IV Simposio International Sao Paulo Nov Hematologic malignant diseases molecular information, present and future
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1 IV Simposio International Sao Paulo Nov Hematologic malignant diseases molecular information, present and future Dr. rer. nat. Alexander Kohlmann, MLL Munich Leukemia Laboratory
2 Spectrum of Methods in Leukemia Diagnostics Cytomorphology Cytogenetics Immunophenotyping Histology FISH Molecular Genetics
3 The Human Genome Sequencing Dimensions Sanger Sequencing ~1,500,000 US$ <5,000 US$ 5 months 1 week 1,000 US$ 1 day Human Genome Project Illumina Genome Analyzer ~2,700,000,000 US$ Ley et al., Nature Mardis et al., N Engl J Med Ley et al., N Engl J Med. 2010
4 Impact of Next-Generation Sequencing on AML IDH1 mutations DNMT3A mutations Mardis E, NEJM 2009 Ley T, NEJM 2010
5 Molecular Diagnostics in Courtesy of Lou Staudt, LLMPP group, NIH
6 Diagnosis and Classification in 2008 (AML) 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
7 MLL Munich Leukemia Laboratory NGS platforms 454 GS FLX [3] 454 GS Junior [4] Illumina MiSeq [2] NimbleGen Fluidigm RainDance [2] Beckman Coulter [3]
8 Accreditation: DIN EN ISO 15189:2007 Example parameters: TET2 CBL KRAS RUNX1
9 Utility of Amplicon (deep-/ultra-deep) Sequencing Disease Characterization / Classification Prognostic Information Predictive Information
10 454 Next-Generation Sequencing at the MLL Amplicon Sequencing Diagnostics Operations Research & Collaborations 1. Kohlmann et al., Amplicons, JCO Grossmann et al., CEBPA, J.Mol.Diagn Klein et al., Toolbox, Bioinformatics Kohlmann et al., IRON, Leukemia Kohlmann et al., NGS review, Sem. Onc Grossmann et al., Blast crisis, Leukemia Grossmann et al., NimbleGen,Leukemia Grossmann et al., EZH2, Leukemia Grossmann et al., RUNX1, Haematologica Grossmann et al., BCOR, Blood Bacher et al., TET2, Br J Haematol Haferlach et al., NF1, Leukemia Tiacci et al., DNMT3A, Leukemia Weissmann et al., TET2, Leukemia Tiacci et al., BCOR, Haematologica Grossmann et al., CALM-AF10, BJH Schnittger et al., MPNs, Haematologica, Fasan et al., GATA2 mutations, Leukemia Schnittger et al., CBL in MPN, Haematologica Grossmann et al., AML prognosis, Blood Grossmann et al., RAS in Myeloma, BCJ Meggendorfer et al. SFSR2 in CMML, Blood Schnittger et al., ASXL1 in AML, Leukemia 2012
11 Mutation Analysis Using the 454 Instrument Deep-Sequencing of Amplicons in routine diagnostics operations Target (gene / region)
12 454 Sequencing Candidate Genes: TET2 13 amplicons 6 amplicons E3 E4 E5 E6 E7 E8 E9 E10 E11 3,409bp 91bp 94bp 209bp 151bp 90bp 138bp 355bp 1,472bp 27 amplicons Median: 343 bp Minimum: 336 bp Maximum: 350 bp bi-directional sequencing
13 PCR Setup: TET2 CBL KRAS 96-well plate with lyophilized primers (Roche Applied Science) 3 Patients / 96-well plate 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
14 Sample Preparation: Non-454 Workflow Sample Entry Entity Sample type Ficoll Gradient Cell count Purity DNA Isolation 12 samples/run [ Quantification ]
15 Overview: Automation Solutions PCR & Purification PCR set-up Pre-configured Plates PCR purification (Beckman) Amplicon pooling (Beckman) Titanium Chemistry empcr Breaking Enrichment (454 REM) Sequencing 8-lane PTP Multiplexing GS Junior PCR set-up: targeted sequencing Fluidigm, RainDance, NimbleGen PCR clean-up: Agencourt solution; implemented in Q Bead enrichment: implemented in Q4-2010
16 Automation of Sample Preparation Steps - I 1. Purification of PCR products 2. Preparation of quantification
17 Automation of Sample Preparation Steps - II Bead - Enrichment
18 GS FLX Run Performance (8-lane PTP) Reactions 799,961
19 NGS Assay: Flowgram of a Single Well T A C G PicoTiterPlate Flow of incorporated nucleotides
20 Comparison of Sequencing Methodologies T A C G Sanger sequencing Next-generation
21 Sequencing Methodologies: NGS T A C G Read: GS6YAAE01AK65W rank= x=124.5 y= Read length=412 TGTACTACTCTACGGTAGCAGAGACTTGGTCTG ACCGGGATCTCCTCTCTGGTTTCTCCTCTTTAG TAATCTCTATGGGCGTGTGTGGTATCAACATGG GATGCACCATGCCCAACCCCAGGGCATCTTGGT AGGTCACAAACTCTGGACGGCCGGTGGGAAGCC CATAGGGCAACCCAGGCTTTGGGGCAAGGTGCC CAGGAAACAGACTGCCATTGGGTAACAAAACTG GGTGAGGGTAGACAGGTCCTTTGCCATGTAAGG AGAGGGGACTTACAGCAATGCCCTCAGGGGCTG GGTAAGGGAGGTAACTCCTGGGGTAGGGAATTG GTGGGGACCTGAATGCCTCATTTGGAGACAGAA ATATAGAGCTTGGTGGAAGGCCTGTAGAACCAT Next-generation Sequencing GTCGTCAGTGTGAGTA
22 454 NGS Data Analysis Workflow Amplicon Pipeline Alignment & Analysis Sequencing Raw Images Image processing Signal processing Read: GS6YAAE01AK65W rank= x=124.5 y= Read length=412 TGTACTACTCTACGGTAGCAGAGACTTGGTCTG ACCGGGATCTCCTCTCTGGTTTCTCCTCTTTAG TAATCTCTATGGGCGTGTGTGGTATCAACATGG GATGCACCATGCCCAACCCCAGGGCATCTTGGT AGGTCACAAACTCTGGACGGCCGGTGGGAAGCC CATAGGGCAACCCAGGCTTTGGGGCAAGGTGCC CAGGAAACAGACTGCCATTGGGTAACAAAACTG GGTGAGGGTAGACAGGTCCTTTGCCATGTAAGG AGAGGGGACTTACAGCAATGCCCTCAGGGGCTG GGTAAGGGAGGTAACTCCTGGGGTAGGGAATTG GTGGGGACCTGAATGCCTCATTTGGAGACAGAA ATATAGAGCTTGGTGGAAGGCCTGTAGAACCAT GTCGTCAGTGTGAGTA Amplicon variants Indel detection SNP calling Coverage plots Quality report Medical validation Report - 9 hours run time - 600,000 reads images - e.g. 128 RUNX1 assays - 30 GB data - >500-fold coverage
23 nonsense mutation missense mutation deletion conserved domain What About Data Analysis? Post-454 GS Amplicon Variant Analyzer Data Processing SFF files Transfer to cluster AVA Software HTML-Report QC, Visualization JSI Software 454 Toolbox HTML-Report JSI Sequence Pilot Klein H.-U. et al., Bioinformatics. 2011;27(8):
24 Detection of Mutations in AVA: TET2 example Reference sequence 454 intensity data Mutation Roche Amplicon Variant Analyzer (AVA) software
25 TET2 Variant Analysis in AVA Software c.??? p.??? Roche Amplicon Variant Analyzer (AVA) software
26 TET2 Variant Analysis in Sequence Pilot c.5183_5187delagatg p.glu1728glyfsx10 JSI SeqPilot software
27 Utility of Amplicon (deep-/ultra-deep) Sequencing Disease Characterization / Classification Prognostic Information Predictive Information
28 Chronic Myelomonocytic Leukemia (CMML) 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
29 CMML Patients (n=81) PCR amplicons Sex Male 57 (70.4%) Female 24 (29.6%) CBL: exons 8 and 9 Age (years) JAK2: exons 12 and 14 MPL: exon 10 NRAS: exons 2 and 3 KRAS: exons 2 and 3 Patient Characteristics and Target Genes RUNX1: complete coding region TET2: complete coding region CMML Patients (n=81) Median 72.8 Range 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 Missing data 27 Peripheral blood blasts (%) Median 3.0 Range 0-35 Missing data 48 White blood cell count (10 9 /liter) Median 13.4 Range Missing data 11 Platelets (10 9 /liter) Median 82.5 Range Missing data 13
30 Variant Frequency Table: Exemplary 9 Cases CBL JAK2 KRAS MPL NRAS
![Molecular Aberrations in 81 CMML Patients TET2 [44.4%] CBL [22.2%] NRAS [22.2%] KRAS [12.3%] JAK2 [9.9%] RUNX1 [8.7%] MPL [0%] no mut. [27.1%] Karyotype CMML normal karyotype (incl.](/docs-images/80/82327948/images/31-3.jpg "-Y) aberrant karyotype data not available CMML-1 CMML-2 59/81 (72.8%) cases mutated In mean, 1.")
31 Molecular Aberrations in 81 CMML Patients TET2 [44.4%] CBL [22.2%] NRAS [22.2%] KRAS [12.3%] JAK2 [9.9%] RUNX1 [8.7%] MPL [0%] no mut. [27.1%] Karyotype CMML normal karyotype (incl. -Y) aberrant karyotype data not available CMML-1 CMML-2 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 Kohlmann A. et al., J Clin Oncol ;28:
32 -(d/dt) Fluorescence (640/530) Sensitivity of Amplicon Deep-Sequencing NGS JAK2 V617F 606 reads 1.16% mutated GTC Melting Peaks codon 617 ==> TTC Standard Melting curve analysis * ~1% mutated V617F and LOH Mutation Melting Peaks wild-type Temperature ( C) Tem perature ( C) Melting Peaks * Schnittger S. et al., Leukemia Dec;20(12):
33 RAS Pathway Alterations in CMML 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 KRAS 107: G/T 9.1% 130: G/T 131: A/T 18.10% 17.28% G12C L19F T20S Patient #34 Clonality?
34 B Resolution of Distinct Subclones (KRAS) KRAS Case #33 exon 2; Gly12Cys exon 2; Leu19Phe exon 2; Thr20Ser
35 454 Hematology Focus Group 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
36 IRON Study: Participants and Laboratories Prof. Haferlach, Munich Leukemia Laboratory, Munich Dr. Timmermann, Max Planck Institute for Molecular Genetics, Berlin Germany Germany Italy Prof. Basso, Università degli studi di Padova, Padova Prof. Young, St. Bartholomews, London GB USA Dr. Simen, 454 Life Sciences, Branford Dr. Gabriel, Blood Bank, Linz Austria Austria Belgium Prof. Vandenberghe, UZ Leuven, Belgium Prof. Martinelli, University of Bologna Italy Netherlands Brazil Dr. Garicochea, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre Dr. JH Jansen, Radboud University Medical Centre, Nijmegen
37 IRON Study: Samples and Study Materials 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: Interlaboratory RObustness of Next-generation sequencing
38 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%
39 IRON: Mutation Load and Coverage Distribution Kohlmann A. et al., Leukemia. 2011;25(12):
40 Utility of Amplicon (deep-/ultra-deep) Sequencing Disease Characterization / Classification Prognostic Information Predictive Information
41 Survival according to Cytogenetics: AML Schoch et al., Blood, 102, , 2003
42 Karyotype vs. Molecular Markers in AML A novel hierarchical prognostic model of AML solely based on molecular mutations 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. Grossmann et. al., Blood 2012
43 Karyotype vs. Molecular Markers in AML Grossmann et. al., Blood 2012
44 Can we detect MRD? RUNX1 Alterations in AML RUNX1: Runt-related transcription factor 1 Regulator of differentiation of hematopoietic stem cells into mature blood cells Various types of alterations: 1) Translocations 2) Intragenic mutations Frequently mutated in de novo AML with NK or non-complex chromosomal imbalances Dicker F. et al., Blood. 2007;110:
45 RUNX1 Mutations in AML 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 total cohort RUNX1wt (n=183; median: n.r.) RUNX1mut (n=97; median: 378 days) intermediate cytogenetic risk RUNX1wt (n=81; median: n.r.) RUNX1mut (n=67; median: 348 days) p= Days Patients with NK and noncomplex chromosomal imbalances p= Days without mutations in NPM1, CEBPA, FLT3-ITD, or MLL-PTD Schnittger S. et al., Blood. 2011; 117(8):
46 RUNX1: First Candidate Gene in Routine Dx Transcript ID: ENST E3 270bp E4 157bp E5 105bp E6 192bp E7 162bp E8 476bp 7 amplicons Median: 342 bp Minimum: 341 bp Maximum: 348 bp Grossmann V. et al., Haematologica. 2011;96(12):
47 GS Junior Performance: Ideal Output RUNX1 107,842 reads
48 Detection of Molecular RUNX1 Mutations Clone #1: 909-fold coverage 11.55% mutated c.1098_1106delaggcccgttinsg p.gly367profsx203 #1 #2 Clone #2: 909-fold coverage 32.34% mutated c.1144_1150delgcctcgginscc p.ala382profsx189
49 Serial Analyses of RUNX1 Mutations 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 November 2010 Mutation at diagnosis Amplicon 8.1 Codons fold coverage p.gly366glyfsx204 p.ser383argfsx188 February 2011 Mutation at 1 st follow-up Amplicon 8.1 Codons ,697-fold coverage
50 Serial Analyses of RUNX1 Mutations Category #2: patients with refractory disease and Transplantation
51 What is the Near-Term Future of Diagnostics? Gene expression DNA copy number Methylation exome or genome sequencing Amplicon deep-sequencing (gene panels)
52 Summary and Conclusions 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 largescale international programs; and (2) the implementation of targeted resequencing of regions/genes of interest in diagnostic workflows
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