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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