Use of Affymetrix Arrays(Gene Chip® Human Transcriptome 2.0 Array

HTA and Cytoscan® HD Array)in hematological malignancy studies

Giovanni Martinelli, MDInstitute of Hematology and Medical Oncology “L.e A. Seragnoli”

University of Bologna, Italy

Finding and Implementing the right clinically actionable cancer biomarker: 360° Tumor profiling

Disclosure

-

-

-

-

Grant and/or MTA from: Novartis, Merck SD, Astrazeneca, Roche, Takeda, J&J,Mundipharma, Lilly, Pfizer, Cepheid, Onconova, Celgene, Amgen

Consultant/advisor of Celgene, Novartis, Merck SD, Astrazeneca, Roche, Takeda,JeJ, Mundipharma, Pfizer, BMS, Ariad, Personal Genomics, Quiagen, IL,Cepheid, Adienne P.

I have no grant or conflict from Affymetrix.

“The statements in this presentation are those of the Author and not of Affymetrix”

Background

• For istace, the genetic hallmark of APL is the t(15;17) (detected by karyotype)resulting in the fusion of the promyelocytic leukemia (PML) gene and retinoic acidreceptor α (RARα) gene (PML-RARα) (detected by RT-PCR).

• PML-RARα is necessary but not sufficient for the development of APL Areadditional cooperating genetic events also required for its pathogenesis?

• The development of single nucleotide polymorphism (SNP) Cyto ScanHD®-arraysnow allows to perform genome-wide screens for submicroscopic genomicalterations with unprecedented informativity and to map all the genes involved inthese alterations.

Nexus Copy NumberTM 7.0PARTEK Genomics SuiteGenotyping Console 3.1 software

Aim and methods

To explore, in the clinical content, the potential of SNP array for a high-resolutionscreening of additional submicroscopic genomic alterations which characterize APLand may be used to better classify genomic subsets.

Genomic DNA from bonemarrow mononuclear cells

Genome-Wide Human SNP AssayCytoScan®HD Array, Affymetrix(1.85 millionSNPs; medianphysical distance

between SNPs: 700 bp)

Leukemia cases in remission(paired and unpaired analysis)

dCHIP

COPY NUMBER ALTERATIONS

SNP ARRAY: by the new potent CytoScan® HD* Array Affymetrix

CytoScan®HD ARRAY2.67 milions di markers750,000 SNP probes1.9 milioni non-polymorphic probes•100% Sanger cancer gene coverage•100% ICCG constitutional gene coverage•12,000 OMIM® genes•36,000 RefSeq genes

FDA version approved fordiagnostic use

*for research use only

Patients VariablePatients (number)Median age, yrs (range)Male/FemaleDe novo AML

M0M1M2M3M4M5Bi-lin

Secondary AMLCytogenetics

NormalComplex*t(15;17)t(8;21)inv(3)inv(16)other**+8NA***

n10550 (18-84)64/4186 (82%)6 (7%)11 (13%)11 (13%)28 (33%)14 (16%)15 (17%)1 (1%)

19 (28%)

35 (33%)8 (8%)28 (27%)3 (3%)3 (3%)1 (1%)22 (21%)2 (2%)3 (3%)

* Presence of at least 3 chr abnormalities in the absence of t(8;21), inv(16)/t(16;16), t(15;17),and t(11q23);** rare traslocations; *** NA: not available

Principal Component Analysis (PCA)Results I

PCA showed an evident separation between APL and other AML subtypes →a peculiar genomeprofile characterizeAPL patients

APLKaryotype

• NA• Normal• Complex• inv(3)• inv(16)• t(15;17)• t(8;21)• other

From PartekGenomicsSuite

Identification of multiple copy number alterations

A wide spectrum of different genetic lesions (gains/losses) involving completechromosome arms or submicroscopic genomic intervals were identified in all cases.

No significant difference in the average number of alterations was detected amongdifferent karyotpye subgroups, except for complex karyotype group.

KaryotypeNormal

Average CNAs* (n)14

Range3-44

Complex

t(15;17)t(8;21)

Trisomy 8

Others

55

812

5.5

9

35-92

1-246-16

5-6

6-18

p < 0.01

* CNAs: Copy Number Alterations; they includingboth deletions and amplifications

Alteration= region which has a copy number state lower or higher than 2.At least 5 probesets have been considered.

Results IIMacroscopic alterations in APL (>1.5 Mbp)

lossgain

-6

For each type of aberration,each line representsa different case (from AffymetrixGenotyping Console v3.1).

+8q

Trisomy 8

-20q

Microscopic alterations (<1.5 Mb)

Chr

1q23.3

6q25.1

7q11.23

8q24.21

11q23.1

12q24.12

Type ofCNA

gain

loss

loss

gain

gain

loss

Median size(Kbps)

258

202

224

1,907

238

226

# samples

1

2

3

3

1

1

Candidate genes*

LMX1A

AKAP12

MLXIPL, BCL7

PVT1, MYC

NCAM1

ALDH2, BRAP, MAPKAPK5

Function (GO**)

Transcription factor

Signal transduction

Negative regulation of transcription

Cell cycle progression

Cell adhesion

Negative regulation of signalingtansduction/MAP Kinase activity

Pts with gain of 8q24 Pts with normal 8q24

**GO: Gene Ontology* After comparisonwith the Database of Genomic Variants(http://projects.tcag.ca/variation/)

p<0.0001

Overexpression of the PVT1 oncogene

Results V

CytoScan® HD Array Affymetrix SNP arrays allow classification ofAcute Promyelocitic Leukemia genomic subgroups

1. No additional chromosomalabnormalities and low burdenofCNAs

3. Additional chromosomalabnormalities and high number ofCNAs

vs

2. Additional chromosomalabnormalities and low burden of CNAs

Results VCopy number alterations (CNAs) worsen outcome

ACA: Additional chromosomal abnormalities

ACA+ CNAs > 10 (GroupIII)

(Group I + Group II)

Event Free Survival Time (months)

Stratification according to additional chromosomal abnormalities and a high number of CNAs isassociated with a highly significant shorterevent-freesurvival

Chr1

2

3

4

56789

BCR-ABL1-positive ALL (n=106)

Trisomy 4

Monosomy 7

Loss of 9p1011121314161822

dChip Log2-ratio copy number heatmap showing the main macroscopic alterations on all chromosomes

Results in Acute Lymphoblastic Leukemia

High resolution genome-wide analysis by CytoScan® HD ArrayAffymetrix SNP arrays

10

0

20

30

50

40

CDKN2A CDKN2B ANRIL

Diagnosis

Relapse

29%

47%

24%29%

40%

43%

%

P = ns

An almost significant increase in the detection rate of CDKN2A loss (47%) wasfound at the relapse compared to diagnosis (p = 0.06).

Iacobucci I et al, Clin Cancer Res 2011Iacobucci I et Al. PLos1 2013

Other Hematological Malignancies: CDKN2A/B deletions in 112 adult Ph+ALL patients

SNP CytoScan HDSNPS array analysis oand GEP profiling

P = 0.06

p=0.0033CDKN2A del: 22.2% (C.I. 95%: 18.8-26.3)

CDKN2A/ARF loss and outcomeDisease Free Survival according to CDKN2A deletion

CDKN2A wt : 55% (C.I. 95%: 47.3-64.1)

Cumulative Incidence of Relapse according toCDKN2A deletion

p=0.0043

CDKN2A wt : 40.4% (C.I. 95%: 39.3-41.6)

Monthssince CR

CDKN2A del: 73.3% (C.I. 95%: 71.6-75.1)

Monthssince CR

Deletions of CDKN2A/ARF aresignificantly associated with pooroutcome both in terms of disease free-survival and cumulative incidence ofrelapse.

Istituto “Seragnoli” Clin Cancer Research 2011

SNP ARRAY: AMPLIFICATION OF LONG ARM OF CHROMOSOME 1… See what’s been missing

Shaughnessy J, Hematology 2005: Amplification and overexpression ofCKS1B at chromosome band 1q21 is associated with reduced levels ofp27 Kip1 and an aggressive clinical course in multiple myeloma

ID 343CHROMOSOME 1

Gene MDM4 1q32.1: 3N

Istitutodi Ematologia“L. e A. Seràgnoli”

Gene CKS1B 1q21.3: 2N

Gene CDKN2C 1p32.3: 2N Gene FAM46C 1p12: 1N

Istitutodi Ematologia“L. e A. Seràgnoli”

SNP ARRAY: DELETIONS OF SHORT ARM OF CHROMOSOME 1… See what’s been missing

Leone PE, Clin Canc Res 2008

ID 293CHROMOSOME 1

SNP ARRAY: DELETION OF SHORT ARM OF CHROMOSOME 17

Chr 17: deletion of gene including TP53

Not ALL the patients have the same quantity of p53!

LOH

DEL

AMP

TP53

MUTATION and+ ALLELIC Burden

Mutational ANALYSIS OF p53 status

… See what’s been missing

Phase 1b Study Of The MDM2 Antagonist RG7112, the ri-activator ofP53 function, in Combination With 2 Doses/Schedules Of Cytarabine

Karen Yee1, Giovanni Martinelli2, Sarit Assouline3, Margaret Kasner4, Norbert Vey5, Kevin R.Kelly6, Mark W. Drummond7, Michael Dennis8, Karen Seiter9, Steven Blotner10,

Lori Jukofsky11, Steven Middleton12, Jianguo Zhi12, Gong Chen13, Hua Zhong13 and GwenNichols12

1Princess Margaret Hospital, Toronto, ON, Canada; 2Department of Hematology and Oncological Sciences “L. e A. Seràgnoli”,University of Bologna, Bologna, Italy; 3Jewish General Hospital, Montreal, QC, Canada; 4Kimmel Cancer Center, Thomas

Jefferson University, Philadelphia, PA; 5Hematology department, Institut Paoli Calmettes, Marseille, France;6University of TexasHealth Science Center at San Antonio, CTRC Institute for Drug Development, San Antonio, TX; 7Beatson West of Scotland

Cancer Centre, Glasgow, United Kingdom; 8Haematology, Christie NHS Foundation Trust, Manchester, United Kingdom; 9NewYork Medical College, Valhalla, NY; 10Biostatistics, Hoffmann-LaRoche, Inc, Nutley, NJ;11Hoffmann-LaRoche, Nutley,

NJ; 12Research and Early Development, Hoffmann-LaRoche, Inc, Nutley, NJ;13Bioinformatics, Hoffmann-La Roche, Nutley, NJ

24

p53 symmetric division

The mdm2-mdm4 inhibition restore P53 activation, MYC silencing and p21down regulation in leukemia

RG7112

oncogeneMDM2

γH2AX

p21 p21-/-

induction of apoptosiscell cycle stopasymmetric divisionsdna repair stopreduced self renewal

The p21 down regulation resembles the mouse model p21-/-

CHROMOTHRIPSIS is frequent associated to p53 structural alteration

Istitutodi Ematologia“L. e A. Seràgnoli”

Tubio J, Nature 2011

Single CATASTROFIC EVENT with breakageof multiple sites of the region of the chromosome• Rearrangements• Deletions

CHROMOTHRIPSIS by SNP ARRAY in MM: CROMOSOMA 16

Marina Martelloet Al. UnpublishedIstitutodiEmatologia“L. e A. Seràgnoli”

FRAGILE SITEFRA16D

Analysis of genes involved in chromosomal translocations

Comparison between AML and ALL subtypes: aneuploidy vs.

euploidy

2. How does this approach enable the better identification and validation ofactionable biomarkers, translating these into clinical utility and personalizedtherapy

Use of Human Transcriptome ®Array

>285,000 full-length transcripts covered:n >245,000 coding transcriptsn >40,000 non-coding transcriptsn >339,000 probe sets covering exon-exon junctions

Probes designed to maximize exon coverage enable you tomeasure all transcript isoforms

Confidence in your results:n Reproducible: Intra-lot correlation coefficient ≥0.99n <6.5% coefficient of variation observed for all tissues tested

Minimum total RNA required: 50 ng

GeneChip®Human Transcriptome Array 2.0

Use ofAffymetrixArrays (Human TranscriptomeArray -HTAand Cytoscan HDArray)

in hematological malignancy studies

Gene expression analysis

Benefits of GeneChip Human TranscriptomeArray 2.0:

ex1 ex2 ex3 ex4 ex5 ex6 ex7 ex8

5’ 3’

The extent of IKZF1 deletions correlated with the expression of dominant-negative oruntranslated Ikaros isoforms

Δ4-7 deletion (65%)

Deletion

Δ2-7 deletion (30%)

ex1 ex8

5’ 3’

ex2 ex3 ex4 ex5 ex6 ex7

Dominant-negativeIk6 isoform

Untranslated isoform

Deletion

Iacobucci I et al, Blood 2009

Additional discovery sequencing essential to identify the full range of

Up to 15% of childhoodALL, 26% of young adults

Patients may be detected by

-

-

phosphosignaling analysis / expression profiling

next generation sequencing

Diverse range of alterations converge onABL1/JAK signaling

kinase-activating alterations

Kathryn G. Roberts, Cancer Cell 2012

C.Mullighan, Hematology 2012

IKZF1 Deletions Are Associated With High Rate Of Cumulative Incidenceof Relapse and with Short Disease Free Survival

Martinelli G, Iacobucci I, et al JCO 2009

Ph+ ALL

Characterization of the molecular basis of acute lymphoblastic leukemiaBCR-ABL-like

in adults

The primary objective of the study is to genetically characterize BCR-ABL1-like ALL, to determine their frequency and

prognosis in adult patients and to find NEW BIOMARKERS FOR TARGET THERAPY

GeneChip®Human TranscriptomeArray 2.0

Acute Myeloid Leukemia: Samples characteristics

Normal karyotypeOne-two abnormalitiesMonosomal karyotypeComplex karyotype

27/506/505/504/50

Other abnormalities 8/50

Raretranslocations

Sample

# 20

FISH

t(6;17)(p21;q11)

RNAseqSTK38 (chr 6p21) – PSMD11 (chr 17q11)

RPL7L1 (chr 6p21) – BC062794 (chr 17q12)ZEB2 (chr 2q22) – BCL11B (chr 14q)

FAM128A/B (chr 2q21) – CDC42BPB (chr 14q32)ANO3 (chr 11p14) – CORO1C (chr 12q24)

# 59810

# 21

t(2;14)t(11;12)

t(3;12)(p22;q24)monosomalkaryotype

AL049692.1 (chr 11p13) – CNOT2 (chr 12q15)HINFP (chr 11q23) – RSRC2 (chr 12q24)

NUMA1 (chr 11q13) – SLC35E3 (chr 12q15)FEZ1 (chr 11q24) – TAOK3 (chr 12q24)WT1 (chr 11p13) – CNOT2 (chr 12q15)

LIPH (chr 3q27) – PCBP2 (chr 12q13)NICN1 (chr 3p21) – SPATS2 (chr 12q13)

Simonetti G.

Reference group Cytogenetics abnormalities - no.

Fold difference

(RNAseq sample/ref samples)

1.28 1.32

0.93

0.8

0.4

0.0

1.2

1.631.6

2.0

STK38 BC062794 RPL7L1 PSMD11

Increased expression of PSMD11 in the STK38-PSMD11 fusion

GENES

••

PSMD11 is upregulated compared to the average of the control group.PSMD11 is a component of the lid subcomplex of the 26S proteasome, involved in the ATP-dependent degradation of ubiquitinated proteins. It is required for proteasome assembly.

Simonetti G.

6p21

17q11

Fold difference

(RNAseq sample/ref samples)

0.87 1.00 1.05 1.06 1.18 1.30 1.36 1.48 1.52 1.63 1.75 1.791.99

3.91

2.0

1.5

1.0

4.0

3.5

3.0

2.5

0.5

0.0

GENESSimonetti G. et Al Unpublished Personal comunication .

Increased expression of ZEB2, BCL11B, NUMA1 and HINFP upon gene fusions

14q324.5

2q222.36

Conclusions

• Despite the overall number of lesions across the patient cohort, novelregions of micro and macro genetic alteration were identified in denovo AML-ALL-MM patients by Gene CHIP Human Transcriptome® 2.0array and by Cyto ScanHD®-arrays :• Molecular karyotyping is easy obtained,• Copy neutral loss of heterozygousity (CN-LOH) are friendlyanalysed

• Chromothripsis could be identified• New expressed biomarkers (Bcr-ABL like) for target leukemiatherapy

The identification of additional cytogenetic abnormalities and a highnumber of microscopic genetic alterations allows to define subgroupswith worse prognosis, and rapidly stratify these patients toindividualized, personalized leukemia therapy.

Guadagnuolo, Stefania Paolini, Sarah Parisi, Cristina Clissa, Ilaria Iacobucci, Anna

Acknowledgments

Institute of Hematology “L. and A. Seràgnoli”, Bologna

Cristina Papayannidis, Chiara Sartor, Maria Chiara Abbenante, Emanuela Ottaviani, Viviana

Ferrari….,………………………………………

……………………………………..

…………………………………….

Supported by: FP7, European LeukemiaNet, AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna eRavenna

Acknowledgments

•••••••••••

Emanuela OttavianiViviana GuadagnuoloStefania PaoliniCristina PapayannidisAnna FerrariGiorgia SimonettiValentina RobustelliCarolina TerragnaMarina MartelloIlaria IacobucciMichele Baccarani

Institute “L. e A. Seràgnoli” Department of Biopathology,University Tor Vergata, Rome, Italy

• Francesco Lo Coco• Hasan Syed Khizer

Department of Clinical and BiologicalSciences, University at Orbassano

• Giuseppe Saglio• Daniela Cilloni