Challenges for use of CTCs as a Diagnostic. Farideh Z. Bischoff, Ph.D. Interim CSO Sr. Director, Translational Clinical Development Biocept, Inc.

Transcription

1 Challenges for use of CTCs as a Diagnostic Farideh Z. ischoff, Ph.D. Interim CSO Sr. Director, Translational Clinical Development iocept, Inc.

2 Current Technology for CTC Testing Existing CTC testing platform is CellSearch by Veridex/J&J CellSearch provides CTC enumeration CellSearch is FDA cleared for 3 cancer types (breast, colorectal, and prostate) but limited to metastatic, late-stage patients Many supportive studies: Positive correlation between pretreatment (baseline) CTC numbers and clinical prognosis/recurrence or response to therapy

3 Ideal CTC (Diagnostic) Test Components CTC Enumeration Protein Marker analysis by ICC (e.g. ER) Gene Amplification by FISH (e.g. HER2) Mutation Analysis by PCR (e.g. EGFR)

4 CTCs as a Surrogate for Tumor Tissue Guide selection of best therapeutic for individual patients Identify patients with minimal residual disease Detect treatment-resistant profiles Improve understanding of mechanisms of biological metastatic processes Discover and identify new targets for therapeutic manipulation

5 Limits of Current CTC Technology CellSearch detects CTCs in 30-60% of blood samples from cancer patients (breast, CRC, prostate) Limited to epithelial CTCs Use of single CTC-specific capture Ab (e.g., just EpCAM) Use of single CTC detection stain (cytokeratin; CK)

6 Many CTC Recovery and Detection Methods Described Enrichment/Isolation Immunomagnetic (MACS; CellSearch ) Centrifugation/Density Separation Filtration (ISET) Red cell lysis/slide preparation (EPIC) Microfluidic (iocept, MGH, On-Q-ity) Electrophoresis Detection Cytometric (IHC, ICC, FISH) Nucleic Acid-ased

7 Inlet Silicone channel Glass slide Cover slip iocept s OncoCEE Microchannel Design Outlet Uniquely designed to maximize rare cell capture by randomizing size/shape and placement of posts: Prevent laminar flow 9,000 posts; 24?L total volume Capture and visualization of tumor cells directly within the microchannel

8 Problem 1: CTC Capture Solution: Antibody Cocktail Surface antigens are additive; enable more sensitive capture of low and high level antigen expressing cells Epithelial and/or mesenchymal-like cell types captured simultaneously (all aspects of EMT epithelial-to-mesenchymal transition) 8

9 Technical Validation Study: CTC Recovery and Her2 Determination Antibody Cocktail: EpCAM, Trop-2, c-met, Folate binding protein receptor, N-Cadherin, CD318, MSC, Her2, Muc-1,and EGFR CTC Detection (CK+/CD45-/DAPI+) 8 of 29 stage II-III cases (28%) 52 of 63 stage IV or recurrent cases (82.5%)? 60 Informative (CK+) cases CD45+ nucleated lymphocytes Standard CTC Definition CK+, CD45-, DAPI+ tumor cell

10 Determination of Her2 Status 56 of 60 CK+ Cases with Known Her2 Status (by tumor tissue) CTC Result (FISH) Patient (Tissue) Her2 Positive Her2 Negative Her2 Positive 20 4 Her2 Negative 1 31 lue = Concordant Red = Discordant Overall, 91% Concordance Her2 FISH: 83% Sensitivity, 97% Specificity

11 CK+ Her2+ CTC Composite Image DAPI Green Orange CK and Centromere 17 Her2

12 Her2 Amplification Confirms CK-negative CTC Chromosome 8 centromere (blue) Her2 gene (red), on chromosome 17 Chromosome 17 centromere (green) 12

13 Correlation of CK+ stain and Her2 Amp Among all Her2+ cells identified (n=76): 36 identified on relocation of CK+ cells (47%) 40 identified on analysis of all CK- cells (53%)

14 Problem 2: CTC Detection Solution: CK & Enhanced staining CTCs must be distinguished from non-specific background cells (e.g., WCs) Today, CTCs are detected (and defined) by cytokeratin (CK) staining; but CTCs may lose CK through EMT Additional target cell types, e.g., endothelial, mesenchymal and stem cells, may require different detection strategies (beyond CK)

15 15 CEE-Enhanced TM stain: Detecting what we catch CK+ cell CK+ cell plus CEE-Enhanced stain CK- cell plus CEE -Enhanced stain (standard method of CTC detection) C Cell Captured On Channel with Antibody cocktail A

16 Weak Staining CK+ CTCs ecome righter Weak CK+ Ca CTC DAPI-stained WCs + CEE-Enhanced WCs unchanged CK+ cell plus CEE- Enhanced stain

17 reast Cancer CTC Study (MDA): Operable Patient Cohort 25 patient blood samples taken pre-surgery Process and capture using antibody cocktail Stain using CK combined with CEE-Enhanced TM CTCs detected in 19 of 25 (76%) cases y stage: y subtype: T1N0 (9/12) Luminal A (15/19) T1N1 (2/3) Luminal (2/3) T2N0 (1/3) Her2 (1/2) T2N1 (2/2) Triple negative (1/1) T2N3 (1/1) T3N0 (1/1) T4N0 (2/2) T4N3 (1/1) 17

18 Summary Methods that enable reliable recovery and detection of CTCs exist FISH-based methods used for secondary analysis (assay development) and/or detection of clinically relevant biomarkers (CLIA; laboratory developed tests) Clinical validation of biomarkers using CTCs needed Longitudinal studies to assess clinical significance

19 FDA & LDTs Current situation Commercial sale of instrument or diagnostic kits (e.g., Veridex) requires FDA clearance Reference Laboratories offering commercial tests (lab developed tests) are under the prevue of CLIA do not require FDA submission FDA office of In Vitro Diagnostic Device Evaluation and Safety stressed that LDT oversight process will take time to develop & will include input from the industry and consumer groups Draft legislation creating a new category for both LDTs and IVDs regulated by CMS and CLIA that would include reimbursement policies Why the Difference between FDA Submission & LDTS In vitro diagnostic kits/instruments are sold across the US. LDTs are a medical service under the control of a medical director, per CLIA, in a defined setting Why the interest by the FDA Response to public health risks created by LDTs that may not provide reasonable assurances of safety and effectiveness Lack of information or accounting on LDTS provided to FDA CLIA only requires analytical validity of LDTs, not clinical validity. FDA Commissioner Margaret Hamburg believes this is needed for personalized medicine to be successful