The Use of Image Analysis in Assessing Biomarkers Implications for Clinical Trials, Drug Development

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The Use of Image Analysis in Assessing
Biomarkers Implications for Clinical Trials,
Drug Development and Patient Treatment

• Jason Hill, Ph.D.
• Targeted Molecular Diagnostics

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Agenda
The role and importance of image analysis from
pre-clinical 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

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What TMD Does
Pharmaceutical Services
Physician and Hospital Services
Novel Approach to Drug Development Using
Biomarkers
Targeted Diagnostics to Guide Cancer Treatment
CLIA
GLP / GCP
CAP
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Pre-Clinical Biomarker Development Through Phase
III and Beyond
Pharma Biotech
Pre-Clinical
Phase I
Phase II
Phase III
FDA
Launch
Research
Master Research Agreement
Master Laboratory Services Agreement
Pathway Elucidation
Efficacy Mechanism Biomarkers, Resistance
Pathways
Dose Selection (PD Biomarkers) Drug Mechanism
(Surrogate Efficacy Biomarkers) Patient Selection
(Predictive Biomarkers)
Companion Dx Development
Technology Transfer, Regulatory Promotion
Support
Development
Testing Support
Targeted Molecular Diagnostics
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
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Complexity of Signaling Pathways in Cancer
Anti-growth factors (e.g. TGFb)
Tubulin
WNT
Dishevelled
Frizzled
GSK-3b
TCF
APC
TGFbR
Cell
b-Cutenin
b-CuteninTCF
E-Cadherin
p16
CdC42
PI3K
Rac
ECM
Integrins
Cycl DCDK
p15
Smads
Fak
Cas
Crk
Src
Rb
HPVE7
p27
PLC
Fyn
Growth factors (e.g. EGF, amphiregulin TGFa)
Shc
E2Fs
Surface Ag
MKKs
JNKs
JUN
Cycl ECDK2
p21
NF1
PKC
Mos
DNA damage sensor
Grb2
Changes in Gene Expression
Ras
Ral
MEK
MAPK
MAPK
ELK
Fos
RTK
Cell Proliferation (cell cycle)
SOS
MaxMax
p53
MEKK
MycMax
Abl
CdC42
Rac
Rho
GPCR ligands
G-Prol
Ad Cycl
PKA
CREB
ARF
MDM2
7-TMR
Nuclear receptors (e.g. oestrogen)
Bax
NHR (e.g. ER)
PKC
NF-kB
NF-kB
Mitochondria
Stat 3.5
Survival factors (e.g. IGF1)
Cell Death (Apoptosis)
Bcl-2
P13K
Akt
Akka
IKB
RTK
Caspase 8
FADD
Stat 3.5
PTEN
?
Caspase 9
Fap
Deathfactors (e.g. FasL)
Fas
Cytochrome C
Bcl XL
Stat 3.5
Bcl-2
Decoy R
Bid
Bad
Mitochondria
Jaks
Abnormality sensor
Bim, etc.
Cytokine R
Cytokines (e.g. ILs, IFNs)
Hanahan D, Weinberg RA. Cell (2000). Vol 100
5770
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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

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Translation of Biomarkers to Xenograft Studies
HN-5 Xenografts Stained for p-ErbB1
Image analysis in pre-clinical studies to measure
dose dependent target inhibition
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Image Analysis of Efficacy Biomarkers for ErbB
Inhibitors
ErbB-y
ErbB-z

• 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

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PTEN IHC Assay Reproducibility
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
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Image Analyzers
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Phospho-Biomarkers in Clinical Development
Pre treatment
Post treatment
ErbB2
p-ErbB2
Pre treatment
Post treatment
Pre treatment
Post treatment
p-Erk1/2
p-Akt
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Frequency of Achieving 75 Inhibition of
p-ErbB1, p-ErbB2, p-Erk1/2, or p-Akt in Tumors at
Day 21 After Lapatinib Treatment
EGF10004 (Phase I Study) Drug Concentration and
Biological Effect
Frequency ()
Range of Patient Response

• 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.
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EGF10004 (Phase Ib Study) Efficacy Predictive
Biomarkers
Heavily Pretreated Subjects With ErbB1 and/or
ErbB2 overexpression

• 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
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EGF103009 (Phase II Study) Predictive Biomarkers
for Response To Lapatinib in IBC
Arm A Biomarker Analysis
Post-Treatment
Pre-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.
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Using PharmacoDynamic (PD) Phospho-Biomarkers 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

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PhosphoGuard
Examples of Clinical Trials That Have Utilized
PhosphoGuard EGF10004 (Ph I) Published in
JCO 23(11) 2502-12. EGF103009 (Ph
II) Published in JCO 26(7) 1066-72. Global
Trial 100 sites (Ph III) Ongoing Phospho-Src
Based Pharmacodynamic Dose Adjustment (Ph I/II)
Ongoing
No PhosphoGuard (regular formalin)
A431 Xenograft Stained for p-ErbB1
PhosphoGuard
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Using PharmacoDynamic (PD) Phospho-Biomarkers to
Monitor Src Inhibition
Can p-FAK and p-Paxillin be used as 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
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Real-Time PharmacoDynamic (PD) Dose Adjustment
Dose Level 0
Real-time pharmacodynamic dose adjustment
depending on inhibition of p-Src, p-FAK,
p-Paxillin
Pre-Treatment Bx Measurement of p-Src, p-FAK,
p-Paxillin (image analysis)
4 wk Bx Measurement of p-Src, p-FAK,
p-Paxillin (image analysis)
Simultaneous analysis
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PD Phospho-Biomarkers in Pre- vs Post-Treatment
Specimens
Pre-Treatment
Post-Treatment
p-Src
p-FAK
p-Paxillin
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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.

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PTEN IHC Staining in Human Tissue Specimens
No tumor PTEN staining, high stromal cell staining
High tumor PTEN staining, high stromal cell
staining
Moderate tumor PTEN staining, high stromal cell
staining
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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)

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Going digital in diagnostics.