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
...
For Research Use Only
Challenges for laboratories
 Growing need for genome wide copy number profiles
 Large and growing ...
For Research Use Only
Pan Indication Copy Number Actionable Genes
AKT2 CTNNB1 JAK2 MPL PTPN11
ALK DNMT3A JUN MPM1 RAB25
AP...
For Research Use Only
 Chromosomal copy
number changes
contribute significantly to
tumor development and
progression in
g...
OncoScan enables previously
inaccessible solid tumor studies
 “There are currently few studies to have
realized this pote...
For Research Use Only
 “…Our results suggest that
this FISH panel would
have missed 6
melanomas in our cohort
that OncoSc...
OncoScan™ FFPE Assay Kit
Key features
• Input DNA of 80ng
• Will work with archived & highly degraded FFPE samples
• 48 hr...
OncoScan™ FFPE Assay
enabled by MIP technology
 High specificity – low false positives and assay noise: unique approach t...
OncoScan™ FFPE Assay
Advances in algorithm and data visualization
Advances in data analysis:
 internal bioinformatics adv...
Benefit and creation of a universal reference
 Inclusion of a universal reference eliminates need to run
normal controls ...
From array data to copy number, LOH and
somatic mutation results
Array signal data are:
 Normalized against the universal...
1
2
Algorithm Output:
% Tumor and ploidy
Integer CN Track for Tumor component
Normal Adjacent
Tissue:
Colorectal Cancer
Si...
For Research Use Only
Algorithm Output:
% Tumor: ~80%
Ploidy: 2
Adenocarcioma
Site: Prostate
An example of a tumor with fe...
For Research Use Only
Algorithm Output:
% Tumor: ~80%
Ploidy: 2
Adenocarcioma
Site: Prostate
CN=2
CN=2
2 31 4 5 6 7 8 9 10...
For Research Use Only
Algorithm Output:
% Tumor: ~80%
Ploidy: 2
Adenocarcioma
Site: Prostate
CN=3
CN=3
CN=3
2 31 4 5 6 7 8...
For Research Use Only
Algorithm Output:
% Tumor: ~80%
Ploidy: 2
Adenocarcioma
Site: Prostate
CN=1
CN=1
CN=1
2 31 4 5 6 7 8...
For Research Use Only
Algorithm Output:
% Tumor: ~90%
Ploidy: 2
Stromal Carcinoma
Site: Stomach
2 31 4 5 6 7 8 9 10 X11 2 ...
An example of a complex tumor
2 31 4 5 6 7 8 9 10 X
2 31 4 5 6 7 8 9 10 X 2 31 4 5 6 7 8 9 10 X
Algorithm Output:
% Tumor:...
2 31 4 5 6 7 8 9 10 X
2 31 4 5 6 7 8 9 10 X 2 31 4 5 6 7 8 9 10 X
Algorithm Output:
% Tumor: ~80%
Ploidy: 2
Endometrioid C...
2 31 4 5 6 7 8 9 10 X
2 31 4 5 6 7 8 9 10 X 2 31 4 5 6 7 8 9 10 X
Algorithm Output:
% Tumor: ~80%
Ploidy: 2
Endometrioid C...
2 31 4 5 6 7 8 9 10 X
2 31 4 5 6 7 8 9 10 X 2 31 4 5 6 7 8 9 10 X
Algorithm Output:
% Tumor: ~80%
Ploidy: 2
Endometrioid C...
2 31 4 5 6 7 8 9 10 X
2 31 4 5 6 7 8 9 10 X 2 31 4 5 6 7 8 9 10 X
Algorithm Output:
% Tumor: ~80%
Ploidy: 2
Endometrioid C...
2 31 4 5 6 7 8 9 10 X
2 31 4 5 6 7 8 9 10 X 2 31 4 5 6 7 8 9 10 X
Algorithm Output:
% Tumor: ~80%
Ploidy: 2
Endometrioid C...
AGENDA
For Research Use Only
Introduction of the OncoScan FFPE Assay Kit
in 2H 2013 represents a very significant new
capa...
For Research Use Only
Large And Growing List Of Actionable
Copy Number Changes!
MD Anderson 2012– 200 genes actionable for...
For Research Use Only
Large and growing List of actionable
copy number changes
Genes Amplification Deletion
1 ALK
2 EGFR
3...
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The OncoScan(TM) platform for analysis of copy number and somatic mutations in cancer

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The OncoScan microarray offers high-quality copy number, genotype, and somatic mutation data with whole-genome coverage and high resolution in cancer genes for use with challenging FFPE samples.

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The OncoScan(TM) platform for analysis of copy number and somatic mutations in cancer

  1. 1. The OncoScan FFPE Assay Kit for analysis of copy number and somatic mutations in cancer Larry Greenfield M.D., Ph.D
  2. 2. Affymetrix’ clinical product portfolio Cancer Hematological malignancies Solid tumors Drug Metabolism Reproductive Health Prenatal Postnatal Mendelian diseases
  3. 3. 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
  4. 4. 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
  5. 5. 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
  6. 6. 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”
  7. 7. 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
  8. 8. 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
  9. 9. 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
  10. 10. 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
  11. 11. 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
  12. 12. 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
  13. 13. 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
  14. 14. 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 31 4 5 6 7 8 9 10 X11 2 31 4 5 6 7 8 9 10 X11 2 31 4 5 6 7 8 9 10 X11
  15. 15. For Research Use Only Algorithm Output: % Tumor: ~80% Ploidy: 2 Adenocarcioma Site: Prostate CN=2 CN=2 2 31 4 5 6 7 8 9 10 X11 2 31 4 5 6 7 8 9 10 X11 2 31 4 5 6 7 8 9 10 X11 CN=2 An example of a tumor with few aberrations detection of genome wide copy number aberrations
  16. 16. For Research Use Only Algorithm Output: % Tumor: ~80% Ploidy: 2 Adenocarcioma Site: Prostate CN=3 CN=3 CN=3 2 31 4 5 6 7 8 9 10 X11 2 31 4 5 6 7 8 9 10 X11 2 31 4 5 6 7 8 9 10 X11 CN=3 An example of a tumor with few aberrations detection of genome wide copy number aberrations
  17. 17. For Research Use Only Algorithm Output: % Tumor: ~80% Ploidy: 2 Adenocarcioma Site: Prostate CN=1 CN=1 CN=1 2 31 4 5 6 7 8 9 10 X11 2 31 4 5 6 7 8 9 10 X11 2 31 4 5 6 7 8 9 10 X11 CN=1 An example of a tumor with few aberrations detection of genome wide copy number aberrations
  18. 18. For Research Use Only Algorithm Output: % Tumor: ~90% Ploidy: 2 Stromal Carcinoma Site: Stomach 2 31 4 5 6 7 8 9 10 X11 2 31 4 5 6 7 8 9 10 X11 2 31 4 5 6 7 8 9 10 X11 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
  19. 19. An example of a complex tumor 2 31 4 5 6 7 8 9 10 X 2 31 4 5 6 7 8 9 10 X 2 31 4 5 6 7 8 9 10 X Algorithm Output: % Tumor: ~80% Ploidy: 2 Endometrioid Carcinoma Site: Ovary
  20. 20. 2 31 4 5 6 7 8 9 10 X 2 31 4 5 6 7 8 9 10 X 2 31 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
  21. 21. 2 31 4 5 6 7 8 9 10 X 2 31 4 5 6 7 8 9 10 X 2 31 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
  22. 22. 2 31 4 5 6 7 8 9 10 X 2 31 4 5 6 7 8 9 10 X 2 31 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
  23. 23. 2 31 4 5 6 7 8 9 10 X 2 31 4 5 6 7 8 9 10 X 2 31 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
  24. 24. 2 31 4 5 6 7 8 9 10 X 2 31 4 5 6 7 8 9 10 X 2 31 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
  25. 25. 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
  26. 26. 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
  27. 27. 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

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