MassARRAY System –a proven technology for translational ... · Lianxing Yang PhD Senior Scientist –Applications and Technology September 2012 The MassARRAY® System is for Research

MassARRAY® System – a proven

technology for translational genomics

and precision medicine

Darryl Irwin PhD

Lianxing Yang PhD

Senior Scientist – Applications and Technology

September 2012

The MassARRAY® System is for Research Use Only. Not for Use in Diagnostic Procedures.

Presentation Outline

.

The MassARRAY® System, iPLEX Pro Sample ID, and LungCarta™ Panels are for Research Use Only.

Not for Use in Diagnostic Procedures.

� Sequenom MassARRAY applications overview

� Application of key types of genomic biomarkers in Cancer� LungCarta – Broad somatic mutation screening panel in lung

cancer

� SampleID – Sample Tracking and QC

� From comprehensive to Focused Panels – OncoFocus: EGFR/KRAS/BRAF/NRAS + Custom content

� Ultra-sensitive techniques for detecting rare somatic mutations

� Translocations – EML4-ALK: a key biomarker class for precision medicine

� ADME panel

� Quantitative DNA methylation analysis in cancer

� Conclusions and Q&A

2 | Improving healthcare through revolutionary genetic analysis solutions

.

Quality of Science

A Company driven by technology and innovation

Over 1,800 citations

used the MassARRAY

System in peer-

reviewed publications

http://www.sequenom.com/literature


MassARRAY® Applications

.

One Platform, Multiple ApplicationsMiniaturized SpectroChip

Data Analysis Packages

Mass Spectrometry

Biochemistry Processes

GenotypingMethylation

Analysis

Quantitative Gene Analysis

Comparative Sequence Analysis

Molecular Diagnostics:

CF, RHD, AMD

……


Matrix Assisted Laser Desorption Ionization Time Of

Flight Mass Spectrometry (MALDI-TOF MS)

.

De

tecto

r+

+

Matrix/AnalyteLaser Desorption and IonizationAcceleration and Detection

+

+

Mass Spectrum m/z

Combines sensitivity of PCR with the accuracy of MS


Why accurate?

.

DNA consists of a four-letter alphabet:A, C, G, T

Each letter (and building block within a DNA strand)has defined molecular mass:

dAMP = 313.2 DadCMP = 289.2 DadGMP = 329.2 DadTMP = 304.2 Da


Biomarker Continuum:

Discovery to Clinical Applications

.

Focused Panels

w/ Clinical UtilityHypothesis

Generating

Validated

Biomarker

Discovery Verification Validation

Discovery

• Genome-wide or targeted

• Archival samples or model system

Technical confirmation

• Orthogonal technology

• Large panels

Clinical validation

• Large sample sets

• Flexible panels

Clinical Deployment

• Focused panels

• Validated IVDs


Targeted Therapies in Cancer

ONCOGENE TARGETED THERAPEUTIC TREATMENT PHARMA

BCR-ABL IMATINIB (GLEEVEC)* LEUKEMIA NOVARTIS

HER2 TRASTUZUMAB (HERCEPTIN) BREAST CANCER ROCHE

(GENENTECH)

C-KIT

PDGFR

IMATINIB GIST NOVARTIS

SUNITINIB (SUTENT) PFIZER

EGFR GEFITINIB (IRESSA) BREAST, NSCLC ASTRAZENECA

ERLOTINIB (TARCEVA) NSCLC

PANCREATIC

ROCHE

(GENENTECH)

CETUXIMAB (ERBITUX) CRC

HEAD & NECK

BMS

MTOR EVEROLIMUS (AFINITOR) MULTI-CANCER NOVARTIS

PIII TRIALS

MET CRIZOTINIB (XALKORI) NEOPLASMS PFIZER

BRAF VEMURAFENIB (ZELBORAF) MELANOMA DAIICHI

EML4-ALK CRIZOTINIB (XALKORI) NSCLC PFIZER

AR MDV3100 (ABIRATERONE) PROSTATE PIII TRIAL

.8 | Improving healthcare through revolutionary genetic analysis solutions

Broad Research Use Panels

OncoCarta™ and LungCarta™

The OncoCarta™ and LungCarta™ Panels are for Research Use Only. Not for Use in

Diagnostic Procedures.

.

OncoCarta™ Panel

• Targeted screening of tumor

samples for >238 hot spot

mutations in 19 key Oncogenes


LungCarta™ Panel

• Targeted screening of tumor

samples for >214 hot spot

mutations in 26 key cancer genes

LungCarta™ Panel derived from large research discovery project

.

Ding, L., et al.: Somatic mutations affect key pathways in lung adenocarcinoma. Nature 455 (7216): 1069-1075 (2008)

1,000 mutations

identified via large scale sequencing

discovery project…

26 genes mutated at significantly high

frequencies


The LungCarta™ Panel is for Research Use Only. Not for Use in Diagnostic Procedures.

� Comprehensive hot spot mutation panel

� >214 somatic mutations in 26 cancer genes

� Single panel designed in 24 wells

� ≥ 240 ng DNA per sample

� 5% mutation frequency

.

Highlights of the LungCarta™ Panel



LungCarta™ Panel Overview



Case Study Success Story at Paul Brousse

Summary of Observations

� Reliable, sensitive, and rapid

� Ideal content with a very small amount of DNA from FFPE

biopsy (< 100 ng)

� Correlated with gene mutations in the literature

� An industry leader who wants to institute this test throughout

the French Cancer consortium


Customer Quotes

“Offers the possibility to use for the first time, a high-throughput

technology in the context of personalized medicine”

“The ideal test at the right time with the

right targets”

HRM

(global detection)

Exon 18-19-21

Fragment size analysis

Applied - exon 19 deletion

Allelic Discrimination Assay

(Roche) - L858R exon 21

Gene EGFR

Domaine Tyrosine Kinase

Exons 18-24

Exon 18 Exon 19 Exon 20 Exon 21Exon 18 Exon 20

+ +

Sequencing if HRM positive and no

exon 19 deletion or L858R

EGFR Testing Schema at Paul Brousse

One gene requires a fair amount of sample from the

FFPE block—not much left for other tests

14 | Improving healthcare through revolutionary genetic analysis solutions .

Testing LungCarta™ Panel at Paul Brousse

A Perfect Match with Other Methods

N % Tumor DNA ng LungCarta Other methods

1 50 29 PIK3CA H1047R 25% PIK3CA H1047R

2 15 5 HER2 M774-A775 14% HER2 ins12pb (fgt analysis)

3 80 5 EGFR Del L747-752 P753S 73% EGFR del-18pb L747-752 P753S 70%, oncoCarta id

50%

4 30 2 EGFR L858R 18%+T790M 23% EGFR L858R + T790M, oncocarta id L858-19%-

T790-36%

5 10 3 KRAS G12D 22% KRAS G12D

6 50 10 EGFRL861Q 40%, STK11 H174R 32% EGFR L861Q, OncoCarta id 20%

7 40 3 Neg (not present) HER2 ins12pb (fgt analysis), OncoCarta neg

8 40 1 EGFR G719C 50%-S768I 54% EGFR G719C + S768I, OncoCarta id G719-53%-

S768 55%

9 20 3 EGFR Del E746-T750 EGFR del-15pb L746-750 K745K 70%

10 30 7 BRAF V600E 23% BRAF V600E

11 90 3 EGFR exon20 ins773-774 EGFR ins20, OncoCarta ins20 H773-V774 insPH

12 70 4 KRAS G12C KRAS G12C, OncoCarta id G12C 22%

13 90 1 HER2 M774-A775 62% + STK11 F354L 57% HER2 M774-A775, OncoCarta neg

Excellent Correlation with

Previous Results using

<240 ng DNA per sample



.

.

• Mutation patterns aligned with literature

• Interesting mutations such as DDR2 in squamous NSCLC

• Plan to expand cohort to > 100 patients

• Squamous carcinoma, Sarcomatoid forms, Small cell lung cancers (SCLC)

LungCarta™ Evaluation at Paul Brousse


LungCarta™ is for Research Use Only. Not for Use in Diagnostic

Procedures.

Clinical Specimen:

Variability in Quality and Quantity

.

Beading et al., J.Mol.Diag.13(5). 2011

Figure 1. Sensitivity of mass-

spectrometry–based mutation

detection.

A: Specimen with 60% KIT K642E

mutant allele frequency was

diluted twofold into

wild-type DNA, with 10 ng total

DNA in each dilution.

B: The amount of input DNA was

titrated from 20 ng to 0.312 ng.

Mutant and wild-type (wt) alleles

are indicated by arrows.


iPLEX® Pro Sample ID Panel*

*iPLEX® Pro Sample ID Panel is for Research Use Only. Not

for Use in Diagnostic Procedures. Not intended for forensic,

parentage, ethnic identification, or monozygotic twins.

� Comprehensive SNP coverage for

sample identification: 40+ SNPs for

a high degree of discriminatory

power > 1.00 x 10-18

� Includes sex determination markers

� Extracted gDNA from FFPE tissue,

blood, NGS libraries

� High-throughput single reaction

panel with <10ng input DNA

� Real competitive PCR assays (5)

assessing DNA quality and absolute

# of amplifiable template copies

� Automated reports for local or

global sample matching.18 | Improving healthcare through revolutionary genetic analysis solutions

The Importance of Sample Identification in

Research Studies

� Laboratory procedures can lead to mistaken identity

� Samples can be mislabeled or contaminated

� Incorrect samples can lead to erroneous data resulting in repeat experiments, time delays, and/or retraction of publications

� By 2008, US researchers stored 270 million specimens in biobankswith a rate of 20 million new samples per year

� The challenge of the research community is to ensure correct samples are used and reported in research studies

.

Lab Mistakes Hobble Cancer

Studies But Scientists Slow to

Take Remedies, July 2012


Quality Control Check Provided for Each Sample

.

Quickly pass or fail samples to reduce time & material costs

� Not enough SNPs for an accurate match or mismatch (N = 30)

� Not enough amplifiable copies (N < 500, 1 ng)

� Key for qualifying formalin-fixed paraffin embedded tissue

� Don’t waste sample, time, or reagents that would not be suitable

for a MassARRAY®* or NGS run


iPLEX® Pro Sample ID Reports

.

� Perfect match: +10

� Penalty scores for any mismatches

by individual SNPs

� Tumor to normal comparison

excludes any germline

heterozygote SNP calls for LOH

Quickly assess sample matches or mismatches for all 44 SNPs at a glance

Amplifiable template copy number

� Real Competitive PCR assays (5) in

stable Albumin gene

� Absolute number of intact DNA

template available for

amplification


iPLEX® Pro Sample ID Panel is not intended for forensic, parentage, ethnic identification, or monozygotic twins.

Applications for Sample Identification

� Cell line authentication

� Chain of Custody events

� Sample replicate pairing

� Tumor vs. non-tumor pairing

� Sample QC

� Library barcoding


Publications using Sample ID


Lung Adenocarcinoma Testing Paradigm

Test for KRAS

Mutations

Cytotoxic

Chemotherapy

(15-30%)

Test for EGFR

Mutations

Test for

EML4-ALK

translocation

Test for other

Mutations ?*

EGFR Tyrosine

Kinase

Inhibitor (10%)

ALK Kinase

Inhibitor

(5-10%)-

+

-

+-

+

Test for acquired

resistance (e.g.

EGFR_T790M)

(*) “Other” includes

BRAF, MEK1, AKT1, PIK3CA,

DDR2……and/or intensive

research to identify

new driver mutations

Image based on Horn L and

Pao W: EML4-ALK: Honing in

on a new target in non-small-

cell lung cancer. J Clin Oncol

27:4232-4235, 2009


OncoFocus: EGFR/KRAS/BRAF/NRAS


4 Genes / 200+ Mutations (SNP&INDEL) / 12 Reactions / 120ng

Gene Mutations Detected with the EGFR/KRAS/BRAF/NRAS Panel EGFR

(Missense)R108K, T263P, A289V, A289D, G598V, E709K, E709Q/H, E709A, E709G, E709V, E709fs*1, G719S, G719C, G719A, G719D, E746K, E746V, L747P, L747S,

T751I, S752P, S752Y, P753Q, P753S, I759N, D761N, D761Y, S768I, S768N, D770N, R776C, R776H, T790M, T854A, L858M/K/R, L858R, L861Q, L861R

EGFR

Exon 19

K745_E749del, E746_E749del, E746_A750del, E746_T751del, E746_T751>I, E746_A750>IP, E746_T751>IP, E746_S752>I, E746_T751>A,

E746del/I744_K745insKIPVAI1, E746_S752del, E746_T751>I, E746_P753>IS, E746_T751>Q, E746_A750>QP, E746_T751>L, E746_P753>LS,

E746_T751>S, E746_T752>A, E746_T751>V, E746_T751>VAorVP, E746_P753>VS, E746_S752>V, E746_A750>VP, E746_T751>VA, E746_T751>VP,

E746_P753>VQ, E746V/K745_E746insVPVAIK1, E746_S752>D, L747_A750>P, L747_T751>Q, E746_A750>DP, L747_T751>P, L747_S752>Q,

L747_S752>QH, L747_E749del, L747_S752del/L747_S752>Q, L747_P753>Q, L747_T751>A, L747_K754del/L747_K754>N1,

L747_T751>S,L747_T751del,L747_P753>S, L747S/L747_K754>ST1, T751_I759>N, T751_I759>REA, T751_I759>S1, T751_I759del1, S752_I759del,

P753_I759del, T751_I759>S1

EGFR

Exon 20

M766_A767insAI, A767_S768insTLA1, S768_V769>IL1, V769_D770insMASVD, D770_P772>ASVDNR, V769_D770insCV1, V769_D770insASV,

D770_N771>AGG, V769_D770insASV1, V769_D770insGSV/V769_D770insGVV/D770>GY1, D770_N771insG, D770_N771insAPW, D770_N771insGL,

N771>GF, N771>GY, D770_N771insG/D770_N771insGD1, D770_N771insSVD, N771_P772>SVQNR, N771>TH, N771>SH, D770_N771insMATP1,

H773_V774insNPH, H773_V774insH, H773_V774insPH, H773_V774insQ1, V774_C775insHV, N771_P772insN1, D770fs*611,

N771_P772insRH/P772_H773insTHP1, P772_H773insV1, P772_H773insHV1, H773>NPY1, V774_C775insHV1

BRAF D594G, D594V, G469S, G469E, G469A, G469V, G469R, G469R/S, L597Q, L597V, L597R, L597S, V600E, V600K, V600M, V600L

KRASG12S, G12R, G12T, G12V, G12F, G12P, G12A, G12C, G12W, G12D, G12N, G12I, G12L, G12Y, G12E, G12D/V, G13C, G13S, G13A, G13V/I, G13D/N, G13R,

A59T, Q61K, Q61E, Q61L, Q61R, Q61P, Q61H

NRAS G12S/N, G12R/P, G12C/Y, G12D/E, G12A, G12V, G13S/N, G13R, G13C/Y, G13D, G13A, G13V, Q61H, Q61L, Q61R, Q61P, Q61K, Q61E, Q61Q/K

1INDELs detected in 1 direction only

OncoFocus + Custom Content


EGFR/KRAS/BRAF/NRAS + IMPACT Extension

• 16-18 Reactions = <200ng/sample

• IMPACT – Individualised Molecular Profiling for Allocation to

Clinical TrialsGene Mutations Detected with the OncoFocus+IMPACT Extension PIK3CA

S326F, N345K, R38H, E418K, C420R, P539R, E542K, E542Q, E542V, E545A, E545G, E545K, E545Q, Q546E, Q546H, Q546K,

Q546P, Q546R, H701P, R88Q, R88Q, C901F, M1004I, G1007R, Y1021C, Y1021H, R1023Q, T1025A, T1025I, T1025S,

A1035T, A1035V, Y1038C, M1043I, M1043V, N1044K, H1047R, H1047Y, G1049R, I1058F, H1065L, N1068fs*4

KIT

Y503_F504>AY, D52N, K550_K558del, P551_V555del, M552L, Y553_Q556del, Y553_Q556del, W557G, W557R,

K558_E562del, K558_V560del, V559_V560del, V559A, V559D, V559del, V559G, V559I, V560D, V560G, V560del, E561K,

Y568D, Y570_L576del, L576P, D579del, F584S, P585P, K642E, D816H, D816V, D816Y, V825A, E839K

PTENR130*, R130fs*4, R130G, R130Q, R173C, R173H, R233*, P248fs*5, K267fs*9, V317fs*3, N323fs*2, N323fs*21, R335*,

K6fs*4

MET R970C, T992I, Y1230C, Y1235D, M1250T

RETF612_C620del, D631_L633>E, D631G, E632_A640>VRP, E632_L633>V, E632_L633del, C634R, C634W, C634Y, A664D,

E768D, A883F, D898_E901del, M918T

ACK1 S985N

OncoFocus


• 12 Reactions = <250ng/sample (FFPE)

• 4 Genes/200+ mutations

• EGFR

– Exon 19 + 20 INDELS - >99% coverage of mutations in COSMIC; >95%

bi-directional confirmation

– 19 Missense mutations in 13 residues

• BRAF/KRAS/NRAS

– BRAF; 14 missense mutations in 4 residues (V600/G649/D594/L597)

– KRAS; 16 missense mutations in 4 residues (G12/G13/A59/Q61)

– NRAS; 18 missense mutations in 3 residues (G12/G13/Q61)

OncoFocus Plus


• 16 Reactions = <300ng/sample (FFPE)

• 10 Genes/>300 mutations

• EGFR/BRAF/KRAS/NRAS

• PIK3CA - 42 missense mutations in 26 residues

• KIT - 21 missense mutations in 14 residues + 12 INDELs

• PTEN - 14 missense mutations in 10 residues

• MET - 5 missense mutations in 5 residues

• RET - 8 missense mutations in 6 residues + 6 INDELs

• ACK1 - S985N

FFPE Clinical Research Study


55 FFPE samples + 13 Cell Lines with known

mutation signatures

58/64 known mutations identified• 31 Unique mutations in sample set

• 3/6 unidentified mutations <10% frequency

• 6/6 unidentified mutations in highly

fragmented samples (<100 intact template)

25 novel mutations identified• 24/25 novel mutations identified in highly

fragmented samples (<100 intact template)

Mutational Spectrum


0

1

2

3

4

5

6

7

8

BRAF G469EBRAF L597Q

BRAF V600E

BRAF V600K

BRAF K601E

BRAF K601N

EGFR G719S

EGFR E746_A750del

EGFR E746_S752>I

EGFR E746_S752>V

EGFR E746del

EGFR E746K

EGFR L747_P753>Q

EGFR L747_P753>S

EGFR T751_I759>S

EGFR D761N

EGFR S768_V769>IL

EGFR S768IEGFR V769_D770insASV

EGFR D770_N771insSVDEGFR T790M

EGFR L858R

KIT W557G

KIT L576P

KIT K642E

KIT D816Y

KRAS G12C

KRAS G12D

KRAS G12V

KRAS G13D/N

KRAS G13S

KRAS Q61R

NRAS G12C/Y

NRAS G13C/Y

NRAS Q61H

NRAS Q61K

NRAS Q61LNRAS Q61R

Series1

Series2

Confirmed

Mutations

All Detected

Mutations

Resistance – A Sensitivity Challenge

• High-sensitivity detection may aid in personalizing therapy based on a patient’s mutational status

• Detection of low-level mutations does not always correlate with later clonal expansion in patients treated with Tyrosine Kinase Inhibitors (TKIs)1:

• Mutant forms of BCR-ABL (T315I), KIT (T670I), and EGFR (T790M) confer resistance TKIs imatinib, gefitinib, and erlotinib

• Need to balance sensitivity vs. clinically relevant load in source tissue

1. Willis SG, Lange T, et al. High-sensitivity detection of BCR-ABL kinase domain mutations in imatinib-naive patients: correlation with clonal cytogenetic evolution but not response to therapy. Blood. 2005;106 (6): 2128-2137.


Single Allele Base Extension Reaction


T C A T C A T C AG CG T C A T G C C C T T C G G

TT790M EXT

G Control EXT

G

T C A T C A C C AG CG T C A T G C C C T T C G G

T C A T C A C AG CG T C A T G C C C T T C G GT

T C A T C A C C AG CG T C A T G C C C T T C G G

Control EXT

No Extension

WT DNA

T790M DNA

X

WT DNA

T790M DNA

SABER

Mass spectrometry

GUEP

Inte

nsity

Control

No peak

UEP

Inte

nsity

WT

TUEP

Inte

nsity

T790M

0

10

20

30

40

50

60

70

80

5100 5150 5200 5250 5300 5350 5400 5450

C T

...0M_Control#1

EGFR_T790M_Control#1

Intensity

Mass

100%

0

5

10

15

20

25

30

35

40

45

50

55

60

65

5100 5150 5200 5250 5300 5350 5400 5450

C T

...0M_Control#1


Intensity

Mass

9.1%

0

10

20

30

40

50

60

70

5100 5150 5200 5250 5300 5350 5400 5450

C T

...0M_Control#1


Intensity

Mass

1.2%

0

10

20

30

40

50

60

5100 5150 5200 5250 5300 5350 5400 5450

C T

...0M_Control#1


Intensity

Mass

0.6%

0

5

10

15

20

25

30

5100 5150 5200 5250 5300 5350 5400 5450

C T

...0M_Control#1


Intensity

Mass

0.3%

0

10

20

30

40

50

60

70

5100 5150 5200 5250 5300 5350 5400 5450

C T

...0M_Control#1


Intensity

Mass

DW

0

10

20

30

40

50

60

70

80

90

5100 5150 5200 5250 5300 5350 5400 5450

C T

...0M_Control#1


Intensity

Mass

gDNA

0

5

10

15

20

25

30

35

40

45

50

55

5100 5150 5200 5250 5300 5350 5400 5450

C T

...0M_Control#1


Intensity

Mass

H1975

Sensitivity Required for Plasma Analysis

• Noninvasive prenatal diagnostics

– On average 1,400 molecules of any DNA sequence present per ml

– 4% fetal DNA is about 60 molecules: hence 4% ± 1.35% detection

sensitivity suffices but quantification is impossible without

haplotyping

• Noninvasive cancer diagnostics

– Early stage cancer assume a comparable number of molecules of

any DNA sequence per ml: 1,400

– Hence 0.21% detection sensitivity will detect almost anything

with 95% confidence but quantification will require haplotyping


Importance of Sensitivity:

Detection of EML4-ALK – Clinical Research

.

A Novel Mass Spectrometry-Based Assay for

Diagnosis of EML4-ALK-Positive Non-Small

Cell Lung Cancer

Kazuko Sakai, Isamu Okamoto, Ken Takezawa, Tomonori Hirashima,

Hiroyasu Kaneda, Masayuki Takeda, Kazuko Matsumoto, Hideharu

Kimura, Yoshihiko Fujita, Kazuhiko Nakagawa, Tokuzo Arao, and Kazuto

Nishio

Vol 7, No 5, 913-918 May 2012

Journal of Thoracic Oncology O R I G I N A L R E P O R T


Detection of EML4-ALK Fusion Variants in

FFPE Samples

• Sensitive single base mass extension assays across the

fusion boundary

• Substrate is total RNA from FFPE samples of non-small-

cell lung cancer patients

• Typically 5-10% of patients are reported to have EML-ALK

fusions and are sensitive to Crizotinib

• MassARRAY System* detected variant 1 in 3/20 FFPE

biopsies

Sakai et. al., J Thoracic Oncology 7:913 (2012) Kinki University

*The MassARRAY® System is for Research Use Only. Not for Use in Diagnostic Procedures.


EML4-ALK


• 5-10% frequency in non-smokers

• EML4-ALK variants 1 and 3 account for ~60% of EML4-ALK-positive cases of NSCLC

• ALK TKIs approved for use – Crizotinib

EML4-ALK – Design Strategy


EML4 WT

EML4-ALK

ALK WT

T

C

iPlex

iPlex

EML4:MUT

MUT SABER

G

X

A

SABER

SABER

iPlex

EML4-ALK – sensitivity test

.

EML4-ALK – FFPE & Cytology Matched Sample

V1: E13;A20

43%

iPlex EML4

SABER ALK

V1: E13;A20

Positive

V1: E13;A20

Cytology Spec

100%

iPlex EML4


EML4-ALK – FFPE Clinical Research Study


SAMPLE Sequenom RTPCR* MUT:EML4 MUT:ALK ALK WT

1 V1 V1 V1 (60%) V1 +

2 V1 V1 V1 (60%) V1 +

3 NR V1 NR NR +/-

4 V2 V2 V2(60%) V2 -

5 V2 V2 V2 (70%) V2 -

6 V1 + L1196M V1 c.4493C>A (46.4%) + V1 (60%) L1196M_C4493A + V1 +

7 NR NR NR NR -

8 NR V2 NR NR -

9 V3a NR V3a V3a +

10 V2 V2 V2 (60%) V2 -

11 V5Alt WT V5Alt(70%) V5Alt -

14 V1 V1 V1 (60%) V1 +

15 V1 TBD V1 (60%) V1 +

17 NR TBD NR NR -

18 NR TBD NR NR -

19 V1 TBD V1 (60%) V1 -

NR=No Result WT=Wild Type

All Samples ALK FISH positive*Kwak EL et al. Anaplastic Lymphoma Kinase Inhibition in Non–Small-Cell Lung

Cancer. N Engl J Med. 2010 October 28; 363(18): 1693–1703

Assays by Sequenom (AbS)*–

Customer Collaborative Panels

*Assays by Sequenom is for Research Use

Only. Not for Use in Diagnostic Procedures.

.

Pancreatic AbSPancreatic Cancer

140+ mutations in

42 genes

Colon AbSColon Cancer

32+ mutations in 7

genes

OncoCarta v3105+ Mutations in

22 genes


MelaCarta AbSMelanoma

70+ Mutations in

17 genes

Gynecology AbSOvarian + Uterine

92+ Mutations in

12 genes

OncoCarta v2152+ Mutations in

18 genes

RW9

Slide 41

RW9 Please only list the Assay by Sequenom that have been custom developed. If Gyencology Abs, Pancreatic AbS and Colon AbS and OncoCarta v3are 'pipeline' products, you must delete from this slide.Robin Weiner, 3/25/2013

Adverse drug reactions a leading cause of

death globally2

Identifying a sub-group of patients likely to

respond can dramatically increase the cost-

effectiveness of a drug1

>$750B annual global drug sales in 20091

$350B annually spent on ineffective

medicines globally1

90% of drugs work in 30-50% of individuals1

Building the Case for Personalized

Medicine

Need for ChangeStrong case for personalized medicine

Many major Pharma have biomarker programs for drug development!1. McKinsey Quarterly: The microeconomics of personalized medicine, February 2010

2. Lazarou J, Pomeranz BH, Corey PN. “Incidence of adverse drug reactions in hospitalized patients: a meta analysis of prospective studies.” JAMA. 1998; 279:1200-1205; Gretchen E. Parker, PhD, RAC,

Usha Srinivasan, MS, RAC and Gretchen M. Bowker, MS, RAC, Overcoming Challenges through Innovation and Regulation


Defining the Gene & Variants in the

iPLEX® ADME Pharmacogenetics (PGx) panel

Absorption

Distribution

Metabolism Excretion


Validated Markers (FDA)

http://www.fda.gov/Drugs/ScienceResearch/ResearchAreas/Pharmacogenetics/ucm083378.htm

Gene Drug Treatment

CYP2C19 Clopidogrel

Warfarin

antiplatelet agent

anticoagulant

Voriconazole treat serious, invasive fungal infections

Prasugrel a novel platelet inhibitor

CYP2C9 Celecoxib treatment of osteoarthritis, rheumatoid arthritis, acute pain, painful

menstruation and menstrual symptoms

Warfarin anticoagulant

CYP2D6 Atomoxetine treatment of attention-deficit hyperactivity disorder (ADHD)

Fluoxetine HCL antidepressant

Codeine sulfate Cough Diarrhea Mild to moderate pain usw.

NAT Rifampin bactericidal antibiotic

TPMT Azathioprine suppresses the immune system(organ transplantation)

UGT1A1 Irinotecan treatment of cancer

Nilotinib chronic myelogenous leukemia (CML)

VKORC1 Warfarin anticoagulant


Haplotypes Detected with iPLEX ADME Pharmacogenetics panel

.

Genes GenesHaplotypes Haplotypes

iPLEX® ADME PGx panel is for Research Use Only. Not for use in diagnostic procedures.


Screening Approach: iPLEX ADME PGx panel

• Quickly and easily screen known, high-value mutations

• 192 mutations in 36 pharmacogenetically relevant genes

• Biologically relevant data with ADME PGx panel covering

>99% of Pharma Core list*

• Identify SNPs, insertions /deletions and copy number

variants (CNVs)

• Rapid genotyping assay

• Cost effective – 8 wells for 200 assays

• Proven technology1,2,3

*list available at http://www.pharmaadme.org/joomla/

1. Elke Schaeffeler et al., Highly Multiplexed Genotyping of Thiopurine S-Methyltransferase Variants Using MALDI-TOF Mass Spectrometry: Reliable Genotyping in Different Ethnic Groups

2. Fabiana BK, et.al., Molecular diversity oat the CYP2D6 locus in healthy and schizophrenic southern Brazilians.

3. .Yan Shi, et.al., Analysis of50SNPsin CYP2D6, CYP2C19, CYP2C9, CYP3A4 and CYP1A2 by MALDI-TOFmass spectrometry in Chinese Han population.



Sequenom EpiTYPER applications

Quantitative DNA methylation

analysis in cancer


Overview of Sequenom’s

EpiTYPER® Technology

• Flexible & scalable for investigating a few or several hundred regions over multiple samples

• Long reads up to 600 bp in one reaction enable discovery of differential methylation within large promoter regions

• Robust analysis from a range of sample types including Formalin Fixed Paraffin Embedded (FFPE) samples


EpiTYPER for Quantitative DNA Methylation

Analysis Using the MassARRAY System

.

Sequenom Application Note

Introduction to EpiTYPER for

Quantitative DNA Methylation

Analysis Using the MassARRAY

System

Mathias Ehrich, Devan Correll, and

Dirk van den Boom

Doc. No. 8876-007 R02

CO 060109


Potential Large Scale Application of EpiTYPER for

Characterizing DNA Methylation in NSCLC

Overall Goal: Present a previously uncharacterized

method for high-throughput DNA methylation

analysis that utilizes MALDI-TOF mass spectrometry

Methodology:

Use the IGF2/H19 region to show that a single

base-specific cleavage reaction is sufficient to

discover methylation sites and to determine

methylation ratios within a selected target region

Results:

• Successfully applied this method under high-

throughput conditions to quantitatively assess

methylation differences between normal and

neoplastic lung cancer tissues from 48

patients in 47 genes

• Demonstrate that quantitative methylation

results allow accurate classification of samples

according to their histopathology


Colorectal Cancer Cases can be Classified

into Distinct Epigenotypes

Overall Goal: Gain insight into epigenotype

development in colorectal carcinogenesis,

especially intermediate-methylation and its

correlation to KRAS-mutation (+) in adenoma

Methodology:

• Use EpiTYPER to analyze the methylation

status of a new cohort of adenoma, crypt

foci, and normal mucosa samples

• Use iPLEX to analyze BRAF 1799 and KRAS

34, 35, and 38 mutation status

Results:

• Significant correlation with intermediate-

methylation epigenotype and positive KRAS

mutation status

• Epigenotype development occurs at an

earlier stage than carcinoma formation and

is already completed at adenoma stage


Use of EpiTYPER Technology to Assess

Methylation Status of KLF5 in AML

Overall Goal: To show that expression of KLF5 is a requirement for

granulocyte differentiation of 32D cells

Methodology:

• qPCR showed reduced KLF5 expression significantly lower in

AML samples compared to controls

• EpiTYPER used to analyze methylation of 150 CpGs in a 4.4 kb

region spanning the proximal promoter to exon2 of KLF5

Results:

• In selected AML cases, reduced expression is associated with

hypermethylation of the KLF5 locus in the proximal promoter

and/or intron 1, suggesting that this may represent a Class II

genetic lesion in the development of AML

• DMR1 in the proximal promoter was hypermethylated in 31%

of AML patients

• DMR2 in the first intron was hypermethylated in 41% of de

novo AML samples

• Inverse correlation between methylation and expression could

be confirmed with demethylating agents


Use of EpiTYPER Technology to Confirm a

Breast Cancer Methylation Biomarker

Overall Goal: Map MicroRNAs (miRNAs) within the previously described promoter (P1) and

a novel promoter Has-mir-200b cluster (P2) and their role in breast cancer

Methodology:

• Use HRM to assess methylation in 15 promoters in breast cancer cell lines

• Use EpiTYPER to analyze methylation in miR-200b promoter in Grade 3 FFPE samples

Results:

• P1 and P2 were differentially methylated and inversely correlated to expression

• Data suggests an association between miR-200b regulation and breast cancer subtypes

• Potential use of DNA methylation in miRNA promoters as a breast cancer biomarker


Use of EpiTYPER Technology to Validate

Methylation Differences in Ovarian Cancer

.

Overall Goal: Identify epigenetic-based biomarkers for diagnosis of ovarian cancer

Methodology:

• Use genome-wide methylation (MeDIP) to discover epigenetic-based biomarkers in A2780 and CaOV3

ovarian cancer cell lines

• Use EpiTYPER to validate methylation differences within 21 gene promoters identified by the MeDIP-Chip

Results:

• A panel of 6 genes (ARMCX1, ICAM4, LOC134466, PEG3, PYCARD & SGNE1) were shown as a potent

discriminator of cancer vs. normal

• A putative pseudogene of ZNF300 was identified as an individual discriminator

• The potential biomarker panel can hold great promise as a diagnostic test for high-grade (Type II) serous

ovarian cancer


miRNA Expression and Methylation Changes

Common in Oral Squamous Cell Carcinoma

.

Overall Goal: Analyze epigenetic regulation and expression of miRNAs in oral squamous cell

carcinoma (OSCC)

Methodology:

• Use TaqMan miRNA low density arrays to perform initial screen of non-metastatic and

metastatic tumors and matched control tissue

• Use EpiTYPER to further evaluate 10 miRNAs in a larger sample set

Results:

• miRNA expression patterns were consistent in healthy oral epithelium and stroma, but

broadly altered in both tumor and adjacent tissue from OSCC patients

• “A broad spectrum of CpG methylation patterns were observed, ranging from

completely methylated to unmethylated, demonstrating the dynamic range of the

MassARRAY® System”

• Five miRNAs were identified as promising candidates for further investigation


Verification of Genes Frequently Silenced by

Methylation in Melanoma

.

Overall Goal: To identify and confirm novel candidate genes frequently methylated in

melanoma

Methodology:

• Integrate gene expression and methylation array analysis to identify candidate

genes

• Use EpiTYPER to validate the methylation status of the most promising tumor

suppressor genes as a follow up to methylation arrays

Results:

• UCHL1, COL1A2, THBS1, and TNFRSF 10D inversely correlated with promoter

methylation

• Identification of candidate TSGs and future research will increase understanding

of etiology of melanoma and provide tools for its early diagnosis


EpiTYPER Summary

• EpiTYPER method provides a research tool for discovering methylation status in targeted regions

• EpiTYPER method provides a highly useful research tool for verifying key regions for their methylation status across larger sample sets

• EpiTYPER method can be used as a cross-platform tool for array validation and/or correlation to expression

• Recent publications suggest the value of methylation status as a potential biomarker or for future diagnostic use


MassARRAY® System* – Proven Research Tool

for Biomarker Validation and Translation

• Very high quality data: quantitation, sensitivity, precision

• Variety of biomarkers: SNPs, somatic mutations,

amplifications, transcript profiles, methylation markers,

Viral/pathogen detection

• Easily design assays against your biomarkers

• Cost effective, high sample throughput capability

.

*The MassARRAY® System is for Research Use Only. Not for Use in Diagnostic Procedures.


• Proven technology for discovery,

confirmation, and mature panels

for screening studies

Documents

MassARRAY System –a proven technology for translational ... · Lianxing Yang PhD Senior Scientist –Applications and Technology September 2012 The MassARRAY® System is for Research