Transcript Slide 1

The Use of Image Analysis in Assessing
Biomarkers; Implications for Clinical Trials,
Drug Development and Patient Treatment
Jason Hill, Ph.D.
Targeted Molecular Diagnostics
Agenda
The role and importance of image analysis from preclinical through clinical development of targeted
therapies
 Pre-clinical
 Effect of dosing on target inhibition
 Dose schedule
 Assay validation
 Examples of phospho-biomarkers in clinical development
 Using image analysis of biomarkers to assess biologically
efficacious dose (BED) and select patients
 Using image analysis of phospho-biomarkers for PD guided real
time dose adjustments
 Measuring complex biomarkers such as tumor suppressors in
clinical specimens
What TMD Does
Pharmaceutical
Services
Physician and
Hospital Services
Targeted
Diagnostics to
Guide Cancer
Treatment
Novel Approach to
Drug Development
Using Biomarkers
GLP / GCP
CAP
CLIA
Pre-Clinical Biomarker Development
Through Phase III and Beyond
Pharma & Biotech
Research
Pre-Clinical
Phase I
Efficacy & Mechanism
Biomarkers,
Resistance Pathways
Phase III
FDA
Launch
Master Laboratory Services Agreement
Master Research Agreement
Pathway
Elucidation
Phase II
Dose Selection (PD Biomarkers)
Drug Mechanism (Surrogate Efficacy Biomarkers)
Patient Selection (Predictive Biomarkers)
Development
Targeted Molecular Diagnostics
Testing Support
Companion Dx
Development
Technology Transfer,
Regulatory & Promotion
Support
Diagnostic Companies
Diagnostic companies need to manufacture and sell
the final test, but frequently have a hard time
understanding therapy companies. We can help.
Manufacturing,
Commercial Development
& Distribution
Complexity of Signaling Pathways in
Cancer
Anti-growth factors
(e.g. TGFb)
Tubulin
WNT
Frizzled
Dishevelled
GSK-3b
TCF
APC
Cell
b-Cutenin
E-Cadherin
p16
CdC42
ECM
TGFbR
b-Cutenin:TCF
PI3K
Integrins
Rac
Cycl D:CDK+
Fak
Src
Cas
Rb
Growth factors (e.g. EGF,
amphiregulin TGFa)
Fyn
Shc
NF1
Grb2
SOS
RTK
Ras
p27
PKC
Mos
MKKs
Ral
MEK
MAPK
Survival factors
(e.g. IGF1)
G-Prol
JNKs
Ad Cycl
Rac
E2Fs
JUN
MAPK
ELK
Max:Max
MEKK
CdC42
7-TMR
Smads
HPVE7
PLC
Abl
GPCR ligands
Nuclear receptors
(e.g. oestrogen)
p15
Crk
Fos
Myc:Max
Cycl E:CDK2
Changes
in Gene
Expression
Cell
Proliferation
(cell cycle)
Surface
Ag
p21
DNA damage
sensor
p53
Rho
PKA
CREB
ARF
MDM2
Bax
NHR (e.g. ER)
PKC
RTK
P13K
Akt
NF-kB
Akka
NF-kB
Mitochondria
Stat 3.5
Bcl-2
Cell Death
(Apoptosis)
IKB
Caspase 8
FADD
Stat 3.5
?
PTEN
Caspase 9
Fap
Cytochrome C
Stat 3.5
Bcl XL
Bad
Jaks
Cytokines
(e.g. ILs, IFNs)
Abnormality
sensor
Decoy R
Bcl-2
Mitochondria
Bid
Fas
Death
factors
(e.g. FasL)
Bim, etc.
Cytokine R
Hanahan D, Weinberg RA. Cell (2000). Vol 100: 57–70
Uses of Morphological Biomarkers in Drug
Development
 Analyze effect on target/downstream pathways in pre-clinical studies
e.g.
 Phosphorylation (TKIs: Iressa, Tykerb, Gleevec)
 Acetylation (HDACi: SAHA, MS-275)
 Methylation (Vidaza)
 Transition assays to clinical specimens, can be used for e.g.
 Selecting/Guiding dose in Phase I/II
 Identifying potential biomarkers of response and/or resistance
 Refine Response/Resistance Biomarkers in Phase II
 Correlating biomarkers with patient response
 Selecting one (or a few) biomarkers for Phase III
 Select Patients and Standardize in Phase III
Translation of Biomarkers to Xenograft Studies
HN-5 Xenografts Stained for p-ErbB1
Vehicle Control
30 mg/kg
100 mg/kg
Image analysis in preclinical studies to measure
dose dependent target
inhibition
Image Analysis of Efficacy Biomarkers for
ErbB Inhibitors
Ki67 % Nuclear Staining in BT474 Xenografts
Cleaved Caspase 3 % Positive Nuclear Staining
90
20
15
ErbB-y1
Inhibitor
Inhibitor
ErbB-z 2
f
10
5
% Positive Nuclear Area
% Positive Nuclear Area
25
80
70
60
50
40
30
20
10
0
0
Control
1Hr
4Hr
8Hr
12Hr
24Hr
Control
1Hr
4Hr
8Hr
12Hr
24Hr
Hr Post Treatment
Efficacy biomarkers can be used in xenograft models to identify
MOA and assess drug efficacy
• Does the drug work through hypothesized mechanism(s)?
• Efficacy biomarkers can be useful for a class of compounds
PTEN IHC Assay Reproducibility
Optical Density
(tumor)
Optical Density
(stroma)
Day 1
25
6
Day 2
26
6
Day 3
25
6
The PTEN IHC assay was run on
an automated staining platform on
3 different days.
Tumor and stromal cells were
measured by image analysis.
This particular tissue exhibits strong staining in the tumor and weaker staining in the
stroma.
PTEN Day 1
PTEN Day 2
PTEN Day 3
Image Analyzers
Phospho-Biomarkers in Clinical Development
Pre treatment
Post treatment
ErbB2
p-ErbB2
Pre treatment
p-Erk1/2
Post treatment
Pre treatment
p-Akt
Post treatment
EGF10004 (Phase I Study):
Drug Concentration and Biological Effect
Frequency of Achieving >75% Inhibition of p-ErbB1, p-ErbB2, p-Erk1/2, or p-Akt in
Tumors at Day 21 After Lapatinib Treatment
80
Frequency (%)
70
60
50
40
Range of Patient
30
20
Response
500
650
900
1200
1600
10
0
Dose (mg/day)
Uses of Pharmacodynamic (PD) biomarkers in Phase I studies:
•Provide rationale for biologically efficacious dose instead of MTD
•Demonstrate in vivo target inhibition and dose response
Spector, et al. (2005). Study of the Biologic Effects of Lapatinib a Reversible Inhibitor of ErbB1 & ErbB2 Tyrosine
Kinases on Tumor Growth and Survival Pathways in Patients With Advanced Malignancies. JCO 23(11): 2502-12.
EGF10004 (Phase Ib Study):
Efficacy & Predictive Biomarkers
Heavily Pretreated Subjects With ErbB1 and/or ErbB2 overexpression
Name
Result (day 0)
Clinical Outcome
TUNEL
TUNEL score = 0
Excludes PR
ErbB2
Elevated
PR
p-ErbB2
Elevated
PR
p-Erk1/2
Not Elevated
PD
IGF-1R
Elevated
PR
p-p70S6K
Elevated
PR
TGFa
Elevated
PR
Observations:
•Increase in TUNEL (apoptosis) correlated with clinical benefit
•Some TUNEL activity was necessary before treatment for clinical benefit
•Biomarkers may offer “early” profile of response or resistance
“Using Biomarkers for the first time in a struggling Phase III trial is like trying to change a flat
tire at 60 mph” – Sr. Director, Big Pharma
EGF103009 (Phase II Study): Predictive
Biomarkers for Response To Lapatinib in IBC
Arm A Biomarker Analysis
Pre-Treatment
Post-Treatment
Observations of Predictive Biomarkers:
•Most patients in Cohort A (HER2
overexpressing) had high p-HER2
•However, co-expression of p-HER2 AND
p-HER3 predicted for response to
lapatinib
Johnston, et al. (2008). Phase II Study of Predictive Biomarker Profiles for Response Targeting Human HER-2
in Advanced Inflammatory Breast Cancer With Lapatinib Monotherapy. JCO 26(7): 1066-72.
Using PharmacoDynamic (PD) PhosphoBiomarkers to Guide Dosing in Real-Time
 Phospho-biomarkers that are drug targets or
downstream signaling molecules can be used to
monitor target inhibition in real-time
 When combined with an analytical method such as
semi-quantitative IHC, degree of target inhibition can
be measured and used to guide patient dosing
PhosphoGuard™
Examples of Clinical Trials That
Have Utilized PhosphoGuard ™
EGF10004 (Ph I):
Published in JCO 23(11): 2502-12.
No PhosphoGuard™ (regular formalin)
EGF103009 (Ph II):
Published in JCO 26(7): 1066-72.
PhosphoGuard™
A431
Xenograft
Stained for
p-ErbB1
Global Trial > 100 sites (Ph III):
Ongoing
Phospho-Src Based Pharmacodynamic
Dose Adjustment (Ph I/II)
Ongoing
Using PharmacoDynamic (PD) PhosphoBiomarkers to Monitor Src Inhibition
P
P
P
FAK
P
Pax
Can p-FAK and p-Paxillin be used as Src
“readouts” of Src inhibition in patients?
FAK becomes active upon recruitment to the plasma membrane and
autophosphorylates itself
Src is recruited to the plasma membrane, autophosphorylates itself and further
phosphorylates and activates FAK
Paxillin is recruited to FAK and is phosphorylated by Src
Real-Time PharmacoDynamic (PD) Dose
Adjustment
Dose
Level 0
Pre-Treatment Bx
Measurement of pSrc, p-FAK, pPaxillin (image
analysis)
4 wk Bx
Simultaneous
analysis
Measurement
of p-Src, pFAK, p-Paxillin
(image
analysis)
Real-time
pharmacodynamic dose
adjustment depending on
inhibition of p-Src, p-FAK,
p-Paxillin
PD Phospho-Biomarkers in Pre- vs PostTreatment Specimens
Pre-Treatment
p-Src
p-FAK
p-Paxillin
Post-Treatment
PTEN IHC Assay
Background: PTEN is a tumor suppressor gene whose expression is
frequently lost in human tumors (2nd only to loss of p53).
Objective: To develop a specific, sensitive and reproducible
immunohistochemistry (IHC) assay for the detection of PTEN in human
tissue specimens.
 To develop an image analysis method to measure PTEN
expression in both tumor and stromal cells in human specimens.
Importance: Comparison of tumor to stromal cell staining may indicate
if a tumor has reduced PTEN expression relative to normal cells.
PTEN IHC Staining in Human Tissue
Specimens
High tumor PTEN staining,
high stromal cell staining
Moderate tumor PTEN staining,
high stromal cell staining
No tumor PTEN staining,
high stromal cell staining
Conclusions
Image analysis enables:
 Exploring dosing and scheduling in pre-clinical models
 Quantitatively assessing consistency / reproducibility of
IHC assay development and check lot to lot variation
 Measurement of PD biomarkers that can be used to
guide patient dosing in real time
 Analysis of complex biological markers (eg. PTEN)
Going digital in diagnostics….