Hematologic malignant diseases – molecular information, present and future IV Simposio International – Sao Paulo Nov 7 2012 Dr. rer. nat. Alexander Kohlmann, MLL Munich Leukemia Laboratory
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Hematologic malignant diseases
– molecular information, present
and future
IV Simposio International – Sao Paulo Nov 7 2012
Dr. rer. nat. Alexander Kohlmann, MLL Munich Leukemia Laboratory
Spectrum of Methods in Leukemia Diagnostics
Cytomorphology Cytogenetics Immunophenotyping
Histology FISH Molecular Genetics
The Human Genome Sequencing Dimensions
Human Genome Project
2003 20071990 2012 2013+
~2,700,000,000 US$
Illumina Genome Analyzer
<5,000 US$~1,500,000 US$
5 months 1 week
Sanger Sequencing
2008
1,000 US$
1 day
Ley et al., Nature 2008. Mardis et al., N Engl J Med. 2009
Ley et al., N Engl J Med. 2010
Impact of Next-Generation Sequencing on AML
Ley T, NEJM 2010Mardis E, NEJM 2009
IDH1 mutations DNMT3A mutations
Molecular Diagnostics in 2012+
Courtesy of Lou Staudt, LLMPP group, NIH
1. Recurrent genetic abnormalities
2. Gene mutations
3. Multilineage dysplasia and
MDS-related cytogenetic
changes
4. History of patient:
de novo, t-AML, or s-AML
Diagnosis and Classification in 2008 (AML)
MLL Munich Leukemia Laboratory
NGS platforms
• 454 GS FLX [3]
• 454 GS Junior [4]
• Illumina MiSeq [2]
• NimbleGen
• Fluidigm
• RainDance [2]
• Beckman Coulter [3]
Example parameters:
• TET2
• CBL
• KRAS
• RUNX1
Accreditation: DIN EN ISO 15189:2007
www.mll.com
Disease
Characterization /
Classification
Predictive
Information
Utility of Amplicon (deep-/ultra-deep) Sequencing
Prognostic
Information
454 Next-Generation Sequencing at the MLL
Amplicon
Sequencing
1. Kohlmann et al., Amplicons, JCO 2010
2. Grossmann et al., CEBPA, J.Mol.Diagn. 2011
3. Klein et al., Toolbox, Bioinformatics 2011
4. Kohlmann et al., IRON, Leukemia 2011
5. Kohlmann et al., NGS review, Sem. Onc. 2012
Diagnostics
Operations
Research
& Collaborations
1. Grossmann et al., Blast crisis, Leukemia 2011
2. Grossmann et al., NimbleGen,Leukemia 2011
3. Grossmann et al., EZH2, Leukemia 2011
4. Grossmann et al., RUNX1, Haematologica 2011
5. Grossmann et al., BCOR, Blood 2011
6. Bacher et al., TET2, Br J Haematol 2011
7. Haferlach et al., NF1, Leukemia 2011
8. Tiacci et al., DNMT3A, Leukemia 2011
9. Weissmann et al., TET2, Leukemia 2011
10. Tiacci et al., BCOR, Haematologica 2012
11. Grossmann et al., CALM-AF10, BJH 2012
12. Schnittger et al., MPNs, Haematologica, 2012
13. Fasan et al., GATA2 mutations, Leukemia 2012
14. Schnittger et al., CBL in MPN, Haematologica 2012
15. Grossmann et al., AML prognosis, Blood 2012
16. Grossmann et al., RAS in Myeloma, BCJ 2012
17. Meggendorfer et al. SFSR2 in CMML, Blood 2012
18. Schnittger et al., ASXL1 in AML, Leukemia 2012
Deep-Sequencing of Amplicons
in routine diagnostics
operations
Mutation Analysis Using the 454 Instrument
Target (gene / region)
E33,409bp
E4 91bp
E10355bp
E11 1,472bp
E594bp
E6209bp
E7151bp
E890bp
E9138bp
13 amplicons 6 amplicons
27 amplicons
• Median: 343 bp
• Minimum: 336 bp
• Maximum: 350 bp
bi-directional sequencing
454 Sequencing Candidate Genes: TET2
PCR Setup: TET2 – CBL – KRAS
96-well plate with lyophilized primers (Roche Applied Science)
3 Patients / 96-well plate
1 2 3 4 5 6 7 8 9 10 11 12
A TET2 TET2 TET2 TET2 TET2 TET2 TET2 TET2 TET2 TET2 TET2 TET2 FastStart
B TET2 TET2 TET2 TET2 TET2 TET2 TET2 TET2 TET2 TET2 TET2 TET2 FastStart
C TET2 TET2 TET2 TET2 TET2 TET2 TET2 TET2 TET2 TET2 TET2 TET2 FastStart
D TET2 TET2 TET2 CBL TET2 TET2 TET2 CBL TET2 TET2 TET2 CBL FastStart
E TET2 TET2 TET2 CBL TET2 TET2 TET2 CBL TET2 TET2 TET2 CBL FastStart
F TET2 TET2 TET2 KRAS TET2 TET2 TET2 KRAS TET2 TET2 TET2 KRAS FastStart
G TET2 TET2 TET2 KRAS TET2 TET2 TET2 KRAS TET2 TET2 TET2 KRAS FastStart
H TET2 TET2 TET2 control TET2 TET2 TET2 control TET2 TET2 TET2 control
MID Plate #1
MID Plate #2
MID Plate #3
4 5 6
7 8 10
1 2 31 2 4
Ficoll Gradient
• Cell count
• Purity
DNA Isolation
• 12 samples/run
• [ Quantification ]
Sample Entry
• Entity
• Sample type
Sample Preparation: Non-454 Workflow
Titanium Chemistry
• emPCR
• Breaking
• Enrichment (454 REM)
Sequencing
• 8-lane PTP
• Multiplexing
• GS Junior
PCR & Purification
• PCR set-up
• Pre-configured Plates
• PCR purification (Beckman)
• Amplicon pooling (Beckman)
PCR set-up: “targeted sequencing” Fluidigm, RainDance, NimbleGen
PCR clean-up: Agencourt solution; implemented in Q1-2010
Bead enrichment: implemented in Q4-2010
Overview: Automation Solutions
1. Purification of PCR products
2. Preparation of quantification
Automation of Sample Preparation Steps - I
“Bead” - Enrichment
Automation of Sample Preparation Steps - II
GS FLX Run Performance (8-lane PTP)
Reactions
799,961
Flow of incorporated nucleotides
PicoTiterPlate
NGS Assay: Flowgram of a Single Well
T A C G
Next-generationSanger sequencing
T A C G
Comparison of Sequencing Methodologies
Next-generation Sequencing
T A C G Read: GS6YAAE01AK65W
rank=0000487
x=124.5
y=1266.5
Read length=412
TGTACTACTCTACGGTAGCAGAGACTTGGTCTG
ACCGGGATCTCCTCTCTGGTTTCTCCTCTTTAG
TAATCTCTATGGGCGTGTGTGGTATCAACATGG
GATGCACCATGCCCAACCCCAGGGCATCTTGGT
AGGTCACAAACTCTGGACGGCCGGTGGGAAGCC
CATAGGGCAACCCAGGCTTTGGGGCAAGGTGCC
CAGGAAACAGACTGCCATTGGGTAACAAAACTG
GGTGAGGGTAGACAGGTCCTTTGCCATGTAAGG
AGAGGGGACTTACAGCAATGCCCTCAGGGGCTG
GGTAAGGGAGGTAACTCCTGGGGTAGGGAATTG
GTGGGGACCTGAATGCCTCATTTGGAGACAGAA
ATATAGAGCTTGGTGGAAGGCCTGTAGAACCAT
GTCGTCAGTGTGAGTA
Sequencing Methodologies: NGS
Sequencing
Raw Images
Amplicon Pipeline
Read: GS6YAAE01AK65W
rank=0000487
x=124.5
y=1266.5
Read length=412
TGTACTACTCTACGGTAGCAGAGACTTGGTCTG
ACCGGGATCTCCTCTCTGGTTTCTCCTCTTTAG
TAATCTCTATGGGCGTGTGTGGTATCAACATGG
GATGCACCATGCCCAACCCCAGGGCATCTTGGT
AGGTCACAAACTCTGGACGGCCGGTGGGAAGCC
CATAGGGCAACCCAGGCTTTGGGGCAAGGTGCC
CAGGAAACAGACTGCCATTGGGTAACAAAACTG
GGTGAGGGTAGACAGGTCCTTTGCCATGTAAGG
AGAGGGGACTTACAGCAATGCCCTCAGGGGCTG
GGTAAGGGAGGTAACTCCTGGGGTAGGGAATTG
GTGGGGACCTGAATGCCTCATTTGGAGACAGAA
ATATAGAGCTTGGTGGAAGGCCTGTAGAACCAT
GTCGTCAGTGTGAGTA
Signal processing
Image processing
Report
Alignment & Analysis
• Amplicon variants
• Indel detection
• SNP calling
• Coverage plots
• Quality report
• Medical validation
454 NGS Data Analysis Workflow
- 9 hours run time
- 834 images
- 30 GB data
- 600,000 reads
- e.g. 128 RUNX1 assays
- >500-fold coverage
nonsense mutation missense mutation deletion conserved domain
Data Processing
• SFF files
• Transfer to cluster
• AVA Software
• HTML-Report
• QC, Visualization
• JSI Software
GS Amplicon
Variant Analyzer
454 Toolbox
HTML-Report
What About Data Analysis? Post-454
JSI
Sequence Pilot
Klein H.-U. et al., Bioinformatics. 2011;27(8):1162-3.
Detection of Mutations in AVA: TET2 example
Roche Amplicon Variant Analyzer (AVA) software
454 intensity data
Reference sequence
Mutation
TET2 Variant Analysis in AVA Software
Roche Amplicon Variant Analyzer (AVA) software
c. ??? p. ???
TET2 Variant Analysis in Sequence Pilot
JSI SeqPilot software
p.Glu1728GlyfsX10c.5183_5187delAGATG
Disease
Characterization /
Classification
Predictive
Information
Utility of Amplicon (deep-/ultra-deep) Sequencing
Prognostic
Information
• Clonal hematopoietic malignancy characterized by
features of both a myeloproliferative neoplasm and a
myelodysplastic syndrome (WHO classification 2008)
• n=81 cases selected for mutation analysis in seven
candidate genes
• Next-generation amplicon deep-sequencing (454)
• CMML-1 (n=45):
Blasts (including promonocytes) <5% in the PB; <10% in the BM
• CMML-2 (n=36):
Blasts (including promonocytes) 5-19% in the PB or 10-19% in the BM, or
when Auer rods are present irrespective of the blast plus promonocyte count
Chronic Myelomonocytic Leukemia (CMML)
• CBL: exons 8 and 9
• JAK2: exons 12 and 14
• MPL: exon 10
• NRAS: exons 2 and 3
• KRAS: exons 2 and 3
• RUNX1: complete coding region
• TET2: complete coding region
PCR amplicons CMML Patients (n=81)
Sex
Male 57 (70.4%)
Female 24 (29.6%)
Age (years)
Median 72.8
Range 40-85.5
CMML
1 45 (55.6%)
2 36 (44.4%)
Karyotype
Normal or -Y 63 (81.8%)
Aberrant 14 (18.2%)
Missing data 4
Bone marrow blasts (%)
Median 10.0
Range 2-19.5
Missing data 27
Peripheral blood blasts (%)
Median 3.0
Range 0-35
Missing data 48
White blood cell count (109/liter)
Median 13.4
Range 2.3-160.0
Missing data 11
Platelets (109/liter)
Median 82.5
Range 16.0-933.0
Missing data 13
CMML Patients (n=81)
Patient Characteristics and Target Genes
CBL
JAK2
KRAS
MPL
NRAS
Variant Frequency Table: Exemplary 9 Cases
• 59/81 (72.8%) cases mutated
• In mean, 1.6 mutations per case were observed (range 1-6)
• In no case were CBL and KRAS aberrations, or JAK2 and RUNX1 mutations,
concomitantly detected
Karyotype
CMML
TET2CBL
NRASKRAS JAK2RUNX1MPL
normal karyotype (incl. -Y) aberrant karyotype data not available
CMML-1 CMML-2
-Y)
-1 -2
no mut.
[22.2%]
[22.2%][12.3%][9.9%][8.7%][0%]
[44.4%]
[27.1%]
Molecular Aberrations in 81 CMML Patients
Kohlmann A. et al., J Clin Oncol. 2010 ;28:3858-65.
• 606 reads
• 1.16% mutated
JAK2 V617F
GTC ==> TTC
codon 617
NGS
StandardMelting Peaks
Temperature (°C)
85807570656055504540
-(d
/dT
) F
luo
resc
ence
(64
0/53
0)
0.101
0.091
0.081
0.071
0.061
0.051
0.041
0.031
0.021
0.011
0.001
Melting Peaks
Temperature (°C)
85807570656055504540
-(d
/d
T) F
lu
orescen
ce (640/530)
0.101
0.091
0.081
0.071
0.061
0.051
0.041
0.031
0.021
0.011
0.001
Melting Peaks
Temperature (°C)
85807570656055504540
-(d
/dT
) F
luo
rescen
ce (
640/5
30)
0.101
0.091
0.081
0.071
0.061
0.051
0.041
0.031
0.021
0.011
0.001
wild-typeV617F
and LOH
Mutation
• Melting curve
analysis *
• ~1% mutated
* Schnittger S. et al., Leukemia. 2006 Dec;20(12):2195-7.
Sensitivity of Amplicon Deep-Sequencing
• NRAS: 10 mutations were found in 18/81 (22.2%) cases
• KRAS: 8 mutations were detected in 10/81 (12.3%) cases
• Only 3/81 (3.7%) cases harbored mutations in both NRAS and KRAS
• 25/81 (30.9%) patients either harbored mutations in NRAS or KRAS
Patient #34
KRAS 18.10%
17.28%
G12C
107: G/T 9.1%
Clonality ?
130: G/T
131: A/T
L19F
T20S
RAS Pathway Alterations in CMML
Resolution of Distinct Subclones (KRAS)
exon 2; Gly12Cys
exon 2; Leu19Phe
exon 2; Thr20Ser
Case #33B
KRAS
Meeting from Monday 10th May - Tuesday 11th May 2010, Munich
• 50 participants from 12 countries, 3 continents
• Topic: Next-generation sequencing applications
• Results: Interlaboratory Robustness Of NGS (IRON) study
454 Hematology Focus Group
IRON Study: Participants and Laboratories
Austria
GB
Belgium
Germany
Germany
Italy
Austria
USA
Nether-
lands
Dr. Gabriel, Blood Bank, Linz
Dr. Garicochea, Pontifícia Universidade
Católica do Rio Grande do Sul, Porto Alegre
Dr. Simen, 454 Life Sciences, Branford
Prof. Vandenberghe, UZ Leuven, Belgium
Prof. Martinelli, University of Bologna
Dr. JH Jansen, Radboud University
Medical Centre, Nijmegen
Brazil Italy
Prof. Haferlach, Munich
Leukemia Laboratory, Munich
Dr. Timmermann, Max Planck Institute for Molecular Genetics, Berlin
Prof. Basso, Università degli studi di Padova, Padova
Prof. Young, St. Bartholomews,
London
• 18 samples (CMML)
• Centrally collected by MLL
• Aliquots prepared (1.6 µg)
• Shipping package
Blinded sample aliquots
454 reagents
Enzymes
96-well primer plates
Study protocol
IRON Study: Samples and Study Materials
IRON: Interlaboratory RObustness of Next-generation sequencing
IRON Study: Consistency of Mutations
Leu34Phe
Median: 49.8%
Minimum: 45.8%
Maximum: 54.7%
Ile1762Val
Median: 49.6%
Minimum: 45.4%
Maximum: 53.9%
IRON: Mutation Load and Coverage Distribution
Kohlmann A. et al., Leukemia. 2011;25(12):1840-8.
Disease
Characterization /
Classification
Predictive
Information
Utility of Amplicon (deep-/ultra-deep) Sequencing
Prognostic
Information
Survival according to Cytogenetics: AML
Schoch et al., Blood, 102, 2395-2402, 2003
A novel hierarchical prognostic model of
AML solely based on molecular mutations
Grossmann et. al., Blood 2012
1,000 AML patients with cytogenetic data
available were investigated for the
following molecular alterations:
• PML-RARA,
• RUNX1-RUNX1T1,
• CBFB-MYH11,
• FLT3-ITD, and
• MLL-PTD, as well as mutations in
• NPM1,
• CEPBA,
• RUNX1,
• ASXL1, and
• TP53
Clinical data was available in 841 patients.
Karyotype vs. Molecular Markers in AML
Grossmann et. al., Blood 2012
Karyotype vs. Molecular Markers in AML
1) Translocations 2) Intragenic mutations
http://AtlasGeneticsOncology.org/Genes/AML1.htm
Dicker F. et al., Blood. 2007;110:1308-16.
Frequently mutated in de novo AML with NK or
non-complex chromosomal imbalances
• RUNX1: Runt-related transcription factor 1
• Regulator of differentiation of hematopoietic stem cells into mature blood cells
• Various types of alterations:
Can we detect MRD? RUNX1 Alterations in AML
RUNX1 Mutations in AML
Schnittger S. et al., Blood. 2011; 117(8):2348-57.
• strong adverse prognostic effect in AML with normal karyotype or noncomplex
chromosomal imbalances
• Especially in those cases that do not carry CEBPA, NPM1, FLT3-ITD, or MLL-
PTD alterations
intermediate cytogenetic risk
without mutations in NPM1,
CEBPA, FLT3-ITD, or MLL-PTD
0 365 730 1095 1460
Days
RUNX1wt (n=81; median: n.r.)
RUNX1mut (n=67; median: 348 days)
p=0.001p=0.003
RUNX1wt (n=183; median: n.r.)
RUNX1mut (n=97; median: 378 days)
0 365 730 1095 1460
Days
total cohort
Patients with NK and noncomplex
chromosomal imbalances
7 amplicons
• Median: 342 bp
• Minimum: 341 bp
• Maximum: 348 bp
E8476bp
E3270bp
E4157bp
E5105bp
E6192bp
E7162bp
Transcript ID: ENST00000344691
RUNX1: First Candidate Gene in Routine Dx
Grossmann V. et al., Haematologica. 2011;96(12):1874-7.
GS Junior Performance: Ideal Output
RUNX1
107,842 reads
• Clone #1:
#1 909-fold coverage
11.55% mutated
c.1098_1106delAGGCCCGTTinsG
p.Gly367ProfsX203
#2
• Clone #2:
909-fold coverage
32.34% mutated
c.1144_1150delGCCTCGGinsCC
p.Ala382ProfsX189
Detection of Molecular RUNX1 Mutations
November 2010
Mutation at diagnosis
Amplicon 8.1
Codons 297-386
909-fold coverage
• Category #1: “responders”
55% (17/31) of patients responded to therapy and were characterized by a
total clearance of the mutated clone at the first time point of follow-up
February 2011
Mutation at 1st follow-up
Amplicon 8.1
Codons 297-386
1,697-fold coverage
p.Gly366GlyfsX204 p.Ser383ArgfsX188
Serial Analyses of RUNX1 Mutations
• Category #2: “patients with refractory disease” and Transplantation
Serial Analyses of RUNX1 Mutations
What is the Near-Term Future of Diagnostics ?
exome or
genome
sequencing
Amplicon
deep-sequencing
(gene panels)
• Gene expression
• DNA copy number
• Methylation
• “Next-generation sequencing” can be renamed into “Now-generation
sequencing”
• Amplicon deep-sequencing is a technically challenging and complex
workflow, but can be applied in an accredited and certified diagnostic
environment
• Laboratory-developed assays allow the characterization of a variety of
hematological malignancies for targeted genomic regions, mostly distinct
genes or exons, e.g. RUNX1, CEBPA, TET2, TP53, EZH2, KRAS, CBL…
• Major achievements thus far: (1) breakthrough of whole-exome
sequencing efforts in cancer patients, predominantly as part of large-
scale international programs; and (2) the implementation of targeted re-
sequencing of regions/genes of interest in diagnostic workflows
Summary and Conclusions
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