Integrative and Functional Characterization of the Soft Tissue Sarcoma Genome
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Integrative and Functional Characterization of the Soft Tissue Sarcoma Genome The Sarcoma Genome Project Jordi Barretina / Barry S. Taylor CTOS 15 th Annual Meeting 6 th November 2009
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Integrative and Functional Characterization of the Soft Tissue Sarcoma Genome. The Sarcoma Genome Project. Jordi Barretina / Barry S. Taylor CTOS 15 th Annual Meeting 6 th November 2009. Soft Tissue Sarcomas. Derived from connective tissues ( mesenchymal tumors). - PowerPoint PPT Presentation
Transcript of Integrative and Functional Characterization of the Soft Tissue Sarcoma Genome
Integrative and Functional Characterization of theSoft Tissue Sarcoma Genome
The Sarcoma Genome Project
Jordi Barretina / Barry S. Taylor
CTOS 15th Annual Meeting
6th November 2009
Derived from connective tissues (mesenchymal tumors).
Rare (<1% of all cancers) and heterogeneous.
STS have not yet been a focus of large-scale genomic efforts (low TCGA, ICGC priority).
Most of them caused by somatic mutations.
Gleevec as a paradigm of targeted cancer therapies.
Soft Tissue Sarcomas
The need:50% of patients with newly diagnosed sarcoma eventually die of metastatic disease.The majority of sarcoma subtypes are not very
responsive to chemotherapy.
The solution:Conduct a detailed genomic characterization of soft tissue sarcoma tumor samples.Selective therapies against genomically altered targets can be highly effective and can exert
fewer side effects.
From Cancer Genomics to Targeted Therapies
Computational analysis
Data integration
Targeted functional validation
Gene selection
The Sarcoma Genome Project
Sample collection
RNA and DNA extraction
U133A Affy Expression arrays (n=141)
Sample collection
RNA and DNA extraction
U133A Affy Expression arrays (n=141)
250K Affy (StyI)SNP arrays (n= 207)
Sequencing (n=48)
Candidate mutation validation (n=48 + 159*)
250K Affy (StyI)SNP arrays (n= 207)
Sequencing (n=48)
Candidate mutation validation (n=48 + 159*)
Elucidate the genetic alterations and signaling pathways associated with specific sarcoma subtypes.
Improve sarcoma classification.
Identify new therapeutic targets in sarcoma through integrative analysis.
TSGP Goals
… + matched normals (205)
Paired expression data for 141 of them.
Samples
Subtype Karyotype Total
Dedifferentiated liposarcoma
complex 50
Myxoid/round cell liposarcoma
simple, t(12;16), t(12;22) 21
Pleomorphic
liposarcoma
complex 24
Myxofibrosarcoma complex 38
GIST simple 22
Synovial sarcoma simple, t(X;18) 25
Leiomyosarcoma complex 27
TOTAL 207
• 48 Soft Tissue Sarcomas:– 21 liposarcomas– 10 myxofibrosarcomas– 6 GISTs– 11 synovial sarcomas
• Genes chosen for exon resequencing:– Group 1: All Tyrosine Kinases– Group 2: Selected Cancer and Sarcoma genes– Group 3: All microRNAs
( 224 genes + 496 microRNAs,
3587 exons, 3830 amplicons)
Sequencing Summary
Title line 1Title line 2
Gene No. of mut.a Frequencyb Subtype mRNA Protein
CDH1 2 2 DDLPS 712A>AG N238D
4.5 GIST 1849G>AG A617Te
CTNNB1 2 2 DDLPS 122C>CT T41Id
4 Synovial 95A>AT D32Vd
EPHA1 1 2 DDLPS 634G>GA A212T
EPHA5 1 4.2 Pleomorphic 2386A>AG Y796H
EPHA7 1 2.6 MYXF 1649C>CT S550N
ERBB4 2 2.6 MYXF 3437A>AT D1146V
4.2 Pleomorphic 1558A>AT C520S
FBXW7 2 2 DDLPS 338_342delTCATC>TC E113fs
4.5 GIST 563G>GT C188F
IRS1 1 4.5 GIST 3406C>CT E1136K
KIT 6 23 GIST 1727T>CT L576Pd
GIST 1961T>CT V654Ad
GIST 1667_1674delAGTGGAAG>AG Q556fs
GIST 1667_1687delc Q556_I563>Q
GIST 1670_1675delGGAAGG W557_V559>Fe
4.8 MRC 2334G>CG K778N
LTK 1 4 Synovial 2243_2244delTT>T C748fs
MOS 1 4.5 GIST 898A>AG S300P
MST1R 1 4.5 GIST 1229G>AG P410L
NF1 7 10.5 MYXF 7972C>CT H2658Y
MYXF 7790C>CT S2597L
MYXF 910C>T R304*d
MYXF 910C>T R304*d
MYXF 7010T>TG L2337R
8.3 Pleomorphic 1105C>CT Q369*d
Pleomorphic 4006C>CT Q1336*
NTRK1 1 2.6 MYXF 2338C>CT R780W
PI4KA 2 2.6 MYXF 4081_4088delTCTTATCT>TCT 1361fs
4 Synovial 4081_4088delTCTTATCT>TCT 1361fs
PIK3CA 6 19 MRC 1633G>AG E545Ke
MRC 1633G>AG E545Ke
MRC 3140A>AG H1047Re
MRC 3140A>AG H1047Re
4.2 Pleomorphic 1660delC H554fs
4 Synovial 1659delT S553fs
PTEN 2 2.6 MYXF G>CG Splice site
4 Synovial 106G>AA G36Re
PTK2B 1 4.2 Pleomorphic G>AG Splice site
RB1 1 4.2 Pleomorphic 1818T>TA Y606*e
SYK 1 4.2 Pleomorphic 52G>AA G18S
TP53 4 16.7 Pleomorphic 404C>AA C135Fe
Pleomorphic 464G>AA T155I
Pleomorphic C>CT Splice site
Pleomorphic C>TT Splice site
We found 37 somatic point mutations and 9 indels (involving 21 genes across 6 sarcoma subtypes).
Several previously described in sarcoma and other cancers (COSMIC).
30 not previously reported.
24 affected kinases.
18 predicted to have a functional effect by in silico analysis
Sequencing Summary
EPHA5 NTRK1
PIK3CA mutations in ~18% of myxoid/round cell liposarcomas
Diverse NF1 alterations in soft tissue sarcoma
Nucleotide and copy number alterations in
soft tissue sarcoma
• Question: Which amplified genes in DDLPS are necessary for cancer cell proliferation/survival?
• Answer: Genomics-driven RNAi screen in DDLPSSystematic knockdown of ~400 significantly amplified genes with shRNAs in 3 genotype-matched cell lines
Functional annotation of the Cancer Genome
Functional annotation of the Dedifferentiated Liposarcoma “Amplicome”
Genes essential for DDLPS cell proliferation.
PD-0332991CDK4/CDK6 inhibitor
CDK4 as a target inDedifferentiated Liposarcoma
Functional annotation of the Dedifferentiated Liposarcoma “Amplicome”
Genes essential for DDLPS cell proliferation.
YEATS4 and MDM2 co-amplification and potential cooperation in p53 pathway regulation
Our study yields the most detailed map of molecular alterations across diverse sarcoma subtypes to date.
Subtype-specific genomic alterations define new targets for soft tissue sarcoma therapy.
- PI3K pathway in PIK3CA-mutant myxoid/round cell liposarcomas
- mTOR pathway in NF1-deficient soft tissue sarcomas
Summary
Next-Gen sequencing is replacing capillary and array-based technologies
DNA Point mutations (substitutions/indels)
Chromosomal aberrations• Copy gains and losses• Loss of heterozygosity (LOH) • Rearrangements & fusion genes
Epigenetic modifications
RNA
Protein
Transcript expression level changes
Differential alternative splicing
Allele-specific expression changes
Protein expression level changes
Protein modification changes
Protein degradation/stability changes
standard technologyMolecular Alterations in Cancer
CANNOT BE DETECTED
NO GENOME-WIDE HIGH THROUGHPUT TECHNOLOGY
new technology
NO GENOME-WIDE HIGH THROUGHPUT TECHNOLOGY
NO GENOME-WIDE HIGH THROUGHPUT TECHNOLOGY
Significantly AmplifiedGenes
Significantly AmplifiedGenes
Genes Essential for Proliferation
Genes Essential for Proliferation
Recurrent Mutations and Fusion Genes
Recurrent Mutations and Fusion Genes
50 Paired Patient Samples
Affymetrix 250K SNP Array
50 Paired Patient Samples
Affymetrix 250K SNP Array
5 Cell Lines54,020 lentiviral shRNA
pool screen(~11,000 genes)
5 Cell Lines54,020 lentiviral shRNA
pool screen(~11,000 genes)
12 Paired Patient Samples
Whole Exome Sequencing~186,000 baits (~16,000
target genes) & RNA-Seq
12 Paired Patient Samples
Whole Exome Sequencing~186,000 baits (~16,000
target genes) & RNA-Seq
Shantanu Banerji
Next steps in DDLPS…
MSKCCPennelope DeCarolisMariana Lagos-QuintanaAlan HoTsuyoshi SaitoNeerav ShuklaChristopher LauComp Biology CenterBarry TaylorJohn MajorBoris RevaNick SocciAlex LashGenomics Core LabAgnes Viale
Biological Samples PlatformScott MahanJennifer FranklinJennifer ChenAlex ThomsonKristin ArdlieGenetic Analysis PlatformBrendan Blumensteil Kristian CibulskisLiuda ZiaugraCarrie SougnezStacey GabrielSequencing PlatformRobb OnofrioJen BaldwinRNAi PlatformHanh LePat LizotteBrian WongAlan DerrJen GrenierSerena SilverDavid Root
AcknowledgementsCancer Genome AnalysisShantanu BanerjiAlex RamosRameen BeroukhimGaddy GetzCraig MermelDerek ChiangBarbara WeirKinjal ShahLauren AmbrogioTzu-Hsiu ChenMegan HannaLaura MacConaillProject ManagementWendy Winckler
Comp Biology & BioinfJim RobinsonDavid TwomeyTed LiefeldMichael ReichPablo Tamayo
Heidi GreulichTodd Golub Bill HahnLevi GarrawayBill SellersEric LanderMatthew Meyerson
Robert MakiGary SchwartzCristina Antonescu Chris SanderMarc LadanyiHarold VarmusSam Singer
