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Emerging Targets for Squamous Cell NSCLC
Ramaswamy Govindan M.D.Alvin J Siteman Cancer Center
Washington University School of MedicineSt Louis
Structural variants• Translocations• Fusions• Inversion
Copy number alterations
• Amplifications• Deletions• LOH
Point mutations & indels
• Missense• Nonsense• Splice site• Frameshift
Gene expression• Outlier expression• Isoform usage• Pathways & signatures
Wild type AGTGA
Mutant AGAGA
Adapted from: Roychowdhury et al. Sci Transl Med; 20122
Epigenetic Changes
Whole Genome Sequencing
Exome Sequencing
Transcriptome Sequencing(RNA Seq)
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TCGA Lung Cancer ProjectStatus
• Adenocarcinoma
• Goal 500
• Accrued so far 500
• Analysis ongoing
• Squamous Cell Cancer
• Goal 500
• Accrued so far 390
• Analysis completed 178
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TCGA Lung Cancer ProjectStatus
• Adenocarcinoma
• Goal 500
• Accrued so far 500
• Analysis ongoing
• Squamous Cell Cancer
• Goal 500
• Accrued so far 390
• Analysis completed 178
9
Patient Characteristics
• Total number of patients 178
• Age (median; range) 68 (40-85)
• Gender
• Male 131 (74%)
• Female 47 (26%)
• Smoking Status
• Never smoker 7 (4%)
• Follow up in months (median; range) 15.8 (0-177)
• Tumor stage
• Stage I 97 (55%)
• Stage II 38 (21%)
• Stage III 38 (21%)
• Stage IV 3 (2%)
• No information 2 (1%)
10
Specimen Processing
Biospecimen Core Repository Characterization
Centers
SequencingCenters
Data Coordinating
Center
Tissue
Source
Sites (TSS)
Preliminary
Pathology
Review
Tumor Pathology QC
•% Tumor Nuclei>60%•% Necrosis <20%•Pathology review
Molecular Analyte QC•RIN> 7•Spectrophotometery•RNA Bioanalyzer•Electrophoresis•Genotyping
Collection of Clinical Data
ElementsQualified
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Methods
• For all 178 samples
• SNP arrays (copy number variation)
• Exome sequencing (somatic mutations in the exons)
• Transcriptome sequencing–”RNA-seq” (gene expression, gene
fusions)
• Microarrays (gene expression)
• DNA methylation (methylation status)
• Additional data
• miRNA sequencing for 159/178 samples
• Whole genome sequencing of 19 samples
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mRNA Expression Analysis
15% 36% 24% 25%
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mRNA Expression Analysis
15% 36% 24% 25%
PI3K
alterations
NF1 loss
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178 tumor/normal DNA pairs, analyzed on Affymetrix
SNP 6.0 arrays and Agilent CGH arrays
Gain of 3q
Gain of 5p
Copy number analysis Chromosomal arm level alterations
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Copy number analysis Focal alterations
Copy number alterations per tumor (mean): Focal- 47; Broad- 23
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Exome and RNA sequence analysis of squamous cell lung cancer
• 178 tumor/germline DNA pairs and 178 tumor RNAs, on
Illumina paired-end sequencing
• Mean sequencing coverage across targeted bases –
121X (83% of bases above 30X coverage)
• Significantly mutated genes were identified using
modified version of MutSig algorithm
• All somatic mutations were verified by a second
independent hybrid-recapture
• Total number of non-silent mutations in 178 samples-
48,690
• Mutations per tumor (mean)- total: 360; non-silent: 228
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Exome and RNA sequence analysis of squamous cell lung cancer
• 178 tumor/germline DNA pairs and 178 tumor RNAs, on
Illumina paired-end sequencing
• Mean sequencing coverage across targeted bases –
121X (83% of bases above 30X coverage)
• Significantly mutated genes were identified using
modified version of MutSig algorithm
• All somatic mutations were verified by a second
independent hybrid-recapture
• Total number of non-silent mutations in 178 samples-
48,690
• Mutations per tumor (mean)- total: 360; non-silent: 228
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Squamous cell lung cancer has a very high rate of somatic mutations
1 / Mb
10 / Mb
100 / Mb
0.1 / Mb
81 64 38 316 100 17 82 28n=109 119 21 40 20
Hematologic
Childhood
Carcinogens
??
Courtesy: Gaddy Getz and Mike Lawrence,
Broad Institute, MIT
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Significantly Mutated Genes in Squamous Cell Lung Cancer
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CDKN2A: Loss of Function Through Multiple Mechanisms
Tumor samples
Three most common
mechanisms
Homozygous deletion 30%
Methylation 21%
Mutation 17%
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Pathway Alterations in Squamous Cell Lung Cancer
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Therapeutic targets in squamous cell lung carcinoma
SCC-Lung and HNSCC
Gene Pathway
CDKN2A*
Cell division, multiple pathways
PIK3CA*
PTEN*
NFE2L2* Oxidative stress response
NOTCH1*
Genes involved in squamous cell differentiation(mutated in 30% HNSCC and 44% squamous cell lung cancer)
MLL2*
TP63
NOTCH2
NOTCH3
SYNE2
IRF6
RIPK4
*significantly mutated in both cancers
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Therapeutic targets in squamous cell lung carcinomas, defined by TCGA
Lung Cancer Mutation Consortium
Incidence of Single Driver Mutations
Mutation found in 54% (280/516) of
tumors completely tested (CI 50-59%)
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Gene Event Type Frequency
CDKN2A Deletion/Mutation/Met
hylation
72%
PI3KCA Mutation 16%
PTEN Mutation/Deletion 15%
FGFR1 Amplification 15%
EGFR Amplification 9%
PDGFRA Amplification/Mutation 9%
CCND1 Amplification 8%
DDR2 Mutation 4%
BRAF Mutation 4%
ERBB2 Amplification 4%
FGFR2 Mutation 3%
Therapeutic targets in squamous cell lung carcinoma
PIK3CA in SCC
• PIK3CA copy number gains frequent in
– Squamous cell histology
– Smokers
– Males
• 545, 1047 mutations represent 12/28 PIK3CA mutations in TCGA
C2
RB
DH
elical
Kin
aseP
85
8D
542545546
1047
-Anchorage independent growth-Cell migration
AA 1
AA 1068
PIK3CA domains and frequently mutated sites
Adapted from Samuels et al. Cell cycle 2004;3(10):1221-4Image: Okudela et al. Pathol Int 2007;57(10):664-71.
FGFR1 in SCC
• FGFR1 amplification known to occur in primary and metastases of SCC
• Potential target: survival of FGFR1 amplification harboring cells sensitive to FGFR inhibition (Ex: PD173074 )
FGFR -unamplified cell line
FGFR- amplified cell line
• FGFR1 amplified in 17% of TCGA tumors
Image: Dutt et al. PLoS One 2011;6(6):e20351.
DDR2 in SCC
• Mutations in DDR2 kinase reported in ~4% of SCC
(mutated in 1% of TCGA samples)
• Regulates cell differentiation, proliferation, and migration
• Studies indicate that DDR2 mutations are targetable by dasatinib
Image: Hammerman et al. Cancer Discov 2011;1(1):78-89.
Open clinical trials with targeted agents in SCC- Lung
Trial ID Sponsor Phase Targeted agent Target class
NCT01491633 Dana-Farber Cancer Institute II
Dasatinib BCR/ABL, SRCNCT01514864 Bristol-Myers Squibb II
NCT01041781Cancer and Leukemia Group B III Celecoxib COX
NCT01702714 Hoffmann-La Roche I RO5083945
EGFR
NCT01485809 Seoul Veterans Hospital II Gefitinib
NCT01523587Boehringer Ingelheim Pharmaceuticals III Afatinib/Erlotinib
NCT01561456 Axelar AB II AXL1717 IGF-1R
NCT01642004 Bristol-Myers Squibb IIIBMS-936558 (anti-PD1)
Immune basedNCT01285609 Bristol-Myers Squibb III Ipilimumab
NCT01519804 Genentech II Onartuzumab MET
NCT01346540 Boehringer Ingelheim I/IIBIBF-1120 (Nintedanib) VEGFR
NCT00998192 Oncolytics Biotech II Reolysin (Reovirus) Virus
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Summary
• Complex genomes with frequent and unique
rearrangements
• A clear and reproducible sub-classification
• Distinct transforming mechanism defined by common
NFE2L2 activation in the classical subtype
• High somatic mutation rates includes near universal
TP53 mutation and frequent loss of CDKN2A function
• Multiple mechanisms for CDKN2A inactivation
• Therapeutic identified in 127 patients (75%) including
FGFRs, PI3 kinase pathway, EGFR/ERBB2 and
Cyclin/CDK complexes
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DNA methylation analysis
Leslie Cope, Johns Hopkins
Ludmila Danilova, Johns Hopkins
Dan Weisenberger, USC
Peter Laird, USC
James Herman, Johns Hopkins
Steve Baylin, Johns Hopkins
Gene expression and transcriptome
Neil Hayes, North Carolina
Matt Wilkerson, North Carolina
Derek Chiang, North Carolina
Chuck Perou, North Carolina
Roel Verhaak, MD Anderson
Gordon Robertson, UBC
Andy Mungall, UBC
Dominick Stoll, UBC
Jinze Liu, U of Kentucky
DNA sequence analysis
Andrey Sivachenko, Broad
Gad Getz, Broad
Mike Lawrence, Broad
Carrie Sougnez, Broad
Stacey Gabriel, Broad
Eric Lander, Broad
Bryan Hernandez, Broad
Marcin Imielinski, Broad
Elena Helman, Broad
Peter Hammerman Dana-Farber/Broad
Copy number analysis
Gad Getz, Broad
Gordon Saksena, Broad
Steve Schumacher, Dana-Farber/Broad
Andy Cherniack, Broad
Peter Hammerman, Dana-Farber/Broad
Marc Ladanyi, Memorial Sloan Kettering
Barry Taylor, Memorial Sloan Kettering
Alexei Protopopov, Brigham and Women’s
Raju Kucherlapati, Brigham and Women’s
Jianhua Zhang, Brigham and Women’s
Panel of lung cancer expert advisors
Bill Travis, Memorial Sloan Kettering
Bruce Johnson, Dana-Farber
William Pao, Vanderbilt
Roman Thomas, Koln
Cross-platform Analysis
Chad Creighton, Baylor
Eric Collisson, UCSF
Igor Jurisica, Toronto
Sam Ng, UCSC
Jacob Kaufman, Vanderbilt
Nam Pho, Broad
Rileen Sinha, MSKCC
Ronglai Shen, MSKCC
Christine To, Toronto
John Weinstein, MD Anderson
Niki Schultz, MSKCC
Biospecimen Core
Joe Paulauskis, IGC
Bob Penny, IGC
Project management
Kenna Shaw, NCI
Laura Dillon, NCI
Margi Sheth, NCI
Ram Iyer, NCI
Brad Ozenberger, NCI
Tissue collaborators
Malcolm Brock, Johns Hopkins
Ming Tsao, Toronto
Dennis Wigle, Mayo
Val Rusch, Memorial Sloan Kettering
Peter Goldstraw, Royal Brompton
Kwun Fong, Prince Charles
Andrew Godwin, Fox Chase
Maria Raso, MD Anderson
Rajiv Dhir, Pitt
Carl Morrison, Roswell Park
Working group Chairs
Matthew Meyerson, Dana-Farber/Broad
Steve Baylin, Johns Hopkins
Ramaswamy Govindan, Washington U
Writing committee chairs
Peter Hammerman, Dana-Farber
Neil Hayes, UNC
Matt Wilkerson, UNC
Key participants in TCGA lung cancer analysis group
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Working group Chairs
Ramaswamy Govindan, Washington U
Steve Baylin, Johns Hopkins
Matthew Meyerson, Dana-Farber/Broad
Special Acknowledgement
Project management
Kenna Shaw, Director, TCGA
Writing committee chairs
Peter Hammerman, Dana-Farber
Neil Hayes, UNC
Matt Wilkerson, UNC
Patients with lung cancer
and their families
