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Jason Hill, Ph.D. Targeted Molecular Diagnostics

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 pre-clinical through clinical development of targeted therapies.

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Jason Hill, Ph.D. Targeted Molecular Diagnostics

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  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

  2. 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

  3. What TMD Does Pharmaceutical Services Physician and Hospital Services Novel Approach to Drug Development Using Biomarkers Targeted Diagnostics to Guide Cancer Treatment GLP / GCP CLIA CAP

  4. Pre-Clinical Biomarker Development Through Phase III and Beyond Pharma & Biotech Research Pre-Clinical Phase I Phase II Phase III FDA Launch Master Laboratory Services Agreement Master Research Agreement Pathway Elucidation Efficacy & Mechanism Biomarkers, Resistance Pathways Dose Selection (PD Biomarkers) Drug Mechanism (Surrogate Efficacy Biomarkers) Patient Selection (Predictive Biomarkers) Companion Dx Development Development Testing Support Technology Transfer, Regulatory & Promotion 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

  5. 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-Cutenin:TCF E-Cadherin p16 CdC42 PI3K Rac ECM Integrins Cycl D:CDK+ p15 Smads Fak Cas Crk Src Rb HPVE7 p27 PLC Fyn Growth factors (e.g. EGF, amphiregulin TGFa) Surface Ag Shc E2Fs MKKs JNKs JUN Cycl E:CDK2 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 Max:Max p53 MEKK Myc:Max 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) Mitochondria PKC NF-kB NF-kB 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 Fas Deathfactors (e.g. FasL) Cytochrome C Bcl XL Bcl-2 Stat 3.5 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: 57–70

  6. 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

  7. Vehicle Control 30 mg/kg 100 mg/kg 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

  8. 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

  9. 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

  10. Image Analyzers

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. PhosphoGuard™ Examples of Clinical Trials That Have Utilized PhosphoGuard ™ EGF10004 (Ph I): Published in JCO 23(11): 2502-12. EGF103009 (Ph II): Publishedin JCO 26(7): 1066-72. Global Trial > 100 sites (Ph III): Ongoing Phospho-SrcBased Pharmacodynamic Dose Adjustment (Ph I/II) Ongoing No PhosphoGuard™ (regular formalin) A431 Xenograft Stained for p-ErbB1 PhosphoGuard™

  17. P P P P FAK Src Pax 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

  18. 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

  19. PD Phospho-Biomarkers in Pre- vs Post-Treatment Specimens Pre-Treatment Post-Treatment p-Src p-FAK p-Paxillin

  20. 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.

  21. 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

  22. 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)

  23. Going digital in diagnostics….

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