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
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)
The Stem Cell Concept
Stem Cells:
2-20 cell
divisions
per year
• Capable of self-renewal
(although this is a rare
event and stem cells are
mainly quiescent)
Differentiation
• 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)
http://www.biochemj.org/bj/404/0169/bj4040169.htm
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
• The production of mature blood cells by HSC
• In adults, primarily occurs in the bone marrow
http://en.wikipedia.org/wiki/Haematopoiesis
http://www.allthingsstemcell.
com/wpcontent/uploads/2009/02/he
matopoiesis_simple1.png
Hematopoiesis
Myeloid Cells
Lymphoid Cells
Our Stem Cells
Accrue Damage
http://www.hematology.org/Publications/Hematologist/2013/9947.aspx
Number of mutations
per HSC
HSC mutations increase with age
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?
Classification of myeloid disorders
(Blast)
Bone Marrow
Failure
TET2,
ASXL1
Blood
Cytopenia(s)
JAK2
Mature cells
Dysplasia
Blasts
AML transformation
Mutations
JAK2, MPL
BCR/ABL, CBL
Myeloproliferative
Neoplasms
Myelodysplastic
Syndromes
Acute Myeloid
Leukemia
MPN
MDS
AML
↑
↓
↓
rare
common
sometimes
Norm (<5%)
<5% or 5-19%
≥20%
rare
common
n/a
TK pathways
self-renewal, epigen
Two hits
Corey et al. Nature Reviews Cancer 7, 118–129
Classification of myeloid disorders
Core binding factors,
PML-RARA,
NPM1, CEBPA
FLT3, RAS
Mature cells
Dysplasia
Blasts
AML transformation
Mutations
MPN
MDS
AML
↑
↓
↓
rare
common
sometimes
Norm (<5%)
<5% or 5-19%
≥20%
rare
common
n/a
TK pathways
self-renewal, epigen
Two hits
Corey et al. Nature Reviews Cancer 7, 118–129
AML diagnosis: bone marrow studies
BM Aspirate:
BM Biopsy:
•Morphology
•Immunohistochemistry
AML: morphologic features
Granulopoiesis
Myeloblast
with Auer Rod
AML diagnosis requires ≥ 20% blasts
in blood or bone marrow
http://www.tau.ac.il/~inter05/g-all.gif
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
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
http://www.asco.org/
AML: Fluorescent in situ
Hybridization (“FISH”)
HOW DO THESE
TRANSLOCATIONS
CAUSE AML?
Core binding factor translocations
impair cellular differentiaton (i.e. maturation)
Normal
Progenitor
Cell
Maturation
Programs Activated
AML/RUNX1
RUNX1T1
t(8;21)
Maturation
Arrest
inv(16)
Maturation
Arrest
MYH11
http://www.elsevierimages
.com/image/28065.htm
The t(15;17) translocation also
impairs cellular differentiation (i.e. maturation)
Maturation Arrest:
‘M3’ Acute
Promyelocytic
Leukemia (APL)
http://www.bioscience.org/2009/v14/af/3333
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
• Kills dividing cells – not particularly
targeted!
• After induction, if <5% blasts, considered
in morphological remission.
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.
MORPHOLOGY, IMMUNOPHENOTYPING,
CHROMOSOMAL ANALYSIS…
PUTTING IT ALL TOGETHER
TO ARRIVE AT A DIAGNOSIS…
AML:
Current (2008)
Classification
WHO
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
M3
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
Only 2 gene
mutations!
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
Old FAB:
M0
M1
M2
M4
M5
M6
M7
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
www.genome.gov/Multimedia/Slides/.../04_Wilson_Fitting.pdf
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 onesize-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?