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PATH 430 MOLECULAR BASIS OF DISEASE
MICHAEL RAUH, MD, PHDJANUARY 18, 2016
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
• 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 1011 – 1012 new blood cells throughout the body each day (i.e. normal hematopoiesis is “polyclonal”)
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 Cells Accrue Damage
With Age
Num
ber o
f mut
atio
nspe
r HSC
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: 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 KaryotypingAML: 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:Current (2008)Classification
WHO
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?