Transcript How to make sense of genetic studies in AML and MDS Elie Traer September 13, 2012

How to make sense of genetic
studies in AML and MDS
Elie Traer
September 13, 2012
Outline
• Genetic tests and methodology
– Cytogenetics, i.e. large chromosomal abnormalities
– FISH, smaller chromosomal changes
– Genetic mutations
• AML
– Evolution of genetic tests in diagnosis and prognosis
– Risk groups and treatment
– Future
• MDS
– Current prognostication in acute leukemias
• What to order
• How it affects treatment
Cytogenetics - Methodology
• Cytogenetics (karyotype)
– Cells arrested in metaphase with
mitotic inhibitors
– Depends upon chromatin
condensation during metaphase
(metaphase spread)
• Most cells are in interphase
– Staining with Geimsa stain produces
distinctive G-banding patterns
Interphase
Metaphase
Of course, not
everything is neatly
arranged in real life…
Normal cytogenetics – cleaned up
22 paired chromosomes + XY = 46 total
(image from NHGRI)
Classic cytogenetics
• Advantages:
– Whole chromosome analysis of individual cells
– Bone marrow aspirate relatively easier
• Compared to solid tumors
• Disadvantages:
– Time consuming
– Need metaphase spreads
– Not sensitive for small genetic deletions or
changes
FISH – a “new” technique for cytogenetics
• FISH = fluorescence in situ hybridization
• Technique developed in part by Joe Gray
• Proc Natl Acad Sci U S A. 1986 May;83(9):2934-8
• Fluorescently labeled probes targeted to known
areas of chromosomes
• Advantages compared to cytogenetics
– Don’t need metaphase
– Sensitive to small changes
– Can target any part of chromosome
• BUT, you have to know what you are looking for
FISH – CML
• Red probe to
chromosome 22
• Green probe to
chromosome 9
• Fusion creates
red-green or
yellow color
Probes come together
Interphase
Metaphase
Cytogenetics in leukemia
• Oldest method for looking at chromosomes
– >300 cytogenetic abnormalities in acute leukemia
• Most famous translocation is t(9;22), or
Philadelphia chromosome
– fusion of BCR and ABL genes
– led to development of imatinib
• Technique demonstrate clonality of leukemia
– CML with specific secondary cytogenetic
abnormalities
• Cytogenetic abnormalities associated with
disease subtypes and prognosis
How important are genetics?
FAB CLASSIFICATION
o M1 myeloblastic
undifferentiated
o M2 myeloblastic with
differentiaion
o M3 promyelocytic
o M4 myelomonocytic
o M5 monoblastic
o M6 erythroleukaemia
o M7 megakaryoblastic
WHO CLASSIFICATION 2008
o AML with recurrent genetic
abnormalities
o AML with multilineage
dysplasia
o AML therapy related
o AML not otherwise categorised
o AML of ambiguous lineage
WHO 2008 recurrent cytogenetics
•
•
•
•
•
•
t(8;21)(q22;q22)
Inv(16)(p13.1q22) or t(16;16)(p13.1;q22)
t(15;17)(q22;q12)
t(9;11)(p22;q23):MLLT3-MLL
t(6;9)(p23;q34)
t(1;22)(p13q13)
*Considered acute leukemias regardless of blast count
Cytogenetic risk groups
• Favorable (~20%)
– CBF: t(8;21)(q22;q22), Inv(16)(p13.1q22) or t(16;16)(p13.1;q22)
– APL: t(15;17)
• Intermediate (~60%)
– Normal cytogenetics (~50% of all AML cases)
– t(9;11)(p22;q23):MLLT3-MLL
– Any cytogenetic abnormality not classified as favorable or adverse
(trisomy 8)
• Adverse (~20%)
–
–
–
–
–
–
–
Inv(3)(q21q26.2) or t(3;3)(q21;q26.2)
t(6;9)(p23;q34)
t(v;11)(v;q23): MLL rearranged
- 5 or del(5q)
-7
Abnl 17p
Complex karyotype
Core Binding Factor Leukemias
• t(8;21) and inv16/t(16;16)
atlasgeneticsoncology.org
t(8;21)(q22;q22) RUNX1-RUNX1T1
•
•
•
•
RUNX1 = AML1 = CBFa = 21
RUNX1T1 = ETO = 8
Approximately 8% of AMLs - predominately in younger patients
Blasts have cytoplasmic hoffs, occasional Auer rods, occasional salmoncolored granules
• Dysplastic features in maturing neutrophils
• Favorable prognosis when presenting with white blood cell count less than
20 x 109/L and NO KIT mutation
Jaffe Hematopathology
Inv(16)(p13.1q22) or t(16;16)(p13.1;q22)
•
•
•
•
•
•
CBFB = 16q22
MYH11 (smooth muscle myosin heavy chain) = 16p13
5-8% of AML
All age groups, predominately in younger patients
Blasts have myelomonocytic features
Abnormal eosinophils with large granules in bone marrow
(M4Eo)
– no peripheral eosinophilia
• Good prognosis when NO KIT mutation is present
• May be missed on routine karyotyping, need FISH
Acute promyelocytic anemia (APL) with
t(15;17)(q22;q12) PML-RARA
• Proliferation of leukemic blasts blocked at the promyelocyte
stage of differentiatoin
– 5-8% of AML
– Abundant cytoplasmic granules and Auer Rods
– Weak or absent HLA-DR and absent CD34 expression
– Low white count
– Disseminated intravascular coagulation (DIC) – Medical
Emergency!
– Treat with all-trans-retinoic acid (ATRA)
Survival associated with cytogenetics
At Risk
Cumulative Percent
100
Favorable
Intermediate
Unfavorable
80
121
278
184
Deaths
53
168
162
Estimate (CI)
at 5 Years
55% (45-64%)
38% (32-44%)
11% ( 7-16%)
60
40
20
Heterogeneity of 3 Groups: p<.0001
0
0
2
4
6
8
Years After Entering Study
Slovak et al. Blood, 2000
Is cytogenetic analysis old news?
• Presence of a monosomy (ie chromosome 7
deletion) with 2 additional chromosomal
deletions or with complex cytogenetics
• Associated with a poor CR rate and OS (4%)
• Many recent studies have confirmed
– phrases such as dismal outcome and very
unfavorable
Breems J Clin Oncol 2008
Effect on overall survival
Breems J Clin Oncol 2008
Cytogenetics summary
• Cytogenetics remains an important risk
classification for acute leukemia, particularly
AML
• Cytogenetics/FISH can only detect large
genetic changes
• However, 50-60% of AML with “normal”
cytogenetics
– Really normal?
Mutations in leukemia (molecular markers)
• Smaller changes to DNA
are not detected with
cytogenetics/FISH
– Mutations
– Smaller duplications
– Deletions
Quick genetic review
• DNA holds all
instructions
– 3 trillion base pairs
• mRNA translated
from DNA
– introns spliced out
– hundreds to
thousands of base
pairs
• mRNA translated into
protein
Mutations – methodology
• PCR
– Method for amplifying known sections of DNA or mRNA
– Can be quantitative (QPCR)
• BCR-ABL, PML-RARA
– Can detect small variations in size (deletions or amplifications)
• e.g. FLT3 ITD
• Sanger sequencing
– Direct sequencing of DNA
• Sequenom
– Multiplexed PCR and MALDI-TOF
• Next generation sequencing
– Whole exome/genome
– Deep sequencing
PCR
• Primers surrounding area
of interest
• Selective area amplified
• Can be sequenced or
analyzed by gel
electrophoresis
FLT3 internal
tandem
duplication
• FLT3 is receptor tyrosine
kinase
• Japanese groups
originally found internal
tandem duplication
• Nakao et al. Leukemia
1996
• ITD leads to activation
of kinase
• Can be detected by PCR
ITD
normal
ASH Education Book January 1, 2001 vol. 2001 no. 1 541-552
FLT3 ITD is unfavorable risk marker
• FLT3 ITD detected in
~30% of normal
cytogenetics AML
• Point mutations, i.e.
D835 mutations found
in ~10%
• Not prognostic
• Associated with higher
relapse and worse
overall survival
Kottaridis et al. Blood 2001
Point mutations:
CEBPa
• Transcription factor
involved in neutrophil
differentiation
• Mutations in multiple
sites but most lead to
early truncation of
protein
• 10% mutations in
normal cytogenetics
AML
Preudhomme et al. Blood 2002
NPM1 mutations
• Most frequent
molecular
abnormality in
normal cytogenetics
AML: 50-60%
• Usually 4 nucleotide
insertion
• Can be detected
with Sanger
sequencing
Chen et al. Arch Pathol Lab Med. 2006
NPM1 mutation
• Nuclear transport protein
• Mutation associated with abnormal
localization of protein in cytoplasm
• C-terminal mutations detected in 85%
– NLS domain
• Frequently occurs with FLT3 ITD
Fallini N Engl J Med 2005; Döhner Blood 2005;
Schnittger Blood 2005; Verhaak Blood 2005
NPM1 and CEBPa are favorable
prognostic markers (without FLT3 ITD)
Schlenk N Engl J Med 2008
c-Kit
• Receptor tyrosine kinase
• Activating mutations in c-Kit have been described
in AML
• Most common mutation in exon 17 (D816V)
• Only has prognosis in core binding factor (CBF)
AML, inv(16) and t(8;21)
• Paschka et al. J Clin Oncol, 24 2006
• However, recent report suggests that this may be
limited to t(8;21)
• Park et al. Leuk Res, 2011
Genetic (cytogenetic and molecular)
abnormalities and prognosis in AML
Sequencing more genes and more
samples is increasing known mutations
• Largely driven by technology
– Dramatic cost reductions
– ~$100,000 to sequence genome in 2008
• Ley et al. Nature 2008
– Now about $2000
• Prognosis still not clear for many of these
genes
Mutations in AML
NPM1
45-64% CN-AML
Good without other mutations present
CEBPA
10-18% CN-AML
Good with both alleles are mutated
FLT3 ITD
28-34% CN-AML
Worse
KIT
25-30% CBF AML
Inferior* in CBF-AML
FLT3 TKD
5-10% all AML/11-14% CN-AML
?
IDH1/2
10-15% CN-AML
Inferior – controversial
WT1
10-13% CN-AML
?
RUNX1
5-13% all AML
few studies – worse
MLL-PTD
5-11% CN-AML
Worse, but not an independent prognostic factor
NRAS
9-14% CN-AML, 40% CBF AML,
25-30% AML with inv(3)
None
KRAS
5-17% CBF-AML
TP53
Complex/monosomal karyotpype
and tx-related AML
Inferior
TET2
23% of CN-AML
?
ASXL1
~15%, Exon 12
Few studies, worse
DNMT3A
20%
inferior
BCOR
6%
?
Others: CBL, JAK2, EZH2
J Clin Oncol. 2011 Feb 10;29(5):475-86
AML and MDS panel at OHSU
Ion Torrent – next generation sequencing
A “torrent” of data
• ~25 base pairs
of data
• Massive
computing to
align sequences
• Deep
sequencing
– average
coverage of
one nucleotide
Ion Torrent panel - GeneTrails
R-tyrosine kinase FLT3
KIT
CSF3R
C-tyrosine kinase
Signaling
molecule
Serine/threonine
kinase
Cytokine receptor
Phosphatase
Epigenetic
Splicing
machinery
Transcriptional
factor
Other
ABL1
CBLB
NRAS
KRAS
MPL
PTPN11
IDH1
IDH2
SF3B1
PRPF40B
DNMT3A
SF1
TET2 MLL
EZH2
SF3A1 ZRSR2 SRSF2
NPM1
GATA1
CEBPA
ETV6 RUNX1 WT-1
ASXL1
SH2B3
(LNK)
TP53
JAK2
CBL
HRAS
BRAF
UTX
U2AF1/
U2AF2
Why do extra testing?
• Clinical
– Help with prognosis and/or treatment
• e.g. AML with multiple MDS-type mutations
• Prepare for the future
– Ion Torrent can sequence multiple genes at once
•
•
•
•
Replace multiple genetic tests
More sensitive
Prepare for future prognostic studies
Cost is going down
Reality check –
How does this influence therapy?
EORTC AML-10 trial
• After induction, all patients < 46y allocated to
- alloSCT if they have a donor
- ASCT otherwise
• Intent-to-treat analysis
• N= overall 1198 pts
• After induction, n=293 with a donor, n=441
without a donor
Suciu et al, Blood 2003
EORTC AML-10: results in cytogenetics
groups
Favorable CG
Intermediate CG
Favorable risk gets no improvement in OS
with allo
Intermediate group as well but good
portion were certainly favorable risk by
molecular studies (just not known at time)
Poor CG
RFS in a donor vs no donor basis:
NPM1+ FLT3-ITD-
Schlenk N Engl J Med 2008
And that’s why we do this
3+7 followed by HiDAC
3+7 followed by allo SCT
if good match available,
consider auto or just
chemo
3+7 followed by allo SCT
Myelodysplastic syndrome
• MDS – classically defined
– Cytopenias
– Abnormal cell maturation (dysplasia)
– Can transform to AML
• Arbitrary line = 20% blasts in marrow is AML
• Shared genetic abnormalities
• Heterogeneous disease (like AML)
– Transplant is only cure, but timing is important
– Genetic studies becoming more important
• Diagnosis/prognosis
• Defining treatment
Old classification - Morphology
Up To Date
WHO 2008 classification
• Genetic abnormalities becoming more
important
– Added MDS with isolated 5q-
• More importantly, risk assessment and
genetics becoming more refined
– Revised-IPSS
Cytogenetics
R-IPSS
Blood. 2012;120(12):2454
Scoring system R-IPSS
Cytogenetics influences prognosis
Blood. 2012;120(12):2454
Like AML,
mutations are
becoming more
important in
MDS
N Engl J Med. 2011 364(26):2496-506
Many concurrent mutations
N Engl J Med. 2011 364(26):2496-506
Mutations can be used to assess risk
N Engl J Med. 2011 364(26):2496-506
Some mutations associate with low
risk MDS
Blood 2011 118(24):6239-46
RARS – refractory anemia with ringed sideroblasts
Lower risk disease by morphology
SF3B1 mutations - OS
Blood 2011 118(24):6239-46
* Not independent of morphology
Ion Torrent panel - GeneTrails
R-tyrosine kinase FLT3
KIT
CSF3R
C-tyrosine kinase
Signaling
molecule
Serine/threonine
kinase
Cytokine receptor
Phosphatase
Epigenetic
Splicing
machinery
Transcriptional
factor
Other
ABL1
CBLB
NRAS
KRAS
MPL
PTPN11
IDH1
IDH2
SF3B1
PRPF40B
DNMT3A
SF1
TET2 MLL
EZH2
SF3A1 ZRSR2 SRSF2
NPM1
GATA1
CEBPA
ETV6 RUNX1 WT-1
ASXL1
SH2B3
(LNK)
TP53
JAK2
CBL
HRAS
BRAF
UTX
U2AF1/
U2AF2
Genetics and treatment
• Isolated 5q
– Lenalidomide
• Epigenetic mutations (TET2, DNMT3A, ASXL1)
– May predict better response to hypomethylating
agents
• Leukemia 2011 25(7):1147-1153
• High risk
– Allogeneic transplant
Summary
• Cytogenetics and genetic tests are important for risk
stratification and treatment
• Important to collect information at diagnosis
– Especially AML
• Cytogenetics and FISH still important
• Mutations also prognostic
• More mutations being discovered
– GeneTrails/Ion Torrent