protein pathway mapping: realizing the promise of...
TRANSCRIPT
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
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!!!!!!!!!!