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