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Traditional Pathology Meets Next-Generation in Acute Myeloid Leukemia

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Presentation on theme: "Traditional Pathology Meets Next-Generation in Acute Myeloid Leukemia"— Presentation transcript:

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”)


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

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?


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