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