© 2010 Illumina, Inc. All rights reserved.

Illumina, illuminaDx, Solexa, Making Sense Out of Life, Oligator, Sentrix, GoldenGate, GoldenGate Indexing, DASL, BeadArray, Array of Arrays, Infinium, BeadXpress, VeraCode, IntelliHyb, iSelect, CSPro, GenomeStudio, Genetic Energy, and HiSeq are

registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.

Platform Approach to

Cancer Discovery and

Diagnostics

Development

© 2009 Illumina, Inc. All rights reserved.

Illumina, illuminaDx, Solexa, Making Sense Out of Life, Oligator, Sentrix, GoldenGate, GoldenGate Indexing, DASL, BeadArray, Array of Arrays, Infinium, BeadXpress, VeraCode, IntelliHyb,

iSelect, CSPro, and GenomeStudio are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.

Emily Winn-Deen, Ph.D.

Vice President

Diagnostics Product Development

2

United States: 2000United States: 2025Male Female

Population (millions)

Why Focus on Cancer?

The Population is Aging

3

Copyright ©2008 American Cancer Society

From Jemal, A. et al.

CA Cancer J Clin 2008;58:71-96.

Annual Age-adjusted Cancer Death Rates* Among Males for Selected Cancers, United States, 1930 to 2004

Cancer Continues to be a Major Health Issue

4

Cancer Therapies Dominate the Biotech Pharma Pipeline

10

11

16

17

23

25

31

36

37

37

39

42

46

51

54

66

79

79

89

96

97

145

Sarcoma

Bladder

Cervical

Stomach

Liver

Head/Neck

Myeloma

Ovarian

Brain

Kidney

Cancer-Related Conditions

Pancreatic

Skin

Other Cancers

Lymphoma

ColoRectal

Breast

Prostate

Leukemia

Lung

Unspecified Cancers

Solid Tumors

4

4

6

7

9

10

13

14

15

17

18

22

22

44

50

210

Transplantation

Growth Disorders

Eye Conditions

Skin Disorders

Genetic Disorders

Blood Disorders

Respiratory Disease

Digestive Disorders

Diabetes

Neurological Disease

Other

HIV

CVD

AutoImmune

Infectious Disease

Cancer

Solid Tumors Lead Cancer Therapies

Source: PhRMA: 2006 Medicines in Development Biotechnology

5

Oncology DiscoveryInitial Targets

Source: WHO

Gastric

Colon/ rectum

Breast

Lung

Percentage of All Cancers

Pro

bab

ilit

y o

f C

ure

(%

)

100

0 6 8 10 12 14

0

10

20

30

40

50

60

70

80

90

2 4

KidneyCervix/ Uteri

Melanoma

Bladder

Oral

NHL

Testis

Thyroid

Prostate

Esophagus

MyelomaLeukemia

Brain

Pancreas

Ovary

CNS

Nasopharynx

Liver

6

Cancer Sequencing Workflow

Visualize in IGV

Sequence

& QC data

Receive

samples

Genotype for

tracking and QC

Call somatic

variants:- substitutions

- indels <100 bp

- SVs >100 bp

- CNVs

Check tracking

and QC

Annotate

variants

Cross-sample

comparison

Build

genome

(CASAVA)

7

Correlate

within

pathways

Call somatic

variantsApply mutational

consequence scores

(optional)

Map mutated genes

to known (cancer)

pathways

Correlate across cancers

Mutated genes

in known

pathways

Disrupted

pathways

Bin by region

Correlate

across

cancers

Variants

with no

annotation

Mutated

regions in

common

Map variants to genes and

other genomic features

Somatic

variant lists

List of

mutated

features

Mutated

features in

common

Correlate

across cancers

List of

mutated

genes

Mutated genes

in common

Map mutated

genes to known

cancer genes

Correlate

across cancers

Mutated genes

known

in cancer

Known cancer

genes in

common

Mutated

pathway genes

in common

Disrputed

pathways in

common

Correlate

across

cancers

Oncology DiscoveryCancer Specific Analyses

8

Melanoma: COLO-829

► Collaboration with Mike Stratton’s team at the Sanger Center (UK)

► Melanoma cell line COLO-829 and its matched normal (>30x depth)

► Male; metastatic tumour; prior to treatment

► BRAF, CDKN2A and PTEN mutations previously confirmed by exon

sequencing

► SKY karyotype indicates pseudotriploidy

Karyotype courtesy of Paul Edwards, University of Cambridge

9

Variant Detection

► Run specifications

– 2 x 75 base runs, 15 Gb / run. 0.2, 1, 2 & 4 kb libraries

► Single base substitutions

– Paired reads aligned with ELAND (non-gapped)

– Requires Q100 per allele, e.g. 3 x Q33 bases or equivalent

► Small indels

– Split reads aligned individually or grouped (Pindel, BWA, Grouper)

– Minimum 3x depth

► Larger structural variants

– Anomalous read pairs from short and long insert libraries

– >3 S.D. (deletions) or >5 S.D. (insertions)

– Minimum 10 reads

► Copy number changes

– Read depth (BWA+) in 10 kb windows

10

Somatic Substitutions in COLO-829

► Subtraction process– High stringency calibrated tumour calls (germline + somatic)

– Subtract if evidence of variant in normal; & dbSNP

► 33,345 somatic substitutions (gain of an allele) in total

– 1 per 100 kb

– 32,325 single base

– 510 double base (adjacent)

► 96.6% specificity

– 454/470 novel somatic calls confirmed by new PCR-capillary sequencing

– 16 may be artefacts of either method

► 88% (98%) sensitivity

– 42/48 previous somatic variants called automatically

– (98%) or 47/48 automatic + manual (five seen only manually as the evidence

was below automatic threshold)

– 1/48 disputed call

11

Somatic Mutational Signature of UV Exposure

► 69.9% of all somatic substitutions are CT or GA

► 92% of somatic CT are in a YC pyrimidine dimer (vs. 53% by chance)

A

T

G

C

12

Complex CNV in tumour on chromosome 3

Normal

Tumor

Tumour (40X)

Normal (30X)

4 genes in this region:

RARB, TOP2B, NGL1,KS

13

Catalogue of COLO-829 Somatic Variants

33,345 Single base substitutions

– 286 coding

1018 small indels

– 14 coding

37 Structural rearrangements

– 34 intrachromosomal:

25 deletions

6 insertions

2 duplications

1 complex

– 3 interchromosomal

– 19 breakpoints in genes

198 changes in copy number

Ideograms / val indels / sn10M / cdsn / cn /loh / sv

14

Oncology Discovery Ovarian Cancer

Deadly

Incurable

Difficult Dx

Patients present with a suspicious/palpable mass

Less than 40% are cured

204,449 New cases annually; 124,860 deaths

Early Dx is Key

Five year survival is good if diagnosed early,

but most patients are diagnosed late stage

Illumina Solution

Develop a diagnostic assay which will diagnose

ovarian cancer at an early stage

19%22%

30%33%

37%40%

47%

54%

72%

100%

92%

85%82%

69%

56%

51%

39%

26%

17%

12%

-10%

10%

30%

50%

70%

90%

110%

Ia Ib Ic Iia Iib Iic IIIa IIIb IIIc IV

Cum % of Cases

5 Yr Survival

15

Ovarian Cancer Discovery

► Establish end-to-end workflow for sequencing, analysis, interpretation

► Refine analysis tools

► Sequence 25 tumour normal genome pairs (>30x depth)

► Identify genome wide catalogues of somatic mutation

► Search for mutational signatures, novel biomarkers, etc.

► Incorporate RNA and methylation data

► Gain experience in line with ICGC and similar projects

16

Illumina’s Oncology Discovery Initiative

Samples Discovery Validation Dx Service Dx Product

Ovarian is here

Clinical Partnership

Obtain consent

Obtain samples

Prep samples

Build libraries

25 Tumor/

Normals

W GS

Methylome

Transcriptome

300 + samples

BeadChip

analysis

LDT in

CLIA lab

Prospective

trials

CE- IVD

US IVD

17

Cancer Genome Summary Statistics

Tumour Tumour

depth

Normal

depth

Somatic

gains

Melanoma 829 40.7x 32.5x 33,345

Breast 100 31.4x 29.0x 4,838

Ovarian A 40.5x 37.3x 7,147

Ovarian B 32.5x 35.1x 1,099,481

Ovarian C 38.0x 39.1x 4,284

Ovarian D 32.7x 37.2x 1,244

Ovarian E 36.2x 35.4x 3,727

Ovarian F 37.2x 29.7x 11,984

Ovarian G 30.4x 32.0x 3,091

Ovarian H 39.9x 35.0x 5,623

Ovarian I 34.8x 34.2x 8,824

Ovarian J 35.8x 39.9x 4,815

Tumour Tumour

depth

Normal

depth

Somatic

gains

Ovarian K 32.3x 33.0x 3,255

Ovarian L 32.8x 39.6x 3,093

Ovarian M 37.6x 42.9x 4,480

Ovarian N 38.3x 37.8x 6,698

Ovarian O 36.7x 30.1x 5,485

Ovarian P 35.9x 39.2x 3,489

Ovarian Q 34.7x 35.5x 5,584

Ovarian R 35.7x 33.9x 5,973

Ovarian S 34.5x 33.6x 2,154

Ovarian T 35.2x 33.3x 2,203

Ovarian U 38.1x 35.6x 2,881

18

IGV visualisation of two variants in TP53

Normal

R

Tumour

R

Normal

L

Tumour

L

19

Summary of TP53 somatic substitutions (SNVs)

► 14/17 high grade serous samples have TP53 somatic substitution variants

(SNVs) – further analysis of remaining samples ongoing

► Review vs. COSMIC:

– All are in known TP53 hotspots

– All are novel observations in ovarian cancer

– Two seen previously in other cancers

► TP53 mutations reported in 97% of high grade pelvic serous carcinoma

samples in a recent study (Ahmed et al 2010)

20

sample100

sample101

sample102

sample103

sample104

sample111

Tumor

Normal

Copy Number: Chr6

C6orf170, FABP7, GJA1, HSF2,

NKAIN2 ,PKIB, RLBP1L2, SERINC1,

SLC25A5P7, SMPDL3A, TRDN

AIM,1 ASC3C, ATG5, BEND3, BVES, C6orf112,

C6orf203, CCNC, GRIK2, HACE1, LIN28B, MCHR2,

MIRN587, POPDC3, PRDM1, PRDM13, PREP,

QRSL1, RPL35P3, RTN4IP1, SIM1, USP45

21

Summary and Future Prospects

► A Genome-Wide Opportunity:

– Comprehensive variant catalogues

– >95% specificity and ~90% sensitivity of somatic substitutions

► System developments enable large-scale studies:

– Chemistry, software improvements (>99.9% raw data accuracy)

– HiSeq2000 platform (>300 Gb per run: two 30x genomes per instrument per

week)

► Implications for individual genome sequencing:

– Understanding disease mechanism, biomarker discovery

– Sequencing individual cancer genomes for personalised treatment

22

…to all our Collaborators…

…and the Illumina teams...

Many Thanks…