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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 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….