path 430 molecular basis of disease michael rauh, md, phd january 19, 2015

PATH 430 MOLECULAR BASIS OF DISEASE

MICHAEL RAUH, MD, PHDJANUARY 19, 2015

Traditional PathologyMeets Next-Generation inAcute Myeloid Leukemia

…and Challenges our Definition of “Acute” Leukemia !!!

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

http://www.biochemj.org/bj/404/0169/bj4040169.htm

The Stem Cell ConceptStem 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 divisionsper 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

• 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

Hematopoiesis

http://en.wikipedia.org/wiki/Haematopoiesis

• The production of mature blood cells by HSC

• In adults, primarily occurs in the bone marrow

http://www.allthingsstemcell.com/wp-content/uploads/2009/02/hematopoiesis_simple1.png

Hematopoiesis

Myeloid CellsLymphoid Cells

http://www.hematology.org/Publications/Hematologist/2013/9947.aspx

Our Stem CellsAccrue Damage

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Increasing age of human subjects

HSC mutations increase with age

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)

MPN MDS AML

Mature cells ↑ ↓ ↓

Dysplasia rare common sometimes

Blasts Norm (<5%) <5% or 5-19% ≥20%

AML transformation rare common n/a

Mutations TK pathways self-renewal, epigen Two hits

JAK2 JAK2, MPLBCR/ABL, CBL

TET2,ASXL1

Bone MarrowFailure

BloodCytopenia(s)

MyeloproliferativeNeoplasms

MyelodysplasticSyndromes

Acute MyeloidLeukemia

Corey et al. Nature Reviews Cancer 7, 118–129

Classification of myeloid disorders

MPN MDS AML

Mature cells ↑ ↓ ↓

Dysplasia rare common sometimes

Blasts Norm (<5%) <5% or 5-19% ≥20%

AML transformation rare common n/a

Mutations TK pathways self-renewal, epigen Two hits

Core binding factors,PML-RARA,

NPM1, CEBPA

FLT3, RAS

BM Aspirate:

BM Biopsy:•Morphology•Immunohistochemistry

AML diagnosis: bone marrow studies

http://www.tau.ac.il/~inter05/g-all.gif

AML: morphologic featuresGranulopoiesis

Myeloblastwith Auer Rod

AML diagnosis requires ≥ 20% blasts in blood or bone marrow

AML: French-American-British(FAB) Classification

M0: with minimaldifferentiation

M1: withoutmaturation

M2: with maturation M3: promyelocytic

M4: myelomonocytic M5: monoblastic/monocytic

M6: erythroid M7: megakaryoblastic

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 thishelps to assign their “lineage” and make the diagnosis of AML

http://www.asco.org/

AML: G-band Karyotyping

AML: recurring chromosomal translocations

AML: Fluorescent in situHybridization (“FISH”)

HOW DO THESE TRANSLOCATIONSCAUSE AML?

http://www.elsevierimages.com/image/28065.htm

AML/RUNX1 RUNX1T1

MYH11

NormalProgenitorCell

t(8;21)

inv(16)

Core binding factor translocationsimpair cellular differentiaton (i.e. maturation)

MaturationPrograms Activated

MaturationArrest

MaturationArrest

http://www.bioscience.org/2009/v14/af/3333

Maturation Arrest:‘M3’ Acute Promyelocytic Leukemia (APL)

The t(15;17) translocation alsoimpairs cellular differentiation (i.e. maturation)

APL: using ATRA to induce blast differentiation

ARE THERE ANYOTHER 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 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

AML:Current (2008)Classification

WHO

Old FAB:M0M1M2M4M5M6M7

M3

Only 2 gene mutations!

AML: cytogenetic risk stratification

“CBF” & “PML-RARA”

The problem:

Traditional diagnostics and treatmentsare reaching their limitations

Where can we turn for novel insights and approaches?

AML: tradition meets next-generation

Success story:

Higher-throughput sequencing technologiesmake 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 alignthe short sequences to areference genome and ‘variants’ are called

Types of DNA Mutations (4 “Tiers”)

www.genome.gov/Multimedia/Slides/.../04_Wilson_Fitting.pdf

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 = cooperativityRed = exclusivity

MovingTowardsRevisedDiagnosticCategoriesAnd targetedtherapeutics

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 leukemiathrough sequential HSC mutations

THANK YOU!

QUESTIONS?