MOLECULAR PATHOLOGY
LABORATORY
Next Generation Sequencing for
High Yield AML and MDS Analysis
Aaron D. Bossler, MD, PhD
Clinical Associate Professor
Director, Molecular Pathology Laboratory
Phone: 319-384-9566
aaron-bossler@uiowa.edu
Disclosure: Grant or research support received from: Iowa Department of Health,
Roche Diagnostics and Cepheid, Inc.
1
Acknowledgements
MOLECULAR PATHOLOGY
LABORATORY
Anup Tilak, PhD
Natasha Guseva, PhD
Aaron Stence
Jon Pruessner
Connie Floerchinger
Deqin Ma, MD, PhD
Anthony Snow, MD
Overview
MOLECULAR PATHOLOGY
LABORATORY
Mutations
Normal
Hematopoiesis
Clinical
Trials
MDS
Treatment
Leukemia
Prognosis
WHO 2008 AML Classification
MOLECULAR PATHOLOGY
LABORATORY
AML with recurrent
genetic abnormalities
AML with
multilinage dysplasia
Therapy related AML
AML not otherwise
classified
Recurrent Mutations in AML
MOLECULAR PATHOLOGY
LABORATORY
NEJM.366.1079.2012.Patel
Recurrent Mutations in MDS
MOLECULAR PATHOLOGY
LABORATORY
Leuk.28.241.2014.Haferlach
Ion Torrent PGM
Semiconductor Sequencing
MOLECULAR PATHOLOGY
LABORATORY
Chips are biosensors with
1-11 million pH meters each
•
•
•
•
•
•
•
pH sensing semiconductor sequencing by synthesis
35–400bp read lengths
PCR-based target capture – requires only 10ng of DNA
Bar-coding to be able to run multiple specimens
Fast sequencing times (< 1 day)
Relatively inexpensive to operate
Detection of substitutions and small insertion/deletion (<30bp) mutations
Ion Torrent PGM:
pH Measurement
MOLECULAR PATHOLOGY
LABORATORY
template
dNTP flow cycle
T
A
C
Ion Torrent PGM Semiconductor
based Sequencing
Library
construction
Template clonal
amplification
Sequencing
by PGM
MOLECULAR PATHOLOGY
LABORATORY
Data
analysis
9
MOLECULAR PATHOLOGY
LABORATORY
Functional Groupings
MOLECULAR PATHOLOGY
LABORATORY
UIHC AML/MDS 30 Gene Panel
MOLECULAR PATHOLOGY
LABORATORY
Pathway / Classification
Genes
1. Nucleophosmin
2. Myeloid Transcription Factors
3. Activated Signaling
NPM1
RUNX1, CEBPA, GATA2
FLT3, KIT, CBL, JAK2, KRAS,
NRAS, HRAS, BRAF
TP53, PHF6, NF1, PTEN,
PTPN11, WT1
DNMT3A, TET2, IDH1, IDH2,
MLL, ASXL1, EZH2
ZRSR2, SF3B1, SRSF2, U2AF1
SETBP1
4. Tumor Suppressors
5. Epigenetic Regulation/
Chromatin Modifiers
6. Spliceosome
7. DNA Replication
12
MOLECULAR PATHOLOGY
LABORATORY
1. NPM1
NPM1 Mutation Associated with
Improved Survival; Meta-analysis
MOLECULAR PATHOLOGY
LABORATORY
Ann.Hematol.93.1279.2014.Port
MOLECULAR PATHOLOGY
LABORATORY
2. Myeloid Transcription Factors
CEBPA
RUNX1
GATA2
CEBPalpha
MOLECULAR PATHOLOGY
LABORATORY
• 7-15% of AMLs have CEBPA mutations (most are single mutations)
• Double mutant/biallelic cases predict a favorable prognosis
– Low frequency of other mutations or other cytogenetic abnormalities
J.Clin.Onc.29.2739.2010.Green
RUNX1
MOLECULAR PATHOLOGY
LABORATORY
• Alpha subunit of core binding factor transcription factor
• 13% of AMLs have RUNX1 mutations
– Associated with poor outcomes in contrast to RUNX1 gene fusions,
t(8;21)
• Prior H/o MDS or radiation exposure frequently
• Will become a WHO defined AML for de novo only
< 60 yrs old
≥ 60 yrs old
J.Clin.Onc.30.3109.2012.Mendler
GATA2
< 60 yrs old
•
MOLECULAR PATHOLOGY
LABORATORY
≥ 60 yrs old
Indispensable transcription factor for hematopoiesis
– Maintains the proliferative progenitor-cell phenotype
•
•
Mutations are associated with bi-allelic CEBPA mutations and mostly favorable
outcomes in cytogenetically normal AML
Found in families with predisposition to MDS and AML
Leukemia (2013) 27, 482–485. Fasan A
Familial AML and MDS Associations;
Germline Mutations
MOLECULAR PATHOLOGY
LABORATORY
• CEBPA – familial AML
• GATA2 – familial MDS/AML
• RUNX1 – familial platelet disorder with
propensity to myeloid malignancy
• All are heterogeneous with regard to their
clinical presentation and progression
MOLECULAR PATHOLOGY
LABORATORY
3. Activated Signaling
FLT3
KIT
CBL
NRAS and KRAS
FLT3 ITD Association with Worse
Survival; Meta-analysis
•
ITD mutations in 37-46% of cytogenetically normal AMLs
•
•
•
MOLECULAR PATHOLOGY
LABORATORY
Tyrosine kinase domain codons 835 and 836 mutated in 10-15%
Constitutive kinase activity  activation of multiple signaling pathways  promotes cell
proliferation and resistance to apoptosis
Clinical Trials: first gen inhibitors not successful, 2nd and 3rd gen inhibitors in trials now
Ann.Hematol.93.1279.2014.Port
KIT Mutation
MOLECULAR PATHOLOGY
LABORATORY
• Mutated in core binding factor
AML (t(8;21), inv or t(16))
– Increased incidence of relapse
and inferior outcome
• Treatment: Imatinib with
standard chemo therapy has
some activity
– Phase 1/2 studies are
investigating combination
dasatinib and standard
chemotherapy in core binding
factor leukemias
Leuk.28.1132.2014.Manara
CBL
MOLECULAR PATHOLOGY
LABORATORY
• Ubiquitin ligase
– Targets a variety of tyrosine kinases for degradation by ubiquitination
– Important for the termination of signaling of receptor tyrosine kinases
• Mutations more common in MDS (~5% of cases) than AML
NRAS and KRAS
•
•
MOLECULAR PATHOLOGY
LABORATORY
GTP binding proteins - act downstream of tyrosine kinase receptors (FLT3 or KIT)
Codon 12, 13, 61, and 146 mutations
– inactivate intrinsic GTPase activity resulting in constitutive activation
– Seen in 5-15% of AML and some MDS
•
•
Hyperactive signaling via pro-proliferative PI3K/AKT/mTOR and RAF/MEK/ERK
pathways is centrally implicated in malignant transformation driven by RAS
mutations
Treatment: several clinical trials for downstream targets including AKT and MEK
inhibitors
RAS mutations lead to
constitutively active RAS
To Summarize
MOLECULAR PATHOLOGY
LABORATORY
Favorable Prognosis
• NPM1 duplication
• CEBPA bi-allelic mutations
– GATA2 mutations
Association with MDS
• GATA2
• CBL
Poor Prognosis
• RUNX1 mutations
• FLT3 ITD and TKD
• KIT exon 8 and 17
Treatment/Clinical Trials
• FLT3
• KIT
• RAS (via MEK or AKT inhibition)
To Summarize
MOLECULAR PATHOLOGY
LABORATORY
Favorable Prognosis
• NPM1 duplication
• CEBPA bi-allelic mutations
– GATA2 mutations
Poor Prognosis
• RUNX1 mutations
• FLT3 ITD and TKD
• KIT exon 8 and 17
Associated with familial MDS/AML
Association with MDS
• GATA2
• CBL
Treatment/Clinical Trials
• FLT3
• KIT
• RAS (via MEK or AKT inhibition)
MOLECULAR PATHOLOGY
LABORATORY
4. Tumor Suppressors
WT1
WT1; Prognostic Importance is
Controversial
Normal karyotype patients
MOLECULAR PATHOLOGY
LABORATORY
• Zinc finger transcription
factor
• Exon 7 and 9 Mutations
– 5-10% in AML
– ~3% in MDS
• Prognosis: CN patients with
WT1 mutations had worse
overall survival
• Treatment: clinical trials of
immunogenic WT1 peptide
(confirmed WT1 expression)
Leuk.29.660.2015.Krauth
MOLECULAR PATHOLOGY
LABORATORY
5. Epigenetic Regulation; DNA
methylation and chromatin modification
DNMT3A, IDH1, IDH2, TET2,
MLL, ASXL1, EZH2
High Frequency of Mutations in
Regulators of Epigenetics
MOLECULAR PATHOLOGY
LABORATORY
2010
NEJM.366.1079.2012.Patel
Epigenetic Regulation of a
Leukemogenic Locus
•
Histone acetyltransferases (HAT) and
methyltransferases (MLL) activate expression
•
•
Polycomb complex (PRC2; EED, EZH2 and SUZ12)
represses
DNMT3A mediates de novo methylation
•
•
•
•
MOLECULAR PATHOLOGY
LABORATORY
involved in formation of 5-mC from cytosine;
interacts with HMTs and PRC2
Loss of function mutations
Oncomorphic mutation R882H disrupts wildtype
function
ASXL1 directs PRC2 to the locus to repress
– Loss-of-function mutations in PRC2 or ASXL1
remove PRC2 repression.
•
TET2 mediates hydroxylation of 5mC to 5hmC
- requires α-ketoglutarate provided by
– Aberrant methylation patterns result from TET2 or
IDH1/2 mutation and produce 2-hydroxyglutarate
instead of αKG.
BMC Cancer.12.304.2012.Murati
Utility of Mutations in Epigenetic
Regulators
•
MOLECULAR PATHOLOGY
LABORATORY
DNMT3A
– Mutations in 18-22% of AML, 29-34% in NK AML
– Prognosis: poorer outcomes especially with R882 mutation, more favorable outcome with hi dose
daunorubicin
•
IDH1 and IDH2
– 6-9% and 8-12% of AML, resp.
– Prognosis:
•
•
IDH1 – worse Overall and Event-free Survival in NK AML with favorable- (NPM1 mutated) or intermediaterisk disease
IDH2 – mostly worse outcome except for R140 mutation
– Clinical trials of mutant selective inhibitors are now beginning
•
TET2
– Mutations are found in MDS (>30%), MPN and AML (~10%)
– Prognosis: usually poorer outcomes
•
ASXL1
– Mutations in chronic myelomonocytic leukemia, myelodysplastic syndrome, and
myeloproliferative neoplasms (20-25% of cases)
– 5–16% of older patients (those aged 60 years and older) with AML have ASXL1 mutations
– Associated with poor outcome in all studies reported to date
Survival Associations
MOLECULAR PATHOLOGY
LABORATORY
NEJM.366.1079.2012.Patel
Risk Classification of Patients
with Intermediate-Risk AML
•
•
•
•
MOLECULAR PATHOLOGY
LABORATORY
ECOG E1900 trial
398 patients
Showed improved outcomes using induction therapy or cytarabine and high dose daunorubicin in patients with
newly diagnosed AML compared to low does daunorubicin
Profiling was performed to identify genetic differences and prognosis
Risk Stratification Based on
Genetics
MOLECULAR PATHOLOGY
LABORATORY
MLL-PTD: partial tandem duplication in MLL
NEJM.366.1079.2012.Patel
MOLECULAR PATHOLOGY
LABORATORY
6. Spliceosome
SF3B1, ZRSR2, SRSF2, U2AF1
RNA Splicing
MOLECULAR PATHOLOGY
LABORATORY
Int.J.Hematol.101.229.2015.Zoi
Spliceosome (ZRSR2, SF3B1,
SRSF2, U2AF1) Mutations
MOLECULAR PATHOLOGY
LABORATORY
• Can be gain of function through exon skipping or
alternative splicing or loss of function by intron retention
of target genes.
• More prevalent in secondary than in de novo AML
• Usually mutually exclusive - suggesting functional
redundancy or combined lethal effect
Spliceosome Mutations
MOLECULAR PATHOLOGY
LABORATORY
• 60 % of CMML cases harbor mutations
– 50% in SRSF2, 20% in ZRSF2, SF3B1, U2AF35, U2AF65 and
SF3A1
• SF3B1 mutations
– Significantly associated with appearance of ring sideroblasts
and in 75% of RARS-T
• SRSF2 mutations cluster to hotspot residue Pro95
– Co-occur with TET2, ASXL1, RUNX1, or IDH mutations
– Predict increased risk of progression from myeloproliferative
neoplasms to secondary AML
SF3B1 in MDS Conveys
Favorable Prognosis
MOLECULAR PATHOLOGY
LABORATORY
Blood.124.1513.2014.Malcovati
7. SETBP3
MOLECULAR PATHOLOGY
LABORATORY
• Possibly involved in DNA
replication
• 25% of aCML
• 10% advanced MDS
• 5% CMML
• Associated with poor
overall survival and high
risk of leukemic evolution
Nat.Gen.45.18.2013.Piazza
Summary
MOLECULAR PATHOLOGY
LABORATORY
Favorable Prognosis
Poor Prognosis
• NPM1 duplication/FLT3wt
• CEBPA bi-allelic mutations
• DNMT3A when treated with
high dose daunorubicin
• IDH1/IDH2 mutations
• SF3B1
•
•
•
•
•
•
•
Association with MDS
Treatment/Clinical Trials
– GATA2 mutations
•
•
•
•
GATA2
CBL
SF3B1, ZRSR2, SRSF2, U2AF1
SETBP1
RUNX1 mutations
FLT3 ITD and TKD
KIT exon 8 and 17 mutations
DNMT3A R882 mutation
ASXL1 mutations
TET2 mutations
SETBP1 mutations
• FLT3
• KIT
• RAS (via MEK or AKT
inhibition)
UIHC AML/MDS 30 Gene Panel
MOLECULAR PATHOLOGY
LABORATORY
Pathway / Classification
Genes
1. Nucleophosmin
2. Myeloid Transcription Factors
3. Activated Signaling
NPM1
RUNX1, CEBPA, GATA2
FLT3, KIT, CBL, JAK2, KRAS,
NRAS, HRAS, BRAF
TP53, PHF6, NF1, PTEN,
PTPN11, WT1
DNMT3A, TET2, IDH1, IDH2,
MLL, ASXL1, EZH2
ZRSR2, SF3B1, SRSF2, U2AF1
SETBP1
4. Tumor Suppressors
5. Epigenetic Regulation/
Chromatin Modifiers
6. Spliceosome
7. DNA Replication
43
Summary
MOLECULAR PATHOLOGY
LABORATORY
Mutations
Normal
Hematopoiesis
Clinical
Trials
MDS
Treatment
Leukemia
Prognosis
MOLECULAR PATHOLOGY
LABORATORY
Mutation Evolution in MDS
MOLECULAR PATHOLOGY
LABORATORY
Blood.122.3616.2013.Papaemmanuil