The Role of Pathology/Molecular

Diagnostics in Personalized Medicine

Ignacio I. Wistuba, M.D.

Jay and Lori Eissenberg Professor in Lung CancerDirector of the Thoracic Molecular Pathology Lab

Departments of Pathology andThoracic/Head & Neck Medical OncologyM. D. Anderson Cancer Center

Conflict of Interest

• Honoraria: Sanofi Aventis, Johnson &Johnson, Genentech, Champions Inc.,Roche, AstraZeneca, and Bristol-MyersSquibbs

• Research Agreements: Genentech,Pfizer, AstraZeneca, Myriad, Eli-Lilly,and Merck.

Traditional

NSCLC Landscape Change - 2011

Adenocarcinoma

Squamous

Large Cell

BRAFAKT

VEGFRHER2

EPHA/B

PDGFR

FGFR

INSR

PI3K

MAPK

KRAS

EGFR

ALK

Unknown

Adenocarcinoma

Unknown

FGFR1

Amp

EGFRvIII

PI3KCA

EGFR TK

DDR2

Squamous Cell Ca

Lung Cancer Targeted TherapyLandscape Change - 2011

• Histology- Non-squamous 60% Bevacizumab

Pemetrexed

• Adenocarcinoma- EGFR mutation 10% Erlotonib/Gefitinib- ALK-EML4 fusion 3% Crizotinib- MET amplification 20%- PI3KCA mutation 5%- HER2 mutation 1%

• Squamous Cell Carcinoma- FGFR1 amplification 22% FGFR TKIs- EGFRvIII mutation 5% EGFR TKIs- PI3KCA mutation 4% PI3KCA inhibitors- DDR2 mutation 3% Dasatinib & Nilotinib

EGFR Mutations in NSCLC

18

19

21

20

!C- helix

P- loop

A- loop

Deletions - 46%

L858R - 39%

Duplications/Insertions - 9%N-lobe

C-lobe

Extracellular domain

Regulatorydomain

ATP binding cleft TK

Domain

Treatment by subgroup interaction test, p<0.0001

EGFR mut ( + ) EGFR Mut ( - )

HR = 0.48P<0.0001

Gefitinib (n=132)Carboplatin/Paclitaxel (n=129)

HR = 2.85

P<0.0001

Pro

bab

ilit

y o

f p

rog

ressio

n-f

ree

su

rviv

al

0 4 8 12 16 20 240.0

0.2

0.4

0.6

0.8

1.0

Pro

bab

ilit

y o

f p

rog

ressio

n-f

ree

su

rviv

al

Gefitinib (n=91)Carboplatin/Paclitaxel (n=85)

0 4 8 12 16 20 240.0

0.2

0.4

0.6

0.8

1.0

Months Months

Mok TS et al. N Engl J Med 2009;361:947-957

IPASS Trial

EML4-ALK in Lung Adenocarcinoma2p23 region

ALK

EML4

Protein (IHC)FISHHistology

60

40

20

0

–20

–40

–60

–80

–100

Progressive disease

Stable disease

Ma

xim

um

Ch

an

ge

in

Tu

mo

r S

ize

(%

)

–30%

Partial response

Complete response

Kwak EL et al. N Engl J Med 2010;363:1693-1703

• ORR: 57%• DCR at 8 weeks: 87%

Crizotinib in EML4-ALK (+)

Lung Adenocarcinoma and NSCLCWhat is new in 2011?

• All KRAS mutations are not the same

• Mutations data of a panel of genes(multiplex analysis) are available

• Gene signatures associated to EGFR TKIresponse in EGFR wild-type tumors

• Mechanisms of EGFR TKI resistance

KRAS Mutations in Lung Adenocarcinoma

Codon 12 Codon 13Base change 1G2G3T 1G2G3C Amino Acid (replacing Gly)G"T (72%) 1Cys (45%) 1Cys (5%)

2Val (20%)G"A (14%) 2Asp (12%) 2Asp (1%)G"C (14%) 2Ala (9%)T"C (2%) 3Cys (2%)

Lodish et al

KRAS Mutation Sequencing

Codon 12 (G12C)Wild Type Codon 13 (G13A)

G G T G G C T G T G G C G G T G A C

All treatments

Months since randomization0 6 12 18 24

Cys/Val n=24Other n=19Wild n=172

P = 0.046

Pro

gre

ssio

n-F

ree S

urv

ival

1.0

0.8

0.6

0.4

0.2

0

Sorafenib

Months since randomization0 6 12 18 24

Cys/Val n=11Other n=9Wild n=66

P = 0.026

Pro

gre

ssio

n-F

ree S

urv

ival

1.0

0.8

0.6

0.4

0.2

0

BATTLE Trial Novel Findings:Not All KRAS Mutations Are the Same

N. Ihle ,… G. Powis, et al, AACR 2011

mut-KRAS C or V other

Gene Expression Analysis

BATTLE Trial Novel Findings:Not All KRAS Mutations Are the Same

Mut-KRAS C or V other

RPPA Analysis NSCLC Cell Lines

K-Ras

Akt Mek

p70S6K

mut-KRas-G12D

K-RAS

Akt Mek

p70S6K

RalGDS

mut-KRas-G12C

K-RAS

Akt MekRalGDS

p70S6K

wt-KRas

N. Ihle ,… G. Powis, et al, AACR 2011

Mutations(8 genes/115 Assays)• AKT1

• BRAF

• EGFR

• HER2

• KRAS

• MEK1

• NRAS

• PIK3CA

FISH• C-MET amplification• EML4-LK fusion

GO Grant Panel1,000 Adenocarcinomas

Lung Cancer Mutation Consortium

51682

294

172

FISH

Pending

July, 20111,234 Consented

1,063 Studied

Mutation

B. Johnsons and the LCMC co-investigators, WCLC 2011

Fine NeedleAspiration (FNA)

Core Needle Biopsy (CNB)

Advanced Tumor

Types of Histology and Cytology Specimens

Surgical Resection

Histology

Formalin-fixed and Paraffin-embedded (FFPE)

Endobronchial Ultrasound(EBUS) or Pleural Fluid

Alcohol-fixed

Alcohol-fixed – Cell Block

Alcohol-fixed

Multiplexed Mutation AssaysMultiplex PCRTumor Tissue

Resected Specimen Core Biopsy

SNaPshot® (Applied Biosystem)

Dias-Santagata, EMBO Mol Med 2:146, 2010

10% Sensitivity and ~20ng DNA/multiplex reaction

Mass ARRAY SNP - Sequenom, Inc

!"#$%&'&("!#

)*&*+&*)

*$,-.',/

01

!"#$%&'

()*+,#%&'

-./%#-(-.*0

1"#2

#*/0

/2'3

451

*672

#891

Lung Cancer Mutation ConsortiumIncidence of Mutations Detected

• Mutation found in 54% (280/516) of tumorscompletely tested

HER 2

B. Johnson. P. Bunn (PI) and the LCMC co-investigators, WCLC 2011

Mutation Analysis by MassARRAY®

Sequenom® - NSCLC

BRAF (G464-G1391)

Wild-typeMutant

(Heterozygous)

Wild-typeMutant

(Homozygous)

BRAF (G469-G1406)

H. Erickson, E. Kim, and I. Wistuba, 2011

Well Spectrum

Data Analysis

BATTLE-1 Clinical TrialPlatform for future research

• Clinical trial program (Phase II)

• Novel trial design

• Biopsy-mandated study in lung cancer

• Biomarker discovery

Accrual (3 yrs for accrual; Nov 2006 to Oct 2009)

• Biopsies obtained 324

• Patients randomized 255

• Patients evaluable 244

Kim et al, Cancer Discovery, April 2011

Molecular Profiling

• Frozen tumor: mRNA Affymetrix & proteomic (RPPA)

• Serum and PBMCs

Erlotinib SorafenibVandetanib Erlotinib + Bexarotene

Randomization:Equal ! Adaptive

Primary end point: 8 week Disease Control (DC)

Patient Enrollment

Personalized Therapy in AdvancedNSCLC: BATTLE-1 Schema

EGFR KRAS/BRAFVEGF RXR/CyclinD1

Core Biopsy

BiomarkerProfile

E. Kim et al, Cancer Discovery 2011

The BATTLE-1 Gene ExpressionSignatures Scorecard

Signature Derived Validation Predictive?

Pathway Signatures

EGFR 3 Clinical sets3 Clinical sets

1 Cell line set

Yes: EGFR mutations

Yes: outcome in resected NSCLC with wt

EGFR (prognostic)

No: erlotinib response in wt EGFR patients

KRAS 2 Clinical sets1 Clinical set

1 Cell line set

Yes: KRAS mutations in BATTLE

No: erlotinib response in wt EGFR patients

EMT Cell lines 2 Clinical sets Yes: erlotinib DC in wt EGFR

Clinical signatures

5 geneWEE

BATTLE Cell lines Yes: erlotinib DC in wt EGFR patients

Heymach, Saintigny, Byers, Kim, et al, AACR 2011

TRIM72

NPR3

C5orf23

LCN2

OGG1

8wDC: Yes

8wDC: No

8wDCYes

8wDCNo

P<0.001

Sig

natu

revalu

e

8w

DC

(%

)Signature +P<0.001

Signature -

P=ns

Sorafenibarm

Erlotinibarms

Test forinteractionP=0.023

Log-rank <0.001

HR=0.1295 CI 0.03-0.46

P=0.001Pro

bab

ilit

y o

fsu

rviv

al

PFS time (months)

Median PFS: 12.5 weeks vs.

7.2 weeks

Signature -

Signature +

Saintigny et al, AACR 2011

5-Gene Signature Predicts 8-wk DC inEGFR Wild-type Tumors

BATTLE-1 mRNA Profiling (n=139)

Fig. 1 The frequency of observed drug resistance mechanisms.

Sequist L V et al. Sci Transl Med 2011;3:75ra26-75ra26

Mechanisms of Resistance to EGFR TKI

Novel Mechanisms

• T790M Mutation – 49%

• MET amplification – 5%

• PI3KCA Mutation - 5%

• EMT Changes – 5%

• SCLC Features – 14%

• Unknown – 30%

Mechanisms of Resistance to EGFR TKI

Sequist L V et al. Sci Transl Med 2011;3:75ra26-75ra26

Adenocarcinoma SCLC

H&E Synaptophysin H&E Synaptophysin

Traditional

Squamous Cell Lung CarcinomaLandscape Change - 2011

Adenocarcinoma

Squamous

Large Cell

Unknown

FGFR1

Amp

EGFRvIII

PI3KCA

EGFR TK

DDR2

2011

New Target Therapy in SquamousCell Carcinoma of the Lung

Gen Frequency Drug

FGFR1 amplification 22% FGFR TKIs

EGFRvIII mutation 5% EGFR TKIs

PI3KCA mutation 3.6% PI3KCA inhib.

EGFR TK mutation 3.4% EGFR TKIs

DDR2 mutation 3.2% DasatanibNilotinib

Okashi and Pao, Cancer Discovery, April 2011

FGFR1 Amplification in Squamous CellCarcinoma (9%)

Weiss J et al. Sci Transl Med 2010;2:62ra93-62ra93

FGFR1 Copy Number Gain (CNG) Analysisby FISH in NSCLC (n=326)

Tang et al, 2011

0

5

10

15

20

25

!4 !10 Amplification

FGFR1 CNG

Fre

qu

en

cy (

%)

Adenocarcinoma (n=217)

Squamous Cell (n=89)

7%

13%

1%

11%

17%

20%

FGFR1 Copy Number Analysisby FISH in NSCLC

Tang et al, 2011

!10 Copies (ADCA) !10 Copies (ADCA)

!10 Copies (SCC) Amplification (SCC)

DDR2 Gene Mutations in Squamous CellCarcinoma of the Lung (3.2%)

Hammerman et al, Cancer Discovery April, 2011

DDR2 Mutant Tumor Sensitive to Dasatinib

Hammerman et al, Cancer Discovery April, 2011

BRAF Inactivating Mutation AssociateSensitive to Dasatinib

B. Sen,…and F. Johnson et al, unpublished, 2011

Tumor BRAF

Y472C Mutation

What’s the problem?I gave you at least 10 cells!

Fine NeedleAspiration (FNA)

Core Needle Biopsy (CNB)

Advanced Tumor

Tissue is the Emperor -For diagnosis, the pathologist needs some!

Pathology Algorithm of Small Tissue Specimens forManagement of Lung Cancer Patients - 2011

Small Biopsy

SCLC NSCLC – NOS?

Adenocarcinoma Squamous Cell Ca Others

IHC: TTF-1CK 5/7

P63

NSCLCNOS

EML4-ALK

EGFR

Panel:MET ampl

PI3KCA

BRFA

HER2

K-, N-, H-

RAS

MEK

AKT…

FGFR1 Amp

DDR2 Mut

Panel:PI3KCA

BRAF

HER2

K-, N-, H-RAS

MEK

AKT…

EML4-ALK

EGFR

FGFR1 Amp

DDR2 Mut

Panel:MET ampl

PI3KCA

BRAF

HER2

K-, N-, H-RAS

MEK

AKT…

Evolution of Genome SequencingTechnology

1986: Sequencing one genome:

Cost = $3 billion

Time = 1 decade

2010: Sequencing one genome:

Costs = $10 thousand

Time = 3 weeks

2012: Sequencing one genome:

Cost = $1 thousand

Time = 1 week

1mm

Frozen CNB: NSCLC

WT

Seq

T/N

lo

g2

Microarray T/N log2

r=0.48P=10-8

Correlation of WT Seq and mRNA Array Profiling of 11 NSCLCs

Percentage of WT Reads Mapped:10 CNB vs. 10 Resected NSCLCs

% R

ead

s M

ap

ped

CNB Resected

Next Generation of SequencingNSCLC Core Needle Biopsies (CNB)

• Exome Seq (DNA)

• Whole Transcriptome (WT-mRNA)

• Small RNA Seq (miRNA)

Wistuba et al, unpublished, 2011

Molecular Pathology of Lung Cancer: 2011

• New actionable genetic abnormalities continued to bediscovered in NSCLC

• In lung adenocarcinoma and NSCLC:

! All KRAS mutations are not the same

! ~50% of tumors have actionable gene mutations

! Gene expression signatures predict response toEGFR TKI in EGFR wild-type tumors

! Novel mechanisms and changes associated to EGFRTKI resistance

• Squamous cell carcinoma shows distinct geneticabnormalities: FGFR1 amp and DDR2 mutations

• Next generation sequencing shows a promise forapplication to clinical settings

Acknowledgements

• MD Anderson Cancer Center

Thoracic Molecular Pathology Lab

Heidi Erickson

Hector Galindo

Nana Hanson

Christina McDowell

Annette Basey

Chi-Wan Chow

Zuoming Chu

Junya Fujimoto

Lakshmi Kakarala

• MD Anderson Cancer Center

Edward Kim (Medical Oncology)

Waun Ki Hong (Medical Oncology)

Roy Herbst (Medical Oncology)

John Heymach (Medical Oncology)

Scott Lippman (Medical Oncology)

Gordon Mills (System Biology)

Katherine Stemke-Hale (System Biology)

Cesar Moran (Pathology)

Neda Kalhor (Pathology)

Jack Lee (Biostatistics)

Support

• US Department of Defense BATTLE andPROSPECT, Stading Family, Cohen-Reinauch BATTLE-2 Fund and VFoundation