PATH 430 MOLECULAR BASIS OF DISEASE MICHAEL RAUH, MD, PHD JANUARY 18, 2016

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)

Differentiation 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

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 Approx. 10,000 to 20,000 HSC in and adult It is estimated that approx. 1,000 to 10,000 HSC contribute to the production of – new blood cells throughout the body each day (i.e. normal hematopoiesis is “polyclonal”)

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

com/wp- content/uploads/2009/02/he matopoiesis_simple1.png Hematopoiesis Myeloid Cells Lymphoid Cells

Our Stem Cells Accrue Damage With Age

Number of mutations per HSC Increasing age of human subjects 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 0.13 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?

Corey et al. Nature Reviews Cancer 7, 118–129 Classification of myeloid disorders (Blast) MPNMDSAML Mature cells↑↓↓ Dysplasiararecommonsometimes BlastsNorm (<5%)<5% or 5-19%≥20% AML transformationrarecommonn/a MutationsTK pathwaysself-renewal, epigenTwo hits JAK2JAK2, MPL BCR/ABL, CBL TET2, ASXL1 Bone Marrow Failure Blood Cytopenia(s) Myeloproliferative Neoplasms Myelodysplastic Syndromes Acute Myeloid Leukemia

Corey et al. Nature Reviews Cancer 7, 118–129 Classification of myeloid disorders MPNMDSAML Mature cells↑↓↓ Dysplasiararecommonsometimes BlastsNorm (<5%)<5% or 5-19%≥20% AML transformationrarecommonn/a MutationsTK pathwaysself-renewal, epigenTwo hits Core binding factors, PML-RARA, NPM1, CEBPA FLT3, RAS

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

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

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

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

AML: G-band Karyotyping AML: recurring chromosomal translocations

AML: Fluorescent in situ Hybridization (“FISH”)

HOW DO THESE TRANSLOCATIONS CAUSE AML?

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

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

APL: using ATRA to induce blast differentiation

ARE THERE ANY OTHER SUCCESSFUL TARGETED AML THERAPIES? No! (not yet…)

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.

PUTTING IT ALL TOGETHER TO ARRIVE AT A DIAGNOSIS… MORPHOLOGY, IMMUNOPHENOTYPING, CHROMOSOMAL ANALYSIS…

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: Current (2008) Classification WHO Only 2 gene mutations!

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

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

AML: tradition meets next-generation

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

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

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

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

Types of DNA Mutations (4 “Tiers”)

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

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

Moving Towards Revised Diagnostic Categories And targeted therapeutics

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?

AML: Darwinian evolution of leukemia through sequential HSC mutations

THANK YOU! QUESTIONS?