1. Acute Myeloid Leukaemia— phenotype and genotype
Barbara J. Bain
Department of Haematology
St Mary’s Hospital, London

2. 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”

3. 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

4. Things moved on further
Further advances improved the phenotypic diagnosis. These were
Cytochemical stains
Immunophenotyping
Integration and codification of phenotypic diagnoses

5. 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

6. 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.

7. And furthermore — Molecular genetics was invented Now we have
FISH, SKY, spectral karyotyping, CGH
PCR and RT-PCR
Microarray analysis

8. What does this mean for leukaemia diagnosis and classification?
Can we move from a MIC to a MIC-M approach
Should we do so?

9. 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

10. 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

11. 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

12. 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)]

13. 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)

14. 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 therapy-induced AML (and ALL)
These cases might also be prognostically worse than de novo cases

15. 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

16. 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.

17. 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.

18. Therapy-related AML with t(15;17)(q22;q21)
n CR rate 4-yr EFS 4-yr OS
34 97% 65% 85%
‘secondary’
% 68% 78%
de novo
‘Secondary’ = 15 surgery, 17 RT, 10 Chemo, 10 Chemo + RT
Pulsoni et al. (2002) Blood, 100, 1972

19. 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.

20. What should we do?
We should follow the WHO classification but should note the presence of relevant cytogenetic abnormalities
We should recognize that topoisomerase II-inhibitor-related secondary AML is quite a different matter from t-AML related to alkylating agents

21. 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

22. 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

23. Acute promyelocytic leukaemia and “variants” (i)
Subtype Gene ATRA PML
response distrib
M3/M3v/t(15;17) PML-RARA Yes Abnormal
M3-like/t(11;17) PLZF-RARA No Normal
(q23;q21)
M3-like/t(11;17) NuMA-RARA Probably Normal
(q13;q21)
M3-like/t(1;17) NPM-RARA Probably Normal

24. 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

25. 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

26. Acute promyelocytic leukaemia and “variants” (iv)
RARA and the leukaemia cell of M3-like/t(15;17)/PLZF-RARA 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

27. 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

28. 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

29. Acute myeloid leukaemia with 11q23/MLL rearrangement
n < 1 y ALL
t(4;11) % 95 %
t(6;11) % 10 %
t(9;11) % 7 %
t(10;11) % 20 %
t(11;19)/MLL-ELL % nil
t(11;19)/MLL-ENL % 66 %

30. Acute myeloid leukaemia with 11q23/MLL rearrangement
FAB t-AML/MDS
t(4;11) M %
t(6;11) M4/M5a nil
t(9;11) M5a %
t(10;11) M5a %
t(11;19)/MLL-ELL M %
t(11;19)/MLL-ENL M4/M5a nil

31. 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

32. 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

33. 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

34. 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-II-interactive drugs or alkylating agents)
Poor prognosis

35. Are there other cytogenetic/ molecular genetic categories of AML we should recognize?
M7/t(1;22)/OTT-MAL fusion
FAB: M7
Mainly infants

36. 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

37. Can we recognize any purely molecular categories of AML?

38. Can we recognize any purely molecular categories of AML? Yes

39. AML with CEBPA mutations (i)
Mutations in the CEBPA gene, encoding the CCAAT/enhancer binding protein a have now been identified in 7-10% of AML
CEBPa is exclusively expressed in myelomonocytic cells and is essential for neutrophilic differentiation

40. AML with CEBPA mutations (ii)
CEBPa negatively regulates MYC and positively regulates the genes encoding the receptors for M-CSF, G-CSF and GM-CSF
CEBPa-deficient mice have granulopoiesis arrested at the myeloblast stage

41. AML with CEBPA mutations (iii)
CEBPa 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

42. What does the future hold?
Is the future micro-arrays?

43. 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 scientifically-based more effective treatment