1. Traditional Pathology Meets Next-Generation in Acute Myeloid Leukemia
…and Challenges our Definition
of “Acute” Leukemia !!!
PATH 430 Molecular Basis of Disease Michael Rauh, MD, PhD January 19, 2015

2. Objectives
Provide an overview of acute myeloid leukemia (AML) pathophysiology, current diagnosis, classification, and clinical management
Describe the emerging role of next-generation sequencing in AML and the detection of occult malignancy
Provide a foundation for the discussion of today’s papers:
Shlush et al. (Nature, 2014)
Jaiswal et al. (NEJM, 2014)

3. The Stem Cell Concept Stem Cells:
Capable of self-renewal (although this is a rare event and stem cells are mainly quiescent)
Are multipotent (i.e. can give rise to a remarkable number of daughter cells by committing to successive differentiation steps, culminating in terminally-differentiated, mature cells)
2-20 cell
divisions
per year
Differentiation

4. Hematopoietic Stem Cells
Hematopoietic stem cells (HSC) are found in the bone marrow, cord blood, and in smaller numbers in the peripheral blood
Long-lived cells that give rise to all blood cells
Comprise approx. 1 in 10,000 bone marrow cells
It is estimated that approx. 1,000 to 10,000 HSC contribute to the production of 1011 – 1012 new blood cells throughout the body each day

5. Hematopoiesis The production of mature blood cells by HSC
In adults, primarily occurs in the bone marrow

6. Hematopoiesis Lymphoid Cells Myeloid Cells
Hematopoiesis
Lymphoid Cells
Myeloid Cells

7. Our Stem Cells Accrue Damage

8. HSC mutations increase with age
Number of mutations
per HSC
Increasing age of human subjects

9. HSC mutations increase with age
Like other cells in our body, HSC have a fidelity rate of about 0.78 × 10−9 mutations per genomic base pair per cell division
Therefore, mutations randomly appear at a rate of about coding mutations per year of life (i.e. approx. one mutation every 7-8 years)
Mutations accumulate with age, and generally do not impact HSC function (i.e. they do not normally cause AML)
However, in some people, will these mutations occur in genes that predispose to leukemia?

10. Classification of myeloid disorders
(Blast)
Bone Marrow
Failure
TET2,
ASXL1
Blood
Cytopenia(s)
JAK2
JAK2, MPL
BCR/ABL, CBL
Myeloproliferative
Neoplasms
Myelodysplastic
Syndromes
Acute Myeloid
Leukemia
MPN
MDS
AML
Mature cells ↑ ↓
Dysplasia
rare
common
sometimes
Blasts
Norm (<5%)
<5% or 5-19%
≥20%
AML transformation
n/a
Mutations
TK pathways
self-renewal, epigen
Two hits
Corey et al. Nature Reviews Cancer 7, 118–129

11. Classification of myeloid disorders
Core binding factors,
PML-RARA,
NPM1, CEBPA
FLT3, RAS
MPN
MDS
AML
Mature cells ↑ ↓
Dysplasia
rare
common
sometimes
Blasts
Norm (<5%)
<5% or 5-19%
≥20%
AML transformation
n/a
Mutations
TK pathways
self-renewal, epigen
Two hits
Corey et al. Nature Reviews Cancer 7, 118–129

12. AML diagnosis: bone marrow studies
BM Aspirate:
BM Biopsy:
Morphology
Immunohistochemistry

13. AML diagnosis requires ≥ 20% blasts
AML: morphologic features
Granulopoiesis
Myeloblast
with Auer Rod
AML diagnosis requires ≥ 20% blasts
in blood or bone marrow

14. AML: French-American-British(FAB) Classification
M0: with minimal
differentiation
M1: without
maturation
M2: with maturation
M3: promyelocytic
M4: myelomonocytic
M5: monoblastic
/monocytic
M6: erythroid
M7: megakaryoblastic

15. AML: flow cytometric analysis
Blasts: express CD45 at dim levels on their surface

16. AML: flow cytometric analysis
CD34 is a blast marker, but can be expressed by both lymphoid & myeloid blasts
Myeloid blasts express other myeloid markers (i.e. CD13, 33, 117), and this
helps to assign their “lineage” and make the diagnosis of AML

17. AML: G-band Karyotyping
AML: recurring chromosomal translocations

18. AML: Fluorescent in situ Hybridization (“FISH”)

19. HOW DO THESE TRANSLOCATIONS CAUSE AML?

20. Core binding factor translocations
impair cellular differentiaton (i.e. maturation)
Normal
Progenitor
Cell
Maturation
Programs Activated
AML/RUNX1
RUNX1T1
Maturation
Arrest
t(8;21)
Maturation
Arrest
inv(16)
MYH11

21. The t(15;17) translocation also
impairs cellular differentiation (i.e. maturation)
Maturation Arrest:
‘M3’ Acute Promyelocytic Leukemia (APL)

22. APL: using ATRA to induce blast differentiation

23. Are there any other successful targeted aml therapies?
No! (not yet…)

24. Standard 3+7 AML “Induction” Chemotherapy
An anthracycline, Daunorubicin interacts with DNA by intercalation and inhibition of macromolecular biosynthesis. This inhibits the progression of the enzyme topoisomerase II, which relaxes supercoils in DNA for transcription.
3 days, IV
Cytosine arabinoside (Ara-C) is similar enough to human cytosine deoxyribose (deoxycytidine) to be incorporated into human DNA, but different enough that it kills the cell.
Kills dividing cells – not particularly targeted!
After induction, if <5% blasts, considered in morphological remission.

25. Putting it all together to arrive at a diagnosis…
Morphology, immunophenotyping, chromosomal analysis…
Putting it all together to arrive at a diagnosis…

26. AML: Current (2008) Classification WHO M3
Acute myeloid leukemia and related neoplasms:
Acute myeloid leukemia with recurrent genetic abnormalities
AML with t(8;21)(q22;q22); RUNX1-RUNX1T1
AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11
APL with t(15;17)(q22;q12); PML-RARA
AML with t(9;11)(p22;q23); MLLT3-MLL
AML with t(6;9)(p23;q34); DEK-NUP214
AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1
AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1
Provisional entity: AML with mutated NPM1
Provisional entity: AML with mutated CEBPA
Acute myeloid leukemia with myelodysplasia-related changes
Therapy-related myeloid neoplasms
Acute myeloid leukemia, not otherwise specified
AML with minimal differentiation
AML without maturation
AML with maturation
Acute myelomonocytic leukemia
Acute monoblastic/monocytic leukemia
Acute erythroid leukemia
Acute megakaryoblastic leukemia
Acute basophilic leukemia
Acute panmyelosis with myelofibrosis
Myeloid sarcoma
Myeloid proliferations related to Down syndrome
Transient abnormal myelopoiesis
Myeloid leukemia associated with Down syndrome
Blastic plasmacytoid dendritic cell neoplasm M3
Only 2 gene
mutations!
Old FAB: M0 M1 M2 M4 M5 M6 M7

27. AML: cytogenetic risk stratification
“CBF” & “PML-RARA”

28. The problem:
Traditional diagnostics and treatments
are reaching their limitations
Where can we turn for
novel insights and approaches?

29. AML: tradition meets next-generation

30. Higher-throughput sequencing technologies
Success story:
Higher-throughput sequencing technologies
make somatic mutation profiling more feasible
enhancing diagnostic and prognostic yield

31. Next generation genomic sequencing
Couples pH changes during DNA synthesis to sequence data
In-house at Queen’s University

32. Ion Torrent next-generation sequencing
pH sensors below the sample wells record digital sequences

33. Ion Torrent next-generation sequencing
Bioinformatics programs align
the short sequences to a
reference genome and ‘variants’ are called

34. Types of DNA Mutations (4 “Tiers”)

35. Tier 1 (coding exons) comprise only 1.3% of the genome
Mutations in Tier 1 (coding exons) are likely very important
However, little is currently know of the function of other genomic tiers

37. The New Genetic Model of AML
Blue = cooperativity
Red = exclusivity

39. Moving
Towards
Revised
Diagnostic
Categories
And targeted
therapeutics

40. SUMMARY
Currently, AML is diagnosed using blast counts, immunophenotyping, chromosomal analysis, and (rarely) mutations
Apart from ATRA in t(15;17) AML, treatment is mainly one- size-fits all
Gene mutation profiling is helping to refine diagnostic risk categories and to guide rational and targeted therapeutics
Paper 1: Mutation profiling unexpectedly reveals evidence of a pre-leukemic state
Paper 2: How common is this pre-leukemic state and what are the implications?

42. AML: Darwinian evolution of leukemia through sequential HSC mutations

44. THANK YOU! Questions?