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

Jason Hill, Ph.D.

Targeted Molecular Diagnostics

AgendaAgenda

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

The role and importance of image analysis from pre-clinical through clinical development of targeted therapies

What TMD DoesWhat TMD Does

Pharmaceutical Services

Novel Approach to Novel Approach to Drug Development Drug Development Using BiomarkersUsing Biomarkers

Targeted Targeted Diagnostics to Diagnostics to Guide Cancer Guide Cancer

TreatmentTreatment

Physician and Hospital Services

CAP CLIAGLP / GCP

Pre-Clinical Biomarker Development Pre-Clinical Biomarker Development Through Phase III and BeyondThrough Phase III and Beyond

Pre-Clinical Phase I Phase II Phase III FDA

Companion Dx Development

Manufacturing, Commercial Development & Distribution

Technology Transfer, Regulatory & Promotion

Support

Diagnostic companies need to manufacture and sell the final test, but frequently have a hard time understanding therapy companies. We can help.

LaunchResearch

Pharma & Biotech

Pathway Elucidation

Efficacy & Mechanism Biomarkers,Resistance Pathways

Dose Selection (PD Biomarkers)Drug Mechanism (Surrogate Efficacy Biomarkers)Patient Selection (Predictive Biomarkers)

Targeted Molecular Diagnostics

Master Research Agreement Master Laboratory Services Agreement

Development Testing Support

Diagnostic Companies

Complexity of Signaling Pathways in Complexity of Signaling Pathways in Cancer Cancer

WNT

Cell

ECM

Growth factors (e.g. EGF, amphiregulin TGF)

Nuclear receptors(e.g. oestrogen)

Survival factors(e.g. IGF1)

Cytokines(e.g. ILs, IFNs)

Deathfactors

(e.g. FasL)

Anti-growth factors(e.g. TGF)

GPCR ligands

Frizzled Dishevelled

GSK-3

APC

Tubulin

TCF

Integrins

-Cutenin -Cutenin:TCFE-Cadherin

CdC42 PI3K Rac

FakCas Crk

Src

Fyn

ShcNF1

RasRTK Grb2SOS

Ral MEK MAPK MAPK

MEKK

PLC

PKC Mos MKKs JNKs

ELK

Myc:Max

Max:MaxFos

JUN

Abl

7-TMR

CdC42 Rac Rho

G-Prol Ad Cycl PKA CREB

PKC NF-B

NHR (e.g. ER)

NF-B

P13K Akt Akka IKB

PTEN?

Stat 3.5

Stat 3.5

Stat 3.5

Bcl XL

Caspase 9

Cytochrome C

Jaks

Bad BidMitochondria

Bim, etc.Abnormalitysensor

Bcl-2

Cell Death(Apoptosis) Caspase 8

Fap

FADD

Bcl-2

Bax

ARF

p53

Mitochondria

MDM2

DNA damagesensorCell

Proliferation(cell cycle)

Changesin Gene

Expression

Cycl E:CDK2 p21

p27

E2Fs

Rb

p16

Cycl D:CDK+ p15 Smads

RTK

Cytokine R

Decoy R

Fas

SurfaceAg

TGFR

HPVE7

Hanahan D, Weinberg RA. Cell (2000). Vol 100: 57–70

Uses of Morphological Biomarkers in Drug Uses of Morphological Biomarkers in Drug DevelopmentDevelopment

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

Vehicle Control

HN-5 Xenografts Stained for p-ErbB1HN-5 Xenografts Stained for p-ErbB1

30 mg/kg

100 mg/kg

Translation of Biomarkers to Xenograft StudiesTranslation of Biomarkers to Xenograft Studies

Image analysis in pre-clinical studies to measure dose dependent target inhibition

Image Analysis of Efficacy Biomarkers for Image Analysis of Efficacy Biomarkers for ErbB Inhibitors ErbB Inhibitors

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

Cleaved Caspase 3 % Positive Nuclear Staining

0

5

10

15

20

25

Control 1Hr 4Hr 8Hr 12Hr 24Hr

% P

os

itiv

e N

uc

lea

r A

rea

Inhibitor 1

Inhibitor 2f

Ki67 % Nuclear Staining in BT474 Xenografts

0

10

20

30

40

50

60

70

80

90

Control 1Hr 4Hr 8Hr 12Hr 24HrHr Post Treatment

% P

ositi

ve N

ucle

ar A

rea

ErbB-zErbB-y

PTEN IHC Assay ReproducibilityPTEN 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.

PTEN Day 1 PTEN Day 2 PTEN Day 3

This particular tissue exhibits strong staining in the tumor and weaker staining in the stroma.

Image AnalyzersImage Analyzers

Phospho-Biomarkers in Clinical DevelopmentPhospho-Biomarkers in Clinical Development

ErbB2

p-ErbB2

p-Erk1/2 p-Akt

Pre treatment Post treatment

Pre treatment Post treatmentPre treatment Post treatment

Frequency of Achieving >75% Inhibition of p-ErbB1, p-ErbB2, p-Erk1/2, or p-Akt in Tumors at Day 21 After Lapatinib Treatment

0

10

20

30

40

50

60

70

80

Dose (mg/day)

50065090012001600F

req

uen

cy (

%)

Range of Patient

Response

EGF10004 (Phase I Study): EGF10004 (Phase I Study): Drug Concentration and Biological EffectDrug Concentration and Biological Effect

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): EGF10004 (Phase Ib Study): Efficacy & Predictive BiomarkersEfficacy & Predictive Biomarkers

Heavily Pretreated Subjects With ErbB1 and/or ErbB2 overexpression

“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

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

TGF 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

EGF103009 (Phase II Study): Predictive EGF103009 (Phase II Study): Predictive Biomarkers for Response To Lapatinib in IBCBiomarkers for Response To Lapatinib in IBC

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.

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

Pre-Treatment Post-Treatment

Arm A Biomarker Analysis

Using PharmacoDynamic (PD) Phospho-Using PharmacoDynamic (PD) Phospho-Biomarkers to Guide Dosing in Real-TimeBiomarkers 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

No PhosphoGuard™ (regular formalin)

PhosphoGuard™

PhosphoGuard™PhosphoGuard™

A431 Xenograft Stained for p-ErbB1

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

Using PharmacoDynamic (PD) Phospho-Using PharmacoDynamic (PD) Phospho-Biomarkers to Monitor Src InhibitionBiomarkers to Monitor Src Inhibition

Src

FAK

Pax

P

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

P

P

P

Can p-FAK and p-Paxillin be used as “readouts” of Src inhibition in patients?

Real-Time PharmacoDynamic (PD) Dose Real-Time PharmacoDynamic (PD) Dose AdjustmentAdjustment

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)

Dose Level 0 Real-time

pharmacodynamic dose adjustment depending on inhibition of p-Src, p-FAK, p-Paxillin

Simultaneous analysis

PD Phospho-Biomarkers in Pre- vs Post-PD Phospho-Biomarkers in Pre- vs Post-Treatment SpecimensTreatment Specimens

Pre-Treatment Post-Treatment

p-Src

p-FAK

p-Paxillin

PTEN IHC AssayPTEN 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 PTEN IHC Staining in Human Tissue SpecimensSpecimens

No tumor PTEN staining, high stromal cell staining

High tumor PTEN staining, high stromal cell staining

Moderate tumor PTEN staining, high stromal cell staining

ConclusionsConclusions

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…. Going digital in diagnostics….