Protein Pathway Mapping: Realizing the Promise

Of Individualized Therapy at the Bedside

Dr. Emanuel Petricoin

George Mason University

Center for Applied Proteomics and Molecular Medicine

Manassas, VA

703-993-864- phone

703-993-4288- fax

epetrico@gmu.edu

Disclosures:

Co-founder of Theranostics Health, Inc

Co-founder of Personalized Cancer Therapy, Inc

Theranostics Health, Inc has licensed IP related to content that will be discussed

DNA: The blueprints

PROTEINS: The Working Machinery

Cancer is driven by hyperactive or defective protein circuits

The components of these circuits contain the drug targets

of the future.

Patient A Patient B

Each patient’s cancer is different. A drug that works for

one patient may not work for another patient with the

same cancer.

Delivering Personalized Therapy Data for Cancer:

Technical Barriers

• Tissue input: Tiny FNA or Core Needle Biopsy: hundreds to thousands of cells not millions to billions

• Tissue preservation and fixation at tertiary sites limited to formalin and lack of proper storage temp.

• Tissue heterogeneity

• Need to deliver both quantitative and multiplexed information about signaling network activation

The Center for

Applied Proteomics

and Molecular

Medicine

Proteomics Tools for

Clinical Medicine

Core needle biopsy 16 gauge needle

12 mm x 2.5 mm tissue

109 cells in tissue section

15,000 – 30,000 tumor cells for microdissection

Fine Needle Aspirate

2 needle passes

1000 – 25,000 cells

Clinical Proteomics:

Some practical issues

CGH

SNP

Affy

Proteomics

Multiple samples/array

One antibody probe/array

Controls, standards, samples on same array

Reverse Phase Protein Arrays: Invented at NIH in 1999 Originating article:

Trained Mills Lab

Trained Nishizuka/Weinstein Lab 100 laboratories

worldwide

• Built-in low and high controls

NEXT GENERATION REVERSE PHASE ARRAY:

CALIBRATED ASSAY FORMAT Each slide contains the following elements:

• Elimination of long dilution curve: lysates printed in

large number of technical replicates: better precision

and accuracy

• Built-in calibrator: Constructed with constant

total protein concentration but with known and

varying analyte concentration

NEW FORMAT PROVIDES ABILITY TO COMPARE

DATA ACROSS EXPERIMENTS AND TIME

EACH SAMPLE REPORTED AS QUANTITATIVE RU VALUE

Flu

ore

scen

ce U

nit

s

Read off value of

unknown patient sample

Patient Samples

Calibrators

Low Control High Control

RU Calibrator

High

Low

Patient X

4E -BP 1 (S65)

4E -BP 1 (T 37 /46 )

4E -BP 1 (T 70 )

4G10 (anti P hosphotyros ine)

c -A bl (T 735)

c -A bl (Y 245)

A cetyl-C oA C arboxylase (S79 )

A dduc in (S662)

A FX (S193)

A kt (S473)

A kt (S473)

A kt (S473) (587F11)

A kt1 /P KB alpha (S473 ) (SK703)

A kt (S473) (736E11 )

A kt (T 308)

A kt (Y 326)

A LK (Y 1604)

A MP Kalpha (T 172)

A MP Kalpha1 (S485)

A MP Kbeta1 (S108)

A MP Kbeta1 (S182)

A rres tin (beta) 1 (S412) (6 -24 )

A SK1 (S83)

A SK1 (T 845)

A T F-2 (T 71 )

A T F-2 (T 69 /71 )

A urora A (T 288)/B (T 232)/C (T 198 )

Bad (S112)

Bad (S112) (7E11)

Bad (S136)

Bad (S155)

Bc l-2 (S70 ) (5H2)

Bc l-2 (T 56 )

Bc r (Y 177)

BLNK (Y 96 )

Btk (S180) (7A 12)

C aspase-3 , c leaved (D175)

C aspase-3 , c leaved (D175) (5A 1)

C aspase-6 , c leaved (D162)

C aspase-7 , c leaved (D198)

C aspase-8 , c leaved (D374)

C aspase-9 , c leaved (D315)

C aspase-9 , c leaved (D330)

C atenin (beta) (S45 )

C atenin (beta) (S33 /37 /T 41)

C atenin (beta) (T 41 /S45)

C aveolin-1 (Y 14 )

c -C bl (Y 731)

c -C bl (Y 774)

C D19 (Y 513 )

C hk1 (S345)

C hk2 (S33/35 )

C hk2 (T 68 ) (80F5 )

C ofilin (S3 )

C ofilin (S3 ) (77G2)

C onnexin 43 (S368)

C REB (S133)

C REB (S133) (1B6)

C rkI I (Y 221)

C yc lin B1 (S147)

DFF45 , c leaved (D224 )

eEF2 (T 56 )

EGFR (S1046/1047)

EGFR (S1047) (1H9)

EGFR (T 654) (3F2 )

EGFR (Y 845)

EGFR (Y 992)

EGFR (Y 1045)

EGFR (Y 1068)

EGFR (Y 1068) (1H12)

EGFR (Y 1148)

EGFR (Y 1148)

EGFR (Y 1173)

pEGFR (Y 1173) (9H2)

EGFR (Y 1173) (53A 3)

eIF2alpha (S51)

eIF2alpha (S51) (119A 11)

eIF4E (S209)

eIF4G (S1108 )

E lk-1 (S383)

eNO S (S113 )

eNO S (S1177)

eNO S (S1177)

eNO S (T 495 )

eNO S/NO S I I I (S116)

Ephrin B (Y 324/329)

E rbB2/HER2 (Y 877)

E rbB2/HER2 (Y 1221 /1222)

E rbB2/HER2 (Y 1248 )

E rbB2/HER2 (Y 1248 )

E rbB2/HER2 (Y 1248 )/EGFR (Y 1173)

E rbB3/HER3 (Y 1222 ) (50C 2)

E rbB3/HER3 (Y 1289 ) (21D3)

ERK 1 /2 (T 202/Y 204)

ERK 1 /2 (T 202/Y 204) (E10 )

Es trogen Receptor alpha (S118)

Es trogen Receptor alpha (S118) (16 JR)

E tk (Y 40)

E zrin (Y 353 )

E zrin (T 567 )/Radixin (T 564 )/M oes in (T 558)

FA DD (S194)

FA K (Y 397) (18 )

FA K (Y 576/577)

FA K (Y 925)

FC gamma Rec I Ib (Y 292)

FGF Receptor (Y 653 /654)

FHIT (Y 114)

FKHR (S256)

FKHRL1 (S253)

FKHR (T 24)/FKHRL1 (T 32)

FLT 3 (Y 591) (54H1 )

alpha-Fodrin, c leaved (D1185)

FRS2-alpha (Y 436)

Gab1 (Y 627 )

Gab2 (S159 )

Gab2 (Y 452 )

GC N2 (T 898)

G lucocortic oid Receptor (S211)

GSK-3alpha (S21) (46H12)

GSK-3alpha/beta (S21/9 )

GSK-3alpha (Y 279)/beta (Y 216)

GSK-3beta (S9 )

H is tone H3 (S10) M itos is M arker

H is tone H3 (S28)

H is tone H3 (T 11)

HSP 27 (S15 )

IGF-1 Rec (Y 1131)/Insulin Rec (Y 1146)

IGF-1R (Y 1135/36)/IR (Y 1150/51) (19H7 )

IkappaB-alpha (S32)

IkappaB-alpha (S32) (14D4 )

IkappaB-alpha (S32/36) (5A 5 )

IkappaB-alpha (S32/36) (39A 1431 )

IKKalpha (S176)/IKKbeta (S180)

IKKalpha (S180)/IKKbeta (S181)

IL-1beta, c leaved (D116)

IRA K1 (S376)

IRS-1 (S302)

IRS-1 (S307)

IRS-1 (S612)

IRS-1 (S636/639)

IRS-1 (S789)

IRS-1 (S1101 )

IRS-1 (Y 612)

Jak1 (Y 1022/1023)

Jak2 (Y 221)

Jak2 (Y 1007/1008)

c -Jun (S63) I I

c -Kit (Y 703 )

c -Kit (Y 719 )

c -Kit (Y 721 )

Lamin A , c leaved (D230)

LA T (Y 171)

LA T (Y 191)

Lc k (Y 192)

Lck (Y 505)

Lc k (Y 505)

LIM K1 (T 508)/LIM K2 (T 505)

LKB1 (S334 )

LKB1 (S428 )

LKB1 (T 189 )

Lyn (Y 507)

M A P K

(pT EpY )

M A P KA P K-2 (T 334)

M A RC KS (S152/156)

M -C SF Receptor (Y 723 )

M DM 2 (S166)

M EK1 (S298)

M EK1/2 (S217/221 )

M et (Y 1234/1235)

M KK3/M KK6 (S189/207)

M nk1 (T 197/202 )

M SK1 (S360)

M s t1 (T 183)/M s t2 (T 180)

mT O R (S2448)

mT O R (S2481)

c -M yc (T 58 /S62)

M yos in L ight C hain 2 (T 18 /S19)

NF-kappaB p65 (S536)

NP M (T 199 )

p27 (T 187)

p27 (T 187) (2B10B7 )

p38 M A P Kinase (T 180/Y 182)

p40 phox (T 154)

p56Dok-2 (Y 351)

p70 S6 Kinase (S371)

p70 S6 Kinase (T 389)

p70 S6 Kinase (T 412)

p70 S6 Kinase (T 421/S424)

p90RSK (S380)

p130 C as (Y 165)

P A K1 (S144)/P A K2 (S141)

P A K1 (S199/204)/P A K2 (S192/197 )

P A K1 (T 423)/P A K2 (T 402)

P A K2 (S20 )

P A K4 (S474)/P A K5 (S602)/P A K6 (S560)

P A RP , c leaved (D214)

P A RP , c leaved (D214) (19F4 )

P axillin (Y 118)

P DGF Receptor alpha (Y 754) (23B2)

P DGF Receptor beta (Y 716 )

P DGF Receptor beta (Y 751 )

P DK1 (S241)

P I3 -Kinase p85 (Y 458)/p55 (Y 199)

P KA C (T 197 )

P KC alpha (S657)

P KC alpha/beta I I (T 638 /641)

P KC (pan) (betaI I S660)

P KC delta (T 505)

P KC theta (T 538)

P KC zeta/lambda (T 410/403 )

P KR (T 446)

c P LA 2 (S505)

P LC gamma1 (Y 783)

P LC gamma2 (Y 759)

P LD1 (S561)

P LK1 (T 210)

P RA S40 (T 246)

P RK1 (T 774)/P RK2 (T 816)

P roges terone Receptor (S190)

P T EN (S380)

P yk2 (Y 402)

Rac1 /cdc42 (S71)

Raf (S259)

A -Raf (S299)

B-Raf (S445)

c -Raf (S338) (56A 6)

Ras -GRF1 (S916)

Ret (Y 905)

RSK3 (T 356/S360)

S6 Ribosomal P rotein (S235/236) (2F9 )

S6 Ribosomal P rotein (S240/244)

SA P K/JNK (T 183/Y 185)

SEK1/M KK4 (S80 )

SGK (S78)

Shc (Y 317)

Shc (Y 317)

SH IP 1 (Y 1020)

SHP 2 (Y 542)

SHP 2 (Y 580)

Smad1 (S/S)/Smad5 (S/S)/Smad8 (S/S)

Smad2 (S465/467)

Smad2 (S245/250/255)

Smad3 (S433/435)

Src Family (Y 416)

Src (Y 527)

SRF (S103)

Stat1 (S727)

Stat1 (S727)

Stat1 (Y 701)

Stat1 (Y 701)

Stat2 (Y 689)

Stat3 (S727)

Stat3 (S727)

Stat3 (Y 705) (9E12)

Stat3 (Y 705) (58E12)

Stat5 (Y 694)

Stat6 (Y 641)

Syk (Y 323)

Syk (Y 525/526)

T A K1 (T 184)

T A K1 (T 184/187)

T ie2 (S1119 )

T ie2 (Y 992)

T pl2 (S400)

T roponin I (C ardiac ) (S23 /24 )

T uberin/T SC 2 (Y 1571)

T yk2

(Y 1054/1055)

V A SP (S157 )

V A SP (S239 )

V EGFR 2 (Y 951)

V EGFR 2 (Y 996)

V EGFR 2 (Y 1175) (19A 10)

WNK1 (T 60 )

Zap-70 (Y 315/319)

Zap-70 (Y 493)

Zap-70 (Y 319)/Syk (Y 352)

MEASURE ALL OF THESE IN

DNA MUTATION STATUS DOES NOT PREDICT PATHWAY ACTIVATION LEVELS:

DIRECT MEASUREMENT OF PROTEIN PHOSPHORYLATION IS REQUIRED!!

CASE STUDY: 260 T-ALL CLINICAL SPECIMENS COLLABORATOR: JULES MEIJERINK AND SANNE HULSPAS, ERASMUS UNIVERSITY

GOOD CONCORDANCE BETWEEN RPMA DERIVED PROTEIN LEVELS AND

PTEN MUTATION STATUS BUT…

NO CONCORDANCE BETWEEN PTEN/AKT MUTATION STATUS AND

DOWNSTREAM PROTEIN ACTIVATION!!!

EGFR WT

I-SPY TRIAL: an overview

• Sample Size

– 244 patients over 3 years (at 8 institutions)

– Assuming 15% drop-out/ non-compliance rate, expect

sufficient data on 207 patients

• Schema

doxorubicin +

cyclophosphamide

A/C

paclitaxel surgery RT tam

MRI, core biopsy (F1&F2) , serum, mammo

MRI, serum, mammo

MRI, core biopsy (F5&F6) , serum MRI

core biopsy (F3&F4)

Tissue (FS)

Weeks 1-12 Weeks 13-25

Week 27 Weeks 31-38 Weeks 39-78

For research purposes only:

4 MRIs, 12 cores, 3 blood

draws, 2 mammograms

Microdissected Patient Samples

Ph

os

ph

o-

an

d T

ota

l P

rote

in E

nd

po

ints

Her2

Akt/mTOR

MAPK

Organization of I-SPY Samples According to Signaling Pathway Activation

Julie Wulfkuhle: Lead Scientist

Triple Negative Tumors

Protein Pathway Mapping of Triple Negative Tumors:

TAILORING THERAPY BASED ON DIRECT

KNOWLEDGE OF DRUG TARGET ACTIVATION

Proposed Drug Targets for Triple Negative Tumors:

PLK1/Aurora, AKT/mTOR, eNOS, IGF/EGF

PROTEIN PATHWAY ACTIVATION MAPPING OF HUMAN BREAST CANCER

ISPY T1 SAMPLE SET

~10% of FISH/IHC- are pHER2+

and are pathway activated

PAIK ET AL, NEJM 2008

Ellis et al- Cancer Discovery

1-2% activating HER2 mutations

without amplification

But higher percent (10%) of

FISH/IHC- were seen as receiving

benefit by HERCEPTIN

~10% were phosphoHER2+ !

Single readout:

Functional activation of the drug

target itself (phosphoHER2)

INDIVIDUALIZED THERAPY OF METASTATIC CANCER

Biopsy of Metastatic Lesion

CONCEPT: Molecular profiling of the patient’s

metastasis (not the primary) to individualize therapy

Molecular Profiling Select Therapy

CONCEPT: At the molecular level metastatic lesions

may be different from the primary tumor: Reasons:

1. A minor subpopulation in the primary tumor is

selected and expanded in the metastasis.

2. The tumor cells in the metastasis have adapted to

grow and survive in a different tissue “soil”.

IHC

Genomics

Proteomics

Predicate: The Bisgrove Trial

A Pilot Study Utilizing Molecular Profiling by IHC, FISH, DNA

Microarray, and Reverse Phase Protein Microarray (RPMA) of Patients’

Tumors to Find Potential Targets and Select Treatments for Patients

with Metastatic Breast Cancer

1. CARIS/MPI Target NOW based profiling for IHC and DNA Array for

Therapeutic Selection

1. Functional pathway activation analysis on FDA approved

kinase-targeted therapeutics by Reverse Phase Protein Microarrays

Sponsor: Side-Out Foundation

Byant Dunetz

Participants:

CARIS – Molecular Profiling Institute

George Mason University

Dr. Stephen Anthony Spokane WA

Co-PI: Gayle S. Jameson

Co-PI : Dr. Nicholas Robert

Gayle Jameson,

NP

Nicholas James Robert, M.D.

1+

2+

3+

0

PATIENT POPULATION

DISTRIBUTION

Phosphory

late

d/a

ctivate

d E

GF

R level

PATIENT B

PHYSICIAN REPORT

PATIENT B

DRUG TARGET ACTIVITY LEVEL DRUG

Phospho-EGFR O TARCEVA

Phospho-c-KIT 3+ GLEEVEC

Phospho-VEGF 0 AVASTIN

Phospho-mTOR 2+ TORISEL

PATIENT A

Compare new patient value to existing

population data for phospho EGFR values PHYSICIAN REPORT

PATIENT A

DRUG TARGET ACTIVITY LEVEL DRUG

Phospho-EGFR 3+ TARCEVA

Phospho-c-KIT 1+ GLEEVEC

Phospho-VEGF 3+ AVASTIN

Phospho-mTOR 0 TORISEL

Side Out Trial Conclusion To Date

1. Proven feasibility of breast cancer patient recruitment, biopsy, tissue

collection and distribution to molecular profiling labs on opposite sides

of the USA. 25 patients enrolled to date.

1. Molecular profiling report can be reliably generated within 10 days.

2. Valuable information is being gathered comparing the physician’s

“choice” to the therapy indications predicted by molecular profiling.

3. Unique set of molecular data comparing candidate drug targets in the

metastasis / recurrent lesions to the matched primary in the same

patient.

1. Assay informed therapy different then what physician would have

chosen in all 25/25 patients.

1. 60% of PT samples had activation of drug targets determined by RPMA

in 3 major clusters: pan-HER-AKT; EGFR/SRC/ERK/mTOR;

IGFR/RAF/MEK/PLK1.

Inflammatory Breast Cancer : Lymphatic

Tropism

Histologic hallmark: Extensive dermal lymphatic

invasion by cancer cells (Cristofanilli 2012)

• Rare ( 1-3% of Breast Cancers)

• Rapid invasion and metastasis progression

• 5 year survival rate is 36%

• Accounts for a disproportionate percentage of

the yearly breast cancer death rate

Chip Petricoin

Julie Wulfkuhle

Rita Circo

George Mason

University

Fredika M. Robertson

Fredika M. Robertson

4E-BP1 S65

Akt S473

Alk Y1586

Aurora A (T288)/B (T232)/C (T198)

B-Raf S445

c-Abl Y245

BAD (S112)

c-ErbB2/HER2

Chk1 S345

Chk2 S33/35

c-KIT Y719

CrkII Y221

EGFR Y1045

EGFR Y1173

EGFR Y992

eIF4G S1108

Elk-1 S383

ErbB2 Y1248

ErbB3 Y1289

ERK 1/2 (T202/Y204)

Estrogen rec alpha 1D5

FAK Y576/577

GSK3 alpha-beta S21/9

Histone H3 (S10)

IGF-1R (Y1135/36)/IR (Y1150/51)

Ikappa B alpha (S32/36)(5A5)

IRS-1 S612

Jak1 Y1022/1023

Jak2 Y1007/1008

MEK1/2 S217/221

mTOR S2448

NF-kB p65 (S536)

p38 MAPKinase (T180/Y182)

p70 S6 kinase

p70 S6 Kinase T389

p90 RSK S380

PARP cl D214

Paxillin Y118

PDK1 (S241)

PDGF Rec Beta Y751

PLK1 T210

PTEN S380

Raf S259

Ret Y905

RSK3 (T356/S360)

Shc Y317

Smad1(S/S)/Smad5(S/S)/Smad8(S/S)

Smad2 S245/250/255

Src Y527

Stat3 S727

Stat5 Y694

Total EGFR

cleaved NOTCH 1

Total PTEN

VEGFR Y996

Cox2

LC3B

Met Y1234/1235

SAPK/JNK T183/Y185

Survivin

XIAP

KI67

cyclin D1

Phospho-TrkA (Tyr490)

Phospho-TrkA (Tyr674/675)

phospho Syk (Y525/526)

Phospho-Tie2 (Tyr992)

Phospho-CDK4 (Thr172)

phosho Rb (S780)

phosho PIM1 (Y309) antibody

Phospho-Axl (Y779)

Phospho-FGF Receptor (Tyr653/654)

phospho FRS2-alpha (Y436)

Phospho-PI3K p85 (Tyr458)/p55 (Tyr199)

Phospho-Rac1/cdc42 (Ser71)

Phospho MDM2

phospho TCTP s46

The Future of Companion Diagnostics:

2015-beyond:

~ 75 analytes measured

> 60 FDA cleared targeted therapies

What assay platform can quantitatively

Measure 75 CDx targets at once

from LCM material from a single core

biopsy?

MS/MRM?

Layered IHC/Mulitplexed IHC?

Nano capillary?

X Reverse Phase Protein Array

Prostate

Lung

Breast

Colorectal

Endometrial

Other solid

Sarcomas

Leukemias

Classification by Location/Type of Cancer

Reclassification of Cancer by Functional Protein Pathway Activation

Prostate

Lung

Breast

Colorectal

Endometrial

Other solid

Sarcomas

Leukemias

JAK-STAT

SRC

AKT-mTOR

Ras-Raf-MEK- ERK

HER Family

Classification by Protein Activation Mapping

ALK/ROS

Adapted from WO 2009076472, US 20090155804 : ‘Blume-Jensen

Luminal A

Luminal B

HER 2 Enriched

Normal-Like

Basal-Like

Classification by Signatures

Reclassification of Breast Cancer by Functional Protein Pathway Activation

Prostate

Lung

Breast

Colorectal

Endometrial

Other solid

Sarcomas

Leukemias

JAK-STAT

SRC

AKT-mTOR

Ras-Raf-MEK- ERK

HER Family

Classification by Protein Activation Mapping

ALK/ROS

Luminal A

Luminal B

HER 2 Enriched

Normal-Like

Basal-Like

Breast Cancer Patient 1 Breast Cancer Patient 2

CDx Report of the Future: Individualized Protein Pathway Activation Maps

Center for Applied Proteomics and Molecular Medicine Co-Directors: Lance Liotta and Emanuel Petricoin

Julia Wulfkuhle, Valerie Calvert, Virginia Espina, Mariaelena Pierobon, Alessandra Luchini, Weidong

Zhou, Paul Russo, Claudius Mueller, Isela Gallagher, Amy VanMeter, Alex Reeder, Sally Rucker,

Jianghong Deng, Bridget Wilson, Maryam Goudarzi, Robin Araujo, Domenico Napoletani, James Cooper,

Peggy Hackett, Patty Theimer, Lindsay Wescott, Vivian Norman

Fellows: Claudia Fredolini, Davide Tamburro, Caterinia Longo, Francesco Meani, Alessandra Silvestri,

Angela Zupa, Guiseppina Improta, Antonella Chiechi, Mattia Cremona, Rita Circo, Alessandra Romano,

Giulia Federici, Valentina Fodale,

RESEARCH FUNDING

THANK YOU!!!!!!!!!!