The OncoScan(TM) platform for analysis of copy number and somatic mutations in cancer
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Transcript of The OncoScan(TM) platform for analysis of copy number and somatic mutations in cancer
The OncoScan FFPE Assay Kit for analysis of copy number and somatic mutations in cancer
Larry Greenfield M.D., Ph.D
Affymetrix’ clinical product portfolio
Cancer
Hematological malignancies Solid tumors
Drug Metabolism
Reproductive Health
Prenatal Postnatal
Mendelian diseases
For Research Use Only
Challenges for laboratories
Growing need for genome wide copy number profiles Large and growing list of actionable copy number
aberrations Detection of clonal populations enables longitudinal patient
assessments Number of copy number aberrations indicative of prognosis
For solid tumor analysis, FFPE is a critical specimen type but is
often highly degraded, and generates limited DNA
Molecular profiles need to be generated rapidly and at an affordable price Multiplex multiple markers with minimal data analysis
burden
For Research Use Only
Pan Indication Copy Number Actionable Genes
AKT2 CTNNB1 JAK2 MPL PTPN11
ALK DNMT3A JUN MPM1 RAB25
APC EGFR KIT MYC RB1
AR ERBB2 KRAS MYCN REL
ASXL1 ESR1 MAP2K1 MYD88 RET
ATM FGFR1 Map2K2 NOTCH1 RUNX1
BRAF FGFR4 MDM2 NRAS TET2
CCND1 FLT3 MET PDGFRA Tp53
CDK4 IDH1 MITF PIK3CA VHL
CEBPA IDH2 MLL PTEN WT1
Leading cancer centers* are developing a growing panel of actionable pan indication copy number aberrations
Examples include MYC amplification (prognostic in colorectal cancer, prostate and breast), Her2 amplification (predictive in breast, ovarian and endometrial cancers)
Large and growing list of actionable copy number changes
*Test panels from Washington University, MD Anderson, MSKCC
For Research Use Only
Chromosomal copy number changes contribute significantly to tumor development and progression in gastrointestinal stromal tumors (GIST)
Analysis evidencing “…genomic complexity as the best predictor of disease relapse”
In addition, pattern of genomic loss may provide differential diagnostic information
Genomic complexity indicative of prognosis GIST as an example
Chromosome copy number changes carry prognostic information independent of KIT/PDGFRA point mutations in gastrointestinal stromal tumors Mara Silva1, Isabel Veiga1, Franclim R Ribeiro1, Joana Vieira1, Carla Pinto1, Manuela Pinheiro1, Bárbara Mesquita1,Catarina Santos1, Marta Soares2, José Dinis2, Lúcio Santos3, Paula Lopes4, Mariana Afonso4, Carlos Lopes4 and Manuel R Teixeira*1,5
1q gain 12q gain
1p loss 14q loss 15q loss 22q loss
OncoScan enables previously inaccessible solid tumor studies
“There are currently few studies to have
realized this potential for FFPE samples, owing to the inherent problems of working with chemically modified and damaged DNA, the requirements for larger amounts of genomic DNA and/or high depth of sequencing coverage to minimize ambiguous read mapping and the subsequent added bioinformatics challenges that this brings”
“The recently developed OncoScan, a high-resolution SNP array based on molecular inversion probes, appears to perform well in comparison with aCGH platforms and so might prove to be a successful method for studying FFPE samples for CNAs”
For Research Use Only
“…Our results suggest that this FISH panel would have missed 6 melanomas in our cohort that OncoScan detected, one of which proved to be lethal to the patient
This implies a single OncoScan test could be used to confirm the diagnosis of malignant melanoma, stratify the risk of recurrence, and direct pharmacologic interventions”
With enormous implications FISH-based misdiagnosis
OncoScan can also be used to multiplex the commonly tested druggable somatic
mutation BRAFv600E along with copy number
information
OncoScan™ FFPE Assay Kit Key features
• Input DNA of 80ng • Will work with archived & highly degraded FFPE samples • 48 hrs from DNA to results • Off the shelf product with kitted reagents including OncoScan™
Nexus Express software • Works with existing Affymetrix instruments • Universal reference to establish normal baseline • Content
– Genome wide copy number determination – High resolution on ~900 cancer genes – Genome wide LOH resolution – Detection of frequently tested somatic mutations
• Clonal population detection • High copy number dynamic range
OncoScan™ FFPE Assay enabled by MIP technology
High specificity – low false positives and assay noise: unique approach to interrogating the genome where the probe is amplified rather than the genomic DNA
Ideal for FFPE samples that often suffer from severe degradation and chemical impurities: assay
requires only a small footprint (150bp) of intact DNA sequence per region
Genetic content extension possible by the addition of new probes with new tag sequences that are already captured by complementary sequences on the array
~40 base pairs
OncoScan™ FFPE Assay Advances in algorithm and data visualization
Advances in data analysis: internal bioinformatics advances in algorithm development data visualization partnership with BioDiscovery (Nexus
software)
Enabling the following novel features: Incorporation of the Affymetrix’ TuScan algorithm that
supports the determination of tumor burden and ploidy to establish a baseline for copy number determination
Corrected for systematic variability (e.g. local and probe GC correction)
Ability to correlate copy number and clinical outcomes data across multiple samples in a clinical cohort
Benefit and creation of a universal reference
Inclusion of a universal reference eliminates need to run normal controls in each batch and allows for single sample analysis
Universal reference will be initially derived from FFPE
samples encompassing: – 11 countries – 11 states within the United States – 23 different tissue types – Block ages ranging from <1 year - 21 years
The universal reference will be further evaluated over
time and is intended to represent the diversity of samples analyzed using the platform
From array data to copy number, LOH and somatic mutation results
Array signal data are: Normalized against the universal reference Corrected for systematic variability (e.g. GC correction)
Next semi “raw data” are provided: log2 ratio B-allele frequency (BAF)
Data conclusion layer: Copy number summary and somatic mutation data Uses Affymetrix’ TuScan algorithm to compute the integer CN of the
cancer component (when possible) Reports Copy Neutral (and other) LOH segments Presence/absence of Somatic Mutation
1
2
Algorithm Output: % Tumor and ploidy Integer CN Track for Tumor component
Normal Adjacent Tissue: Colorectal Cancer Site: Liver % Tumor: Normal Ploidy = 2
Algorithm Input: : B-allele frequency (~signal(B)/(signal(A) + signal(B)) : log2 ratio
1
2 3
3
CN conclusion: A normal male sample
For Research Use Only
Algorithm Output: % Tumor: ~80% Ploidy: 2
Adenocarcioma Site: Prostate
An example of a tumor with few aberrations detection of genome wide copy number aberrations
2 3 1 4 5 6 7 8 9 10 X 11 2 3 1 4 5 6 7 8 9 10 X 11
2 3 1 4 5 6 7 8 9 10 X 11
For Research Use Only
Algorithm Output: % Tumor: ~80% Ploidy: 2
Adenocarcioma Site: Prostate
CN=2
CN=2
2 3 1 4 5 6 7 8 9 10 X 11 2 3 1 4 5 6 7 8 9 10 X 11
2 3 1 4 5 6 7 8 9 10 X 11
CN=2
An example of a tumor with few aberrations detection of genome wide copy number aberrations
For Research Use Only
Algorithm Output: % Tumor: ~80% Ploidy: 2
Adenocarcioma Site: Prostate
CN=3
CN=3
CN=3
2 3 1 4 5 6 7 8 9 10 X 11 2 3 1 4 5 6 7 8 9 10 X 11
2 3 1 4 5 6 7 8 9 10 X 11
CN=3
An example of a tumor with few aberrations detection of genome wide copy number aberrations
For Research Use Only
Algorithm Output: % Tumor: ~80% Ploidy: 2
Adenocarcioma Site: Prostate
CN=1
CN=1
CN=1
2 3 1 4 5 6 7 8 9 10 X 11 2 3 1 4 5 6 7 8 9 10 X 11
2 3 1 4 5 6 7 8 9 10 X 11
CN=1
An example of a tumor with few aberrations detection of genome wide copy number aberrations
For Research Use Only
Algorithm Output: % Tumor: ~90% Ploidy: 2
Stromal Carcinoma Site: Stomach
2 3 1 4 5 6 7 8 9 10 X 11 2 3 1 4 5 6 7 8 9 10 X 11
2 3 1 4 5 6 7 8 9 10 X 11
CN=2
CN=2
CN=1
CN=1
CN=1
CN=3
CN=3
CN=3
A slightly more complex tumor detection of genome wide copy number aberrations
CN=3
CN=2
CN=1
An example of a complex tumor
2 3 1 4 5 6 7 8 9 10 X
2 3 1 4 5 6 7 8 9 10 X 2 3 1 4 5 6 7 8 9 10 X
Algorithm Output: % Tumor: ~80% Ploidy: 2
Endometrioid Carcinoma Site: Ovary
2 3 1 4 5 6 7 8 9 10 X
2 3 1 4 5 6 7 8 9 10 X 2 3 1 4 5 6 7 8 9 10 X
Algorithm Output: % Tumor: ~80% Ploidy: 2
Endometrioid Carcinoma Site: Ovary
CN=2
CN = 2 CN = 2
An example of a complex tumor
2 3 1 4 5 6 7 8 9 10 X
2 3 1 4 5 6 7 8 9 10 X 2 3 1 4 5 6 7 8 9 10 X
Algorithm Output: % Tumor: ~80% Ploidy: 2
Endometrioid Carcinoma Site: Ovary
CN = 1
CN=1
CN = 1
CN=1
An example of a complex tumor
2 3 1 4 5 6 7 8 9 10 X
2 3 1 4 5 6 7 8 9 10 X 2 3 1 4 5 6 7 8 9 10 X
Algorithm Output: % Tumor: ~80% Ploidy: 2
Endometrioid Carcinoma Site: Ovary
CN = 3
CN = 3
CN = 3
CN = 3
An example of a complex tumor
2 3 1 4 5 6 7 8 9 10 X
2 3 1 4 5 6 7 8 9 10 X 2 3 1 4 5 6 7 8 9 10 X
Algorithm Output: % Tumor: ~80% Ploidy: 2
Endometrioid Carcinoma Site: Ovary
CN = 2
Copy Neutral
LOH
CN = 2
Copy Neutral
LOH
An example of a complex tumor
2 3 1 4 5 6 7 8 9 10 X
2 3 1 4 5 6 7 8 9 10 X 2 3 1 4 5 6 7 8 9 10 X
Algorithm Output: % Tumor: ~80% Ploidy: 2
Endometrioid Carcinoma Site: Ovary
CN = 4
CN=4 +LOH
CN = 4
CN=4 +LOH
An example of a complex tumor
AGENDA
For Research Use Only
Introduction of the OncoScan FFPE Assay Kit in 2H 2013 represents a very significant new
capability
Why?
Only solution for solid tumor genome wide copy number profiling of a typical clinical sample
offering results in 48 hours
For Research Use Only
Large And Growing List Of Actionable Copy Number Changes!
MD Anderson 2012– 200 genes actionable for SM MD Anderson 2013- Same 200 genes now actionable for CN as well!
ABL1 BAP1 CEBPA EP300 FGFR1 GNAS KDM6A MPL NOTCH4 PIK3CG RB1 TGFB1
AKT1 BRAF CHEK2 EPHA3 FGFR2 HNF1B KIT MSH2 NPM1 PIK3R1 RET TGFBR2
ALK BRCA1 CPAMD8 ERBB2 FGFR3 HRAS KRAS MSH6 NRAS PKHD1 RUNX1 TNFAIP3
APC BRCA2 CREBBP ERBB3 FGFR4 IDH1 LAMA1 NAV3 NSD1 PPP1R3A RUNX1T1 TOP1
ARAF CARD11 CSMD3 ERCC3 FLT3 IDH2 LPHN3 NF1 PALB2 PPP2R1A SETD2 TOP2A
ARID1A CASP8 CTNNB1 ERCC4 FOXL2 IKZF1 LRP1B NF2 PAX5 PRDM1 SMARCB1 TP53
ASXL1 CBL CYLD ERCC5 GABRB3 IRS1 MAP2K4 NFKB2 PBRM1 PTCH1 SMO TSC1
ATM CDH1 DNMT3A ETV5 GATA1 JAK1 MEN1 NOTCH1 PDGFRA PTEN SOS1 TSC2
ATR CDH11 ELN EZH2 GATA3 JAK2 MLH1 NOTCH2 PDGFRB PTPN11 SYK TSHR
AURKA CDK6 EML4 FBXW7 GNAQ JAK3 MLL3 NOTCH3 PIK3CA RAD51 TET2 VHL
Partial List Of MD Anderson 200 genes
For Research Use Only
Large and growing List of actionable copy number changes
Genes Amplification Deletion 1 ALK 2 EGFR 3 FGFR1 4 FGFR2 5 KRAS 6 MET 7 PTEN 8 AKT1 9 AXL
10 FGFR3 11 ERBB2 12 MAP2K1 13 PIK3CA 14 PDGFRB 15 TP53
CRUK 1st List – Only SM , no CN CRUK 2nd List- 15 CN Genes