;11)
t(10;11)
t(11;19)/MLL-ELL
t(11;19)/MLL-ENL
FAB
M4
M4/M5a
M5a
M5a
M4
M4/M5a
t-AML/MDS
5.5 %
nil
10 %
1%
7%
nil
Acute myeloid leukaemia with
11q23/MLL rearrangement
Conclusion
• Acute leukaemia with 11q23/MLL involvement is
many disease not one
• This does not yet have much therapeutic
significance but maybe it will have in the future
Are there other cytogenetic/
molecular genetic categories of
AML we should recognize?
M1/t(9;22)/BCR-ABL fusion
• FAB: M1 (occasionally M0, M2, M4)
• Usually no Auer rods
• Mainly de novo but occasionally after
topoisomerase-II-interactive drugs
• Poor prognosis
Are there other cytogenetic/
molecular genetic categories of
AML we should recognize?
M2 or M4Baso/t(6;9)/DEK-CAN fusion
• FAB: M2 (occasionally M4 or M1)
• May show basophilic differentiation and Auer rods
• Mainly de novo but occasionally after
topoisomerase-II-interactive drugs or alkylating
agents
• Poor prognosis
Are there other cytogenetic/
molecular genetic categories of
AML we should recognize?
M5/t(8;16)/MOZ-CBP fusion
• FAB: M5 or M4 with haemophagocytosis
• Abnormal coagulation
• de novo or secondary (topoisomerase-IIinteractive drugs or alkylating agents)
• Poor prognosis
Are there other cytogenetic/
molecular genetic categories of
AML we should recognize?
M7/t(1;22)/OTT-MAL fusion
• FAB: M7
• Mainly infants
Are there other cytogenetic/
molecular genetic categories of
AML we should recognize?
Various/inv(3) or t(3;3)/EVI1 dysregulation
• Any category except M3; M7 over-represented
• Platelet count often normal or even increased
• Trilineage myelodysplasia
• De novo, secondary to alkylating agents or
transformation of a MPD
Can we recognize any purely
molecular categories of AML?
Can we recognize any purely
molecular categories of AML?
Yes
AML with CEBPA mutations (i)
Mutations in the CEBPA gene, encoding the
CCAAT/enhancer binding protein  have now
been identified in 7-10% of AML
CEBP is exclusively expressed in
myelomonocytic cells and is essential for
neutrophilic differentiation
AML with CEBPA mutations (ii)
CEBP negatively regulates MYC and positively
regulates the genes encoding the receptors for
M-CSF, G-CSF and GM-CSF
CEBP-deficient mice have granulopoiesis
arrested at the myeloblast stage
AML with CEBPA mutations (iii)
CEBP mutations were found in 15 of 134
patients studied prospectively and were an
independent good prognostic feature
Such mutations, in the absence of FLT3 ITD,
could lead to a patient being classified and
treated as ‘good prognosis’
Preudhomme et al (2002) Blood, 100, 2717
What does the future hold?
Is the future micro-arrays?
What does the future hold?
• Clinical assessment and morphology will
remain crucial, particularly for diagnosis
• Cytochemistry will continue to decline in
importance, but should not be neglected
• Immunophenotyping will hang on
• Molecular diagnosis will lead to more
accurate classification and scientificallybased more effective treatment