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Development and Validation of a
Scalable Next-Generation Sequencing
System for Assessing Relevant
Somatic Variants in Solid Tumors
Raphael Fonseca, M.Sc.
Thermo Fisher Scientific
Proprietary & Confidential
The world leader in serving science
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Development and validation of the Oncomine™ Cancer Research
Panel (OCP), a scalable next-generation sequencing system for
assessing recurrent somatic alterations in solid tumors.
Kate Rhodes2, Andi Cani1, Dan Hovelson1, Geoff Bien2, Sophie
Rozenzhak2, Cristina Van Loy2, Denise Topacio2, Natalia Jun2, Andrew
McDaniel1, Albert Liu1, Paul Choppa2, Jeoffrey Schageman2, Guoying Liu2,
Fiona Hyland2, Rajesh Gottimukkala2, Jim Veitch2, Santhoshi Bandla2, Paul
Williams2, Bryan Johnson2, Melvin Wei2, Miroslav Dudas2, Adam Broomer2,
Peter Wyngaard2, Seth Sadis2, Dan Rhodes2, Scott Tomlins1
1Department
of Pathology, University of Michigan, Ann Arbor, MI, USA;
2Life Sciences Solutions, Thermo Fisher Scientific
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Personalized Medicine in Oncology
Worldwide: 14M cancer cases diagnosed
8.2M deaths in 20121
Personalized medicine is the most exciting change in
cancer treatment since the invention of chemotherapy2
As a growing number of biomarkers become clinically
actionable, the single-diagnostic/single-drug paradigm
is becoming a challenge to manage3
Sources:
1Cancer fact sheet. World Health Organization, Updated February 2014. Annals of Oncology 25:
2Peter Johnson, CRUK, http://www.cancerresearchuk.org/support-us/donate/become-a-major-donor/how-you-can-give/the-catalyst-club/personalised-medicine
3Delivering precision medicine in oncology today and in future—the promise and challenges of personalised cancer medicine: a position paper by the European
Society for Medical Oncology (ESMO)1673–1678, 2014 doi:10.1093/annonc/mdu217 Published online 20 June 2014
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Changing the Paradigm: Cancer is a Molecular Disease
From Anatomical to Molecular Approach
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Breast Cancer
Cervical Cancer
Colorectal Cancer
Liver Cancer
Lung Cancer
Ovarian Cancer
Pancreatic
Cancer
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Prostate Cancer
Skin Cancer
Stomach Cancer
Thyroid Cancer
Source: Nature Medicine, volume 18, number 3, March 2012
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ALK
AKT1
BRAF
EGFR
ERBB2
KRAS
NRAS
MAP2K1
PIK3CA
RET
ROS1
Undefined
Example: Many Alterations Are Aligned to Therapies
Evidence
FDA Approved
Labels
NCCN
Guidelines
Indication
Alteration
Drug (s)
Breast Cancer
ERBB2 amplification
pertuzumab, trastuzumab
Colorectal Cancer
KRAS mutation
cetuximab, panitumumab
contraindicated
Gastric Cancer
ERBB2 amplification
trastuzumab
Melanoma
BRAF mutation
dabrafenib, trametinib,
vemurafenib
Non-Small Cell Lung Cancer
ALK fusion
ceritinib, crizotinib
Non-Small Cell Lung Cancer
EGFR mutation
afatinib, erlotinib
Gastrointestinal Stromal Tumor
PDGFRA mutation
dasatinib
Colorectal Cancer
NRAS mutation
cetuximab, panitumumab
contraindicated
Melanoma
KIT mutation
imatinib mesylate
Non-Small Cell Lung Cancer
BRAF mutation
dabrafenib, vemurafenib
Non-Small Cell Lung Cancer
ERBB2 mutation
afatinib
Non-Small Cell Lung Cancer
MET amplification
crizotinib
Non-Small Cell Lung Cancer
RET fusion
cabozantinib
Non-Small Cell Lung Cancer
ROS1 fusion
crizotinib
12 different alterations aligned to 14 different approved therapies
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Example: More Alterations Are Under Investigation
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Alteration
Indication
Investigational drug(s)
AKT1 mutation
CCND1 amplification
CDK4 amplification, mutation
CDK6 amplification
DDR2 mutation
KRAS mutation
ERBB3 mutation
FGFR1-4 mutation, amplification, fusion
GNA11 mutation
GNAQ mutation
HRAS mutation
IDH1 mutation
KIT amplification
NRAS mutation
MET mutation
MTOR mutation
MYCN amplification
PDGFRA amplification
PIK3CA mutation
PPARG fusion
PTCH1 mutation
RET mutation
SMO mutation
STK11 mutation
Multiple
Multiple
Melanoma, NSCLC
NSCLC
Multiple
Multiple
Multiple
Multiple
Melanoma
Melanoma
Multiple
Multiple
Melanoma
Multiple
Multiple
Multiple
Multiple
Glioblastoma
Multiple
Thyroid Cancer
Multiple
NSCLC, Thyroid Cancer
Multiple
Multiple
MK-2206, MSC-2363318A
palbociclib
palbociclib
palbociclib
crizotinib + dasatinib
various MEKi combinations
neratinib
BGJ-398, JNJ-42756493
vorinostat
vorinostat
binimetinib + panitumumab, BVD-523
AG-120
dasatinib
various MEKi combinations
AMG-337, crizotinib, INCB-028060
MSC-2363318A
GSK-525762
nilotinib, sorafenib
various PI3K pathway combinations
pioglitazone
vismodegib
ponatinib, sunitinib
vismodegib
MSC-2363318A
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Routine Biomarker Analysis
Today’s Challenges
• Limited sample material for analysis
• Multiple biomarkers for one disease indication
• Need to test multiple biomarkers simultaneously
• Growing number of biomarkers to address
Current methods to test samples against various
biomarkers are slow and require more tumor
sample than available
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Routine Biomarker Analysis
Multiple Markers as a challenge
• Which markers to select?
• Which classes to analyze?
• DNA mutations
• RNA fusion genes
• Copy Number Variation (CNV) events
• Multiple markers/classes => Multiple assays?
• How to standardize analyses throughout labs?
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The Oncomine Knowledgebase – which markers?
The world’s largest curated cancer genomic database, gathered from
public sources, peer reviewed literature, and published clinical trials
Hotspot Mutations
High-Level CNVs
8,000 exomes + COSMIC database
Hotspot enrichment analysis
30,000 array-based genomes
Minimal common region analysis
The Oncomine
Knowledgebase
Deleterious Mutations
8,000 exomes, COSMIC database
Indel + nonsense enrichment analysis
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Gene Fusions
6,000 transcriptomes + COSMIC
Proprietary fusion analysis
Oncomine Comprehensive Assay - Content Summary
143 genes: Several used in multiple applications
(hotspot, CDS, driver fusion)
Categorized by somatic alteration type
73 Hotspot genes
49 Focal CNV gains
26 Full coding sequencing
for deletions & CNV
22 Fusion drivers
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143
Categorized by relevance
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Labels
Guidelines
Drug Targets
Methods
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Prostate Tumor Variant Map – DNA mutations
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Prostate Tumor Variant Map – RNA Fusions
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Copy Number from Oncomine Focus Sequencing
Validation of Oncomine Focus for Copy Number Alterations
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Lab 1
Lab 2
Lab 3
Lab 4
R2 = 0,93
Copy Number from FISH
Conclusões
• Painel genético com todas as classes de mutações cobertas
(mutações em DNA, fusões em RNA e CNV)
• Seleção de genes/mutações com relevância para tumores
sólidos
• Alta sensibilidade (>95% para EGFR, KRAS, BRAF and
ALK)
• Útil na busca de marcadores para companion diagnostics
• Pode ser usado como padrão em ensaios clínicos por ser um
ensaio único e com parâmetros de protocolo e análise prédefinidos
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