MAST BOLOGNA, 25-26 OCTOBER, 2016 DEPArray User Meeting DEP - PowerPoint PPT Presentation

MAST • BOLOGNA, 25-26 OCTOBER, 2016

DEPArray™ User Meeting DEP DEPArray CT CTC Isolation from Samples Pr Prepared with Different Enrichment Te Technologies Nikolas Stoeck cklein Experimental Surgical Oncology Dept. for General, Visceral and Pediatric Surgery Medical Faculty of the Heinrich-Heine-University Düsseldorf, Germany MAST • BOLOGNA, 25-26 OCTOBER, 2016

CTC Profiling Peripheral Blood - CellSearch – DEPArray - CTC isolation Diagnostic Leukapheresis – CellSearch / Alternative Technologies à Innovative improvement of CTC-based liquid biopsy MAST • BOLOGNA, 25-26 OCTOBER, 2016

Isolation of CTCs Banking / Analysis CTC enrichment and enumeration CTC Isolation Cellular Single CTCs suspension Cell sorting CTC enrichment DEPArray CellSearch system 7,5 ml Blood Celltrack cartridges Aphaeresis Micromanipulator (Eppendorf) MoFlo XDP (Beckman Coulter) DEPArray (Silicon Biosystems) Scan of cells in the chip with automated fluorescence microscopy CK DAPI CD45 Event ID CK/DAPI Bright field Group ID CTC CK-PE CTC Single cells 18,5µl buffer WBC Volume CD45-APC reduction

Isolation of CTCs Banking / Analysis CTC enrichment and enumeration CTC Isolation Cellular Single CTCs suspension Cell sorting CTC enrichment DEPArray CellSearch system 7,5 ml Blood Celltrack cartridges Aphaeresis Micromanipulator (Eppendorf) MoFlo XDP (Beckman Coulter) DEPArray (Silicon Biosystems) Dedicated (trained) team Technical Staff Elina Bongers Maria Wecker Scientific Staff Christiane Driemel Rui Neves Rosa Guglielmi

Genomic Analysis of individual CTCs Analysis WGA / Ampli1 Cellular Single CTCs suspension Figure by Bianca Behrens Klein et al 1999 PNAS Stoecklein et al 2002 AJP Stoecklein et al 2008 Cancer Cell Möhlendick et al 2013 Plos One Single Cell Whole genome amplification WGA pos Control WGA neg Control QC pos Control QC neg Control CTCs from peripheral blood 50bp ladder Ampli1 WGA kit (Silicon Biosystems) Cell #1 2 3 4 5 6 7 8 9 10 QC Copy number profiles mutations

Ampli1 WGA of single genomes à Amplify from 6 pg to ~4 µg DNA Fidelity for CNA detection?

NGS whole genome sequencing of single cell WGA products run 1 * run 2 run 3 * * Resolution: 10 kbp! genomic REH DNA Vera Binder & Christoph Bartenhagen et al 2014

NGS whole genome sequencing of single cells Vera Binder & Christoph Bartenhagen et al Hum Mutat 2014

aCGH analysis of single cell genomes (Agilent 4x180K) REH cell line (n=9) PMNC (n=7) Chromosomal Gains Chromosomal Losses Möhlendick PlosOne 2013 and Behrens et al 2016 unpublished

Experience CellSearch à DEPArray à Ampli1 à Genomic Profiling - allows reliable single cell isolation (as reported by Polzer et al EMBO 2014 and others) / profiling - slower than our published FACS approach (Neves Clin Chemistry 2014), BUT offers morphologcal control à improves QC & more reliable cell selection - Deterministic Ampli1 WGA enables precise CNA-profiling in single cells (...also on the sequence level à low ADO, Binder et al 2014) - Robust workflow, which can be implemted into clinical studies à CTC-based liquid biopsy

Problem of CTC-based Liquid Biopsy Negativity Rate in cM1 patients: 29-45% (<1 CTC in CellSearch TM ) Source: www.veridex.com

CTC-based “liquid biopsy”: 10 mL sample is insufficient! In 1961, Roberts and colleagues noted: ‘ In addition to loss of cells during processing, a large sampling error contributes to distortion of the total figures . A 10-cc. aliquot of blood can hardly be regarded as a fair index of total blood volume. Blood samples must be taken at frequent intervals to detect isolated showers of cancer cells .’ Roberts S et al Ann Surg:154; 362–371 (1961)

Detection Efficacy of 1000 CTCs in 5 L Blood (CellSearch) Probabilty of 95% to detect at least 1 CTC in one out of five 7.5 mL blood samples if 1000 CTCs circulate in 5L blood Taken from Tibbe A et al, Cytometry Part A 71A:154–162 (2007)

Strategy 1. to overcome „frequency problem“ 2. to enable more reliable CTC detection à Increase significantly the analyzed blood volume

Physical Characteristics of CTCs allow selection: Density RBC Gra CTCs MNCs Mon Lym PLT Stoecklein, ..., Terstappen, Expert Review Mol Diagnostics 2015

Diagnostic Leukapheresis (DLA) improves CTC detection Density based cell separation - Target-cells: 1.05 – 1.088 g/mL - 2-2.5 Liters of blood - Duration ~ 1 hour - 29 DLA products of 23 patients (PDAC, Breast CA and GI CA) à Majority cM0 Patients 40 mL DLA - 2.3 mL in CS (DLA) Product 25 x 10 8 ~60 mL blood equivalent MNCs Detection rate 72% (vs. 28% in PB) à Median of 612 CTCs (29-13102) in Circulation (4.5 L) Fischer J C et al. PNAS 2013;110:16580-16585

~2 mL DLA product analyzed in CellSearch TM (EpCAM + CK + DAPI + CD45 - ) GOAL Processing the whole DLA product (~40 mL) will further significantly increase CTC yield à Challenge...

Use of complementery CTC detection methods in DLA ✔ 40 mL DLA 2 mL Product 25 x 10 8 CellSearch MNCs DLA - CellSearch – DEPArray isolation ACCEPT à validated by independent group Waste Filtration/Direct Filtration VyCAP Cell Isolation 36 mL Ampli1 DLA – alternative methods delivering cell suspensions: WGA DEPArray à Cell loss during staining in suspension too high (~ 50%) Angle/Parsortix Genomic Profiling Stained Single Cell (aCGH/NGS) Suspension (confirmed by indpendent groups) CD45 Depletion à Methods preventing this cell loss are under development Clearbridge/ ClearCellFX del Monaco V et al 2016

Summary Diagnostic Leukapheresis (DLA) - co-enrichment of CTCs with MNCs - improves significantly CTC detection vs PB sample à Complimentary method in M1 patients à Guide adjuvant therapy in M0 patients à comprehensive CTC analysis (liquid biopsy) à DLA might be further improved for CTC enrichment à Basis for more effective high-volume technologies

Institute for Transplantation Department for Obstetrics Dept. for General, Visceral Diagnostics and Cellular and Gynecology and Pediatric Surgery Therapy University Hospital University Hospital Düsseldorf University Hospital Düsseldorf Düsseldorf Wolfram T. Knoefel Johannes Fischer Tanja Fehm Stefan Topp Katharina Raba Hans Neubauer Alexander Rehders Dieter Niederacher Christian Vay Ellen Honisch Georg Flügen Mira Research Institute Franziska Meier-Stiegen Faculty of Science and Lab Technology Medical Informatics *Bianca Behrens University of Twente, NL University Münster *Elina Bongers Leon Terstappen Christoph Bartenhagen Guus van Dalum Kiki Andree *Christiane Driemel Joost Swennenhuis Dept. for Pediatric Hematology, Rosa Guglielmi Leonie Zeune Oncology, and Immunology *Birte Möhlendick University Hospital Düsseldorf *Rui Neves Vera Binder The Institute for Cancer Constantin Pixberg Arndt Borkhardt Research (ICR) London Swetlana Seidschner Bayer Healthcare UK *Anna Streit Global Biomarker Johann de Bono Maria Wecker Wuppertal/Berlin Maryou Lambros Jun Hao Wu Germany Gunther Boysen Thomas Krahn Penny Flohr Antje Stresemann CTCTRAP