Next Generation Sequencing:i progressi nella caratterizzazionedelle leucemie acute
11 maggio 2012I SESSIONE
Dipartimento di Ematologia e Scienze Oncologiche“L. e A. Seràgnoli”, Università di Bologna
Ilaria Iacobucci
Background
Identification of specificmolecular alterations- “driver” mutations- progression- classification in subgroups
Driven by technological advances, recent years have witnessed a deluge of new methods for interrogating different properties of a cell on a genome-wide scale.
Human Molecular Genetics, 2010, Vol. 19
Next Generation Sequencing Technologies
Chip-Seqmethylation
Transcriptome Small RNA
Genome
Background
Next Generation Sequencing Technologies
Genome
NGS and acute leukemia
Identification of somatic mutations in coding sequences (Illumina/Solexa)
Timothy J Ley, Nature. 2008
98%
84%
Except for FLT3 and NPM1 mutations, the other identified somatic mutations are all single base changes, and none has previously been detected in an
AML genome.
Timothy J Ley, Nature. 2008
Next Generation Sequencing Technologies
WholeGenome
Sequencing
WholeExome
Sequencing
Jing Yan X et al. Nature Genetics 2011*Vera Grossman et al. Blood 2011**
DNMT3A*BCOR**
Timothy J Ley et al. Nature 2008Mardis ER et al N Engl J Med 2009
IDH1
- Novel tumor molecular markers- Novel prognostic markers- Novel targets for new therapies??
NGS and acute myeloid leukemia
Normal karyotype AML
Next Generation Sequencing Technologies
WholeGenome
Sequencing
IDH1
NGS and IDH1 mutations
• Enzyme cytosolic isocitrate dehydrogenase 1
• Mutations occur at a single amino acid residue of IDH1, arginine 132 (R132H/R132C)
• Mutations result in a new ability of the enzyme to catalyse the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2HG).
Next Generation Sequencing Technologies
WholeGenome
Sequencing
IDH1
NGS and acute myeloid leukemia
- Grade II–III gliomas and secondary glioblastomas 80%
- Polycythemia Vera 2%
- Essential Thrombocythemia 1%
- De novo AML (3-17%)
- Secondary AML to MPN (25%)
- Secondary AML to MDS (15%)
Next Generation Sequencing Technologies
WholeExome
Sequencing
Jing Yan X et al. Nature Genetics 2011
DNMT3A
NGS and DNMT3A mutations
• Catalyzes the conversion of cytosine to 5-methylcytosine.
• The majority of mutations occur in patientswith CN-AML (27-30%) and FAB-M5 (20%) or M4 (13%-15%).
• DNMT3A mutations are often found in leukemias with mutations in NPM1, FLT3, IDH1or IDH2.
• Poor prognosis.
Next Generation Sequencing Technologies
WholeExome
Sequencing
Vera Grossman et al. Blood 2011
BCOR
NGS and BCOR mutations
• BCL6 corepressor.
• Mutations include nonsense or conserved splice-site mutations and out-of-frame insertions/deletions introducing premature stop codons.
• Mutations occurred in 3.8% of unselected CN-AML patients.
• Poor prognosis.
NGS and Acute Lymphoblastic Leukemia
Hyperdiploid50 chromosomes
(25%)
Hypodiploid< 44 chromosomes
(2%)
ETV6-RUNX1t(12;21)(22%)
MYC rearrangement(2%)
TCF3-PBX1t(1;19)(5%)MLL-ENL
(0.3%)
HOX11 (0.7%)
TAL1 (7%)
LYL1 (1.5%)
HOX11L2 (2.5%)
MLL rearrangements
(8%)
BCR-ABL1t(9;22) (3%)
Others(22%)
B-ALL
T-ALL
NGS?
Next Generation Sequencing (NGS) in B/T ALL
Iacobucci I et al, Current Hematologic Malignancy Reports 2012
NGS and Acute Lymphoblastic Leukemia
NGS and Acute Lymphoblastic Leukemia: Bologna’s experience
reads for eachgene
BCR-ABL1+ ALL
diagnosisrelapse
RNA
Genome Analyzer II (Illumina/Solexa)/Roche 454
Verona-Prof. Massimo Delledonne
Napoli-Prof. Francesco Salvatore, Prof. Fabrizio Pane
Patients
ID pt Disease Sex Age Karyotype Phase Platform
Pt 1 Ph+ ALL M 56 46 XY, del(9)(p13p22),t(9;22)(q34;q11)
DiagnosisRelaspe
Illumina/Solexa
Pt 2 Ph+ ALL F 71 46 XX, t(9;22)(q34;q11) Diagnosis Illumina/Solexa
Pt 3 Ph+ ALL M 40 46 XY, t(9;22)(q34;q11) Diagnosis Illumina/Solexa
Pt 4 Ph+ ALL F 47 46, XX, t(9;22)(q34;q11), t(3;22)(p25;q11), del(9)(p22)der(9)t(9;22)(q34;q11)
DiagnosisNormalRelapse
Roche 454
Pt 5 Ph+ ALL F 81 46 XX, 45, XX, t(9;22)(q34;q11), -13, del(16q22)
DiagnosisNormalRelapse
Roche 454
Putative novel non-synonymous mutations in BCR-ABL1-positive ALL
T315I BCR-ABL
Diagnosis Relapse
Iacobucci I et al. BCJ 2012.
Differences in mutational patterns suggest that the leukemia clone from which relapsed cells have been developed was not the predominant one at diagnosis and that relapse specific variants were mutations probably acquired during Ph+ ALL progression.
Diagnosis
BCR-ABL
additionalmutations
Remission
BCR-ABL1
Diagnosis mutations T315I BCR-ABL1
Relapse mutations
progression
Relapse
T315I BCR-ABL1
relapsemutations
Iacobucci I et al. BCJ 2012.
Identification of Novel Gene Fusions
Gene 1 Gene2
Screening for the presence of reads showing partial alignment to exon
boundaries from two different genes
Intra chromosomal gene fusions Inter chromosomal gene fusions
BCR-ABL1
transcriptome sequencing to “re-discover” the BCR-ABL1 gene fusion
BCR-ABL1 gene fusion
ACCAACTCGTGTGTGAAACTCCAGACTGTCCACAGCATTCCGCTGACCATCAATAAGGAAGCCAACTCGTGTGTGAAACTCCAGACTGTCCACAGCATTCCGCTGACCATCAATAAGGAAG
GCTGACCAACTCGTGTGTGAAACTCCAGACTGTCCACAGCATTCCGCTGACCATCAATAAGGAAGCCACTCGTGTGTGAAACTCCAGACTGTCCACAGCATTCCGCTGACCATCAATAAGGAAG
ACCACTCGTGTGTGAAACTCCAGACTGTCCACAGCATTCCGCTGACCATCAATAAGGAAG
ex 13 ex 2
AAGCCCTTCAGCGGCCAGTAGCATCTGACTTTGAGCCTCAGGGTCTGAGTGAAGCCGCTCGTTGGAAAAGCCCTTCAGCGGCCAGTAGCATCTGACTTTGAGCCTCAGGGTCTGAGTGAAGCCGCTCGTTGGAACTCCAAAGCCCTTCAGCGGCCAGTAGCATCTGACTTTGAGCCTCAGGGTCTGAGTGAAGCCGCTCGTTGGAACTCCAAGAAGCCCTTCAGCGGCCAGTAGCATCTGACTTTGAGCCTCAGGGTCTGAGTGAAGCCGCTCGTTGGAACTCCAAAGCCCTTCAGCGGCCAGTAGCATCTGACTTTGAGCCTCAGGGTCTGAGTGAAGCCGCTCGTTGGAA
ex 13ex 12 ex 2 ex 3
BCR ABL1
Philadelphia chromosome
5’ 3’
Sequence reads having partial alignment on both chromosomes 22 and 9
Genome/exome/transcriptome
sequencing
DISCOVERY
Genome wide-based NGS
Identification of specificmolecular alterations- SNVs- Gene expression profiles- Fusion transcripts- Novel molecular markers- Novel prognostic markers
Next-Generation Amplicon-Based Deep Sequencing
DIAGNOSIS
• specific candidate genes• high sensitivity
Modified from Next-Generation Amplicon-Based Deep Sequencing and Its Application to Characterize Hematological Malignancies. Cancer Research Application Note No. 7
DNA Input Library
■ PCR Amplicons■ PCR Purification■ PCR Quantitation■ Equimolar Pooling
454 Chemistry 454 Sequencing System
■ emPCR Amplification■ Breaking■ Enrichment
■ 8-Lane PicoTiterPlate device
Next-Generation Amplicon-Based Deep Sequencing
Genetic region of interest
Primer antisense
Primer sense
MID-1MID-1Pt1
Genetic region of interest
Primer antisense
Primer sense
MID-2MID-2Pt2
Genetic region of interest
Primer antisense
Primer sense
MID-3MID-3Pt3
AMPLICON POOLING
IRON I StudyParticipants / Status - Broad coverage across Europe
The Interlaboratory RObustness of Next-generation sequencing (IRON) study
IRON study: Scheme of technical Setup
The Interlaboratory RObustness of Next-generation sequencing (IRON) study
PCR concept
IRON I: Workflow Amplicon Generation
Amplicon Purification(Agencourt AMPure XP beads)
Amplicon Quantification(PicoGreen Fluorescence)
Amplicon Pooling and Dilution
Emulsion PCR Sequencing (454 Roche)
IRON I: Results
• Median high-quality sequencing reads: 430,402 (range, 140,399-601,776).• Median coverage per amplicon: 689-fold (range, 541-872-fold).
Kohlmann et al. Leukemia 2011
IRON I: Results Both the forward and reverse strands were successfully and homogeneously sequenced.
Kohlmann et al. Leukemia 2011
IRON I: Results
In comparison to variant data available from Sanger sequencing, 454 amplicon deep sequencing detected all mutations and SNPs that were previously known.
Kohlmann et al. Leukemia 2011
IRON I: Results
A total of 6/14 of low-level variants with frequencies below the Sanger sequencing detection limit of 20% (median values ranging from 1.6 to 11.6%) were consistently detected in all laboratories.
Kohlmann et al. Leukemia 2011
• IRON I study demonstrated the robustness, precision and reproducibility of 454 amplicon deep sequencing across 10 laboratories in 8 countries.
• Although for individual amplicons the coverage was highly different between the participating centers, the mutational burden in the detected variants was remarkably comparable.
IRON I: Conclusions
http://454.com/products/assays/tet2-cbl-kras.asp
Conclusions
Amplicon Gene candidate
Screening, follow-up, clonal evolution, drug-resistance, risk-assessment, etc…..
Giovanni Martinelli, Michele Baccarani
Dpt of Hematology and Medical Sciences “L. and A. Seràgnoli”, Bologna
Anna Ferrari, Claudia Venturi, Annalisa Lonetti, Cristina Papayannidis, Emanuela Ottaviani, Viviana Guadagnuolo,Simona Soverini, Margherita Perricone, Maria Chiara Fontana, Maria Chiara Abbenante, Sarah Parisi
CEINGE, University of Naples Federico II, NaplesValeria D’Argento, Giorgio Casaburi, Concetta Quintarelli, Anna Lucia Peluso, Fabrizio Pane, Francesco Salvatore
Department of Experimental and Evolutionary Biology, Bologna, Italy
Marco Sazzini, Alessio Boattini, Donata Luiselli
Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna
Dpt of Mother and Child, and Biology-Genetics, Verona
Alberto Ferrarini, Enrico Giacomelli, Luciano Xumerle, Giovanni Malerba, Massimo Delledonne
Acknowledgments
Chair of Hematology and BMT unit, University Of Brescia, ItalyFederica Cattina, Michele Malagola, Domenico Russo
Roche Applied Science, Roche Diagnostics SpA
Katia Accorsi, Francesca Dal Pero, Michele Iacono, Sauro Lamberti, Cinzia Pazzi
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