Cell Signaling

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High-throughput oncogene mutation profiling
Massimo Loda ESMO Milano, 2008
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Somatic mutations

• Genetic and epigenetic (in)activation of
  oncogenes and tumor suppressor genes

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Critical evaluation of mutations

• Where does the mutation occur? Heterogeneity,
  cancer stem cells
• Genetic and epigenetic assessment
• Passenger mutation, oncogene-induced senescence,
  oncogene addiction (context is important)
• Lineage-survival genes (MITF, AR)
• Metabolic oncogenes

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Biological/clinical Heterogeneity
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Techniques to detect genomic and proteome
alterations

• High throughput sequencing (point mutations)
• High density SNPs/aCGH (amplifications,
  deletions)
• Gene expression profiling (signatures)
• miRNA profiling
• Immunohistochemistry, ISH, FISH
• Mass Spectrometry

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From Cancer Genome Discovery to Targeted
Therapeutics?

• BCR-ABL ? Gleevec
• KIT ? Gleevec
• PDGFR-alpha ? Gleevec
• EGFR ? Tarceva
• ERBB2 ? Herceptin
• mdm2 ? nutlin
• PIK3CA ? PI3K inhibitors
• mTOR ? rapamycin

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High throughput sequencing
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Sequencing methodologies

• Sanger sequencing
• 1 M bp/24 hrs low sensitivity low cost
• Single molecule sequencing by synthesis (Solexa)
• 100 M bp/3 days high sensitivity 3K/100 M bp
• Massively parallel sequencing by pyrosequencing
  (454)
• 100 M bp/8 hrs high sensitivity 10K/100M bp

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Genome sequencing in microfabricated high-density
picoliter reactorsMargulies et al., 2005.
454 picolitre-scale sequencing
Sanger DNA sequencing electrophoresis

• 100X throughput improvement 20M-40M bp / 4hr run
• Need 10-12 times oversampling to get same
  accuracy as Sanger

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454 pyrosequencing (massively parallel
sequencing)

• 100 Mbp/8hrs, 10K
• Accuracy gt99.99

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Flowgram
TTCTGCGAA
Cont.
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Solexa sequencing
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(No Transcript)
14

• Other sequencing technologies resources
• 454 www.454.com
• Solexa www.solexa.com
• Helicos tSMS www.helicosbio.com
• ABI seq by ligation on a solid surface
  www.appliedbiosystems.com
• Agilent nanopore sequencing

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High-Throughput Genotyping to Identify Oncogene
Mutations
Each allele yields a different mass extension
product
Run on MALDI-TOF mass spec.
Removes excess dNTPs
C
G
C
G
C
G
Genotype GG CG
CC
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Sample with no mutation
wildtype
mutant
Sample with mutation
wildtype
mutant
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OncoMap Proof-of-Principle

• OncoMap pilot study
• 238 assays
• 17 oncogenes
• 60 multiplexed assays per sample
• 1000 tumors, 17 lineages
• 65/sample

• ABL1
• AKT2
• BRAF
• CDK4
• EGFR
• ERBB2
• FGFR1
• FGFR3
• FLT3

• HRAS
• JAK2
• KIT
• KRAS
• NRAS
• PDGFRa
• PIK3CA
• RET

R. Thomas, A. Baker et al., Nature Genetics, 2007
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gt 70 cancer enrichment --gt WGA --gt multiplex PCR
--gt mass spec
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OncoMap results

• Mutations events are rare and generally do not
  hit the same codon
• Of 17 oncogenes analyzed, 14 were mutated at
  least once and 30 of samples carried at least
  one mutation
• Driver or passenger mutation?

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Hybrid capture of all exons

• high-density microarrays can capture any desired
  fraction of the human genome, gt 200,000
  protein-coding exons. Up to 98 of intended exons
  can be recovered.

Hodges et al Nat Genet. 2007 Dec39(12)1522-7
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Polymorphisms, passenger mutations or driver
mutations?
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Integration of genomic and expression analyses
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Oncogene mutation vs pathway activation
How do we identify activated pathways in order to
target them therapeutically and monitor response?
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What can we monitor?
From Engelman Liu and Cantley 2006
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Mutations in PI3k-Akt pathway in human cancer
PTEN Loss of heterozygosity Glioblastoma 54
(98/180) Prostate 35 (88/250) Breast 23
(37/164) Melanoma 37 (53/143) Gastric 47
(14/30) PTEN Mutations Glioblastoma 28
(122/432) Prostate 12 (26/218) Breast 0
(0/164) Melanoma 8 (15/185) Gastric 0 (0/30)
AKT Amplifications Ovarian 12 (18/147)
Pancreatic 20 (7/35) Breast 3 (3/106)
Gastric 20 (1/5) Head and neck 30 (12/40)
PIK3R1 (p85a) Mutations Ovarian 4 (3/80)
Colon 2 (1/60)
p110a Mutations Breast 26 (176/684) Colon 26
(88/337) Glioma 8 (14/182) Hepatocellular 36
(26/73) Ovarian 10 (35/365) Lung 2 (4/253)
Gastric 7 (24/338) p110a Amplifications Head
and neck 42 (54/128) Thyroid 9 (12/128)
Lung Squamous cell 66 (46/70) Adenocarcinoma
5 (4/86) Breast 9 (8/92) Gastric 36 (20/55)
Esophageal adenocarcinoma 6 (5/87) Cervical
69 (11/16)
From Engelman, Luo, Cantley, 2006
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PTEN -/- pAKT pS6 PPRAS 40
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How to define the shades of gray?
80
PTEN / pAKT - pS6- PPRAS40-
10
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High Throughput
Gene Expression
aCGH
SNP
Validation is low throughput and rate limiting
TMA
FISH/ISH
IHC
Integration of all of these is virtually
nonexistent
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Integration of IHC and gene expression data
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IHC
Tumor
Normal
Only Tumor
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Mutation detection current status

• Next generation sequencing is here and works
  (single molecule sequencing)
• Hybrid capture of all exons is feasible
• Currently 100 discovery 150 validation
  tumor/normal pairs would currently cost 3M
  dollars
• Best done in collaboration/consortium

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How will genomic tests reach patients?

• Genomic facilities in individual cancer centers
• Pharmaceutical companies
• Start up companies
• Diagnostic companies
• Combination of the above

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Objectives towards the development of a new
molecular classification of tumors and targeted
therapy

• How will sarcomas be treated and studied over the
  next ten years?
• What kind of science should be pursued?
• What should the balance/interaction be between
  clinical work and basic research?
• What new technologies or concepts can best be
  developed and applied?

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Goals

• Achieve a more detailed classification of tumors
  by adding detailed molecular annotation to
  existing pathologic and clinical data, that will
  guide therapeutic options in real time
• Predict inhibitors in the pipeline and
  concentrate on developing tools to a) select
  appropriate patients (for clinical trial and in
  routine diagnostics) and b) monitor response to
  therapy

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Acknowledgments

• Center for Cancer Genome Discovery - CCGD (Dana
  Farber Cancer Institute)
• Laura MacConaill
• Levi Garraway
• Center for Molecular Oncologic Pathology - CMOP
  (Dana Farber Cancer Institute), Univ. of Milan
• Giuseppe Fedele Silvano Bosari
• Michelangelo Fiorentino Valentina Vaira
• Elisa Benedettini Giorgia Zadra