# Presentation: 473183

## Patterns of Variation in The Cancer Genome

- David A. Wheeler,
- Human Genome Sequencing Center,
- Baylor College of Medicine
- HIV, Cancer and Public Health
- St. Petersburg, May 26-30

## Acknowledgements

- MIT/HARVARD
- Broad Institute,
- 	Stacey Gabriel
- 	Carrie Sougnez
- 	Kristian Cibulski
- 	Gaddy Getz
- Dana Farber Cancer Institute
- 	Mathew Myerson
- 	Lynda Chin
- Washington University
- Genome Sequencing Center
- 	Li Ding
- 	Eliane Mardis
- 	Rick Wilson
- Memorial Sloan Kettering Cancer Center
- 	Nicholas Schultz
- 	Chris Sanders
- Cancer Genome Characterization Centers of The Cancer Genome Atlas project

- Baylor College of Medicine
- Human Genome Sequencing Center
- 	Donna Muzny
- 	Steve Scherer
- 	Lara Bull
- 	Bea Santillan
- 	Maggi Morgan
- 	Richard Gibbs
- Department of Molecular Virology
- 	Larry Donehower

- National Cancer Institute
- National Human Genome Research Institute

## Human Genome Sequencing Center: Large-scale Cancer Projects

- Tumor Sequencing Project (TSP)
- 188 lung adenocarcinoma tumors
- 630 genes (205 HGSC)
- The Cancer Genome Anatomy (TCGA)
- 96/500 glioblastoma multiforme
- 1,330 genes (450 HGSC)
- Copy Number, Expression, Methylation

## Modeling background mutations

**:  Non-synonymous background mutations rate
*m*: Total number of mutations in *b* bases
- Background, *m,* is binomial distribution ( Poisson)

- Example:
- Gene size  = 1500 bases
- Samples  =  500 tumors
- Total bases  =  750,000
- Mutation rate,   = 1.5x10-6
- Expect:
- Background mutations ~ 1
- Significantly mutated:
- 	~25 => 5%,
- 	~15 => 3%
- 	~10 => 2%

*G. Getz et al., Broad Institute*

## Mutation Discovery Pipeline

**Notes:**
Two arms:
	Genes (exons)
	Specimens

## Mutation Discovery Pipeline

- Variant Detection and Annotation

**Notes:**
Two arms:
	Genes (exons)
	Specimens

## Mutation Discovery Pipeline

- Genotyping

- TUMOR +
- MATCHED NORMAL

**Notes:**
Two arms:
	Genes (exons)
	Specimens

## Mutation Discovery Pipeline

**Final Mutation List**

- Genotyping

**Notes:**
Two arms:
	Genes (exons)
	Specimens

## Glioblastoma multiforme

- Molecular properties not well understood

- Multiform: color, histology, texture and shape.
- Etiology:
- “Unknown but has to do with several mutations in proto-oncogenes and tumor suppressor genes.
- “Changes or loss of chromosome 17 and inactivation of a tumor suppressor gene, p53, play a role. Thus far, we do not know what precipitates these changes.”
*...**U. Connecticut, Pathology Museum*

## Glioblastoma multiforme

- High mortality, treatments palliative

## Top 20 mutated genes in glioblastoma

*L. Ding, Washington U.;  G. Getz, Broad Institute*

- 86 tumor samples, 603 genes sequenced

## Core growth control pathways

- EGFR

- ERBB2

- PI-3Kclass 1

- PDGFRA

- MET

- RAS

- NF-1

- AKT

- FOXO

- PTEN

- Proliferation

- Activated Oncogenes

- MDM4

- TP53

- MDM2

- CDKN2A
- (ARF)

- RB1

**RTK/RAS/PI-3K signaling**

**p53 signaling**

- Senescence

- Apoptosis

- CDK4

- CDKN2A
- (INK4A)

- CDKN2B

- CDKN2C

- G1/S progression

**RB signaling:**

**Notes:**
94 patients
p53 signaling: 86% 
RB signaling: 77%
RTK/RAS/PI-3K: 84%
However there are 5 patients with no genetic alteration in any of these 3 pathways! 

## Mutations in core pathways

- EGFR

- ERBB2

- PI-3Kclass 1

- PDGFRA

- MET

- RAS

- NF-1

- AKT

- FOXO

- PTEN

- Proliferation

- Activated Oncogenes

- MDM4

- TP53

- MDM2

- CDKN2A
- (ARF)

- RB1

**RTK/RAS/PI-3K signaling**
**57% mutated**

**p53 signaling:**
**37% mutated**

- Senescence

- Apoptosis

- CDK4

- CDKN2A
- (INK4A)

- CDKN2B

- CDKN2C

- G1/S progression

**RB signaling: **
**13% mutated**

**Notes:**
94 patients
p53 signaling: 86% 
RB signaling: 77%
RTK/RAS/PI-3K: 84%
However there are 5 patients with no genetic alteration in any of these 3 pathways! 

## Mutations within Rb and p53 pathways rarely involve two hit in one person

- p53

- RB

- RTK-RAS

- PI3K-AKT

- p53
- EGFR
- NF-1
- PTEN
- PI-3K family

## Mutations alone frequently knock out two or more pathways

- RTK-RAS-PI3K pathway
- (growth/proliferation)

- p53 pathway
- (apoptosis/ senesence)

- Rb pathway
- (cell cycle progression)

- 1

- 16

- 7

- 0

- 9

- 4

- 26

## Focal amplification and deletion fill in DNA damage story

- p53

- RB

- RTK-RAS

- PI3K-AKT

- Deleted
- CDKNA2 (ARF/p16)
- CDKN2B
-  Amplification
- EGFR
- PDGFRA

## Large-scale knockout of core growth control

- EGFR

- ERBB2

- PI-3Kclass 1

- PDGFRA

- MET

- RAS

- NF-1

- AKT

- FOXO

- PTEN

- Proliferation

- Activated Oncogenes

- MDM4

- TP53

- MDM2

- CDKN2A
- (ARF)

- RB1

**RTK/RAS/PI-3K signaling:**
**84% of patients**

**p53 signaling:**
**86% of patients**

- Senescence

- Apoptosis

- CDK4

- CDKN2A
- (INK4A)

- CDKN2B

- CDKN2C

- G1/S progression

**RB signaling:  77% of patients**

**Notes:**
94 patients
p53 signaling: 86% 
RB signaling: 77%
RTK/RAS/PI-3K: 84%
However there are 5 patients with no genetic alteration in any of these 3 pathways! 

## Significantly mutated genes in glioblastoma

*Li Ding, G. Getz*

- 86 tumor samples

## Therapeutic implications of NF1 loss in GBM

*NF1 *inactivation via Ras-Raf-MAP kinase pathway
- MAP kinase kinase (MEK) inhibitors
- Promising for growth inhibition of *NF1*, *RAS* and *RAF *mutant tumor cells
- MEK inhibition: a candidate therapeutic strategy for patients whose glioblastomas lack functional *NF1*

*Matthew Myerson, Dana Farber CC & Broad Inst.*

## ERBB2 mutations in GBM:extracellular and kinase domains

- receptor domain
- kinase domain

- missense
- splice

- Extracellular domain

- ERBB2

- L49H

- exon2

- D326G

- C334S

- V750E

- V777A

- No focal copy number variation in 95 samples

*Wendy Winckler, Broad Inst.*

## Therapeutic implications of ERBB2 mutation

- 	GBM mutations are predicted to be activating
- 		Experimental confirmation in progress
- 	Small molecule kinase inhibitors of Erbb2 can be tested for GBM treatment
- 	Lapatinib, HKI-272, others
- 	Dual EGFR/Erbb2 kinase inhibition
- 	Combinations with PI3 kinase pathway inhibitors

*Matthew Myerson, Dana Farber CC & Broad Inst.*

## Lower survival with FOXO mutation

- 1.0
- 0.75
- 0.50
- 0.25
- 0.00

- 0             1.0            2.0            3.0            4.0
-                    Days (x 1000)

- Fraction Surviving

- FOXO (5 events)

- All genes

## Lower survival with PI-3K pathway mutations

- 1.0
- 0.75
- 0.50
- 0.25
- 0.00

- Fraction Surviving

- 0                   1.0                   2.0                 3.0
-                         Days (x 1000)

- 2 mutations

- 1 mutation

- 3 mutations

## Growth proliferation pathway mutations are associated with poor outcomes in glioblastoma multiforme

- EGFR

- ERBB2

- PI-3Kclass 1

- PDGFRA

- MET

- RAS

- NF-1

- AKT

- FOXO

- PTEN

- Proliferation

**RTK/RAS/PI-3K signaling:**
**84% of patients**

**Notes:**
94 patients
p53 signaling: 86% 
RB signaling: 77%
RTK/RAS/PI-3K: 84%
However there are 5 patients with no genetic alteration in any of these 3 pathways! 

## <40 vs. >40

- P = 0.0004

- P = 0.0026

- P = 0.645

## Data Available to Public and to Research Community

- DNA sequence data at the Trace Archive at the National Center for Biotechnology Information.
- Copy number, expression, methylation and mutation data, as well as clinical information is available at the TCGA data portal.
- Data that is potentially identifying is available only to research community.

## tcga data portal

- _[http://cancergenome.nih.gov/dataportal](http://cancergenome.nih.gov/dataportal)_
- see also _[http://cgwb.nci.nih.gov](http://cgwb.nci.nih.gov/)_, click on TCGAGSC_GBM_VALID tab

## Summary

- The TCGA consortium has comprehensively characterized genome variation in ~100 glioblastoma multiforme patients.
- DNA sequencing of cancer genes reveals a relatively small set of genes that are recurrently mutated.
- Other genes preferentially undergo amplification or deletion.
- Systematic gene sequencing reveals at least 2 genes not previously known to be involved in this cancer: NF-1 ERBB2.
- Patients accumulate multiple damage events in each pathway progressively shutting off apoptosis, control of cell cycle and increase growth.
- Preliminary analysis appears to link increased damage in members of the PI-3K pathway with poor prognosis, and EGFR and CDKN2A with later onset (and poor prognosis.

## Future sequencing plans

- Sequence 3 tumor genomes using “next generation sequencers” (454, 12X coverage at BCM;  and Illumina, 33X coverage at Wash U. and Broad Institute)
- Sequence 12 “transcriptomes”
- Transition to next generation sequencing technology
- Ovarian and lung cancer are next tumor types