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

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

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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,

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MAST • BOLOGNA, 25-26 OCTOBER, 2016

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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

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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

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Cell sorting DEPArray

CTC Isolation

Isolation of CTCs

Banking / Analysis

Single CTCs Cellular suspension

CTC enrichment and enumeration

7,5 ml Blood

CTC enrichment

Aphaeresis

CellSearch system

Celltrack cartridges

CK DAPI CD45 CK/DAPI Bright field

CTC

Event ID Group ID

WBC CTC Single cells 18,5µl buffer Volume reduction

Scan of cells in the chip with automated fluorescence microscopy

CK-PE CD45-APC MoFlo XDP (Beckman Coulter) DEPArray (Silicon Biosystems) Micromanipulator (Eppendorf)

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MoFlo XDP (Beckman Coulter) DEPArray (Silicon Biosystems) Micromanipulator (Eppendorf)

Dedicated (trained) team Technical Staff Elina Bongers Maria Wecker Scientific Staff Christiane Driemel Rui Neves Rosa Guglielmi

Cell sorting DEPArray

CTC Isolation Banking / Analysis

Single CTCs Cellular suspension

CTC enrichment and enumeration

7,5 ml Blood

CTC enrichment

Aphaeresis

CellSearch system

Celltrack cartridges

Isolation of CTCs

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Single Cell Whole genome amplification Ampli1 WGA kit (Silicon Biosystems)

Copy number profiles mutations QC WGA / Ampli1

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

WGA neg Control WGA pos Control QC neg Control QC pos Control 50bp ladder

CTCs from peripheral blood

Cell #1 2 3 4 5 6 7 8 9 10

Genomic Analysis of individual CTCs

Analysis

Single CTCs Cellular suspension

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

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Vera Binder & Christoph Bartenhagen et al 2014

run 1 run 3 run 2 genomic REH DNA Resolution: 10 kbp!

NGS whole genome sequencing of single cell WGA products * * *

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NGS whole genome sequencing of single cells

Vera Binder & Christoph Bartenhagen et al Hum Mutat 2014

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REH cell line (n=9) PMNC (n=7)

aCGH analysis of single cell genomes

(Agilent 4x180K)

Möhlendick PlosOne 2013 and Behrens et al 2016 unpublished

Chromosomal Gains Chromosomal Losses

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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

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Problem of CTC-based Liquid Biopsy Negativity Rate in cM1 patients: 29-45% (<1 CTC in CellSearchTM)

Source: www.veridex.com

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In 1961, Roberts and colleagues noted: ‘In addition to loss

  • f

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.’

CTC-based “liquid biopsy”: 10 mL sample is insufficient!

Roberts S et al Ann Surg:154; 362–371 (1961)

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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)

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  • 1. to overcome „frequency problem“
  • 2. to enable more reliable CTC detection

à Increase significantly the analyzed blood volume

Strategy

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Physical Characteristics of CTCs allow selection: Density

RBC Gra

CTCs

Mon Lym PLT

Stoecklein, ..., Terstappen, Expert Review Mol Diagnostics 2015

MNCs

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Diagnostic Leukapheresis (DLA) improves CTC detection

  • 29 DLA products of 23 patients

(PDAC, Breast CA and GI CA) à Majority cM0 Patients

  • 2.3 mL in CS (DLA)

~60 mL blood equivalent 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

Density based cell separation

  • Target-cells: 1.05 – 1.088 g/mL
  • 2-2.5 Liters of blood
  • Duration ~ 1 hour

40 mL DLA Product 25 x 108 MNCs

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GOAL

Processing the whole DLA product (~40 mL) will further significantly increase CTC yield à Challenge...

~2 mL DLA product analyzed in CellSearchTM (EpCAM+CK+DAPI+CD45-)

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Genomic Profiling (aCGH/NGS)

36 mL 2 mL 40 mL DLA Product 25 x 108 MNCs CD45 Depletion Waste Filtration/Direct Filtration VyCAP CellSearch

Cell Isolation

DEPArray

Ampli1 WGA

del Monaco V et al 2016

Clearbridge/ ClearCellFX Angle/Parsortix

Stained Single Cell Suspension

ACCEPT

Use of complementery CTC detection methods in DLA DLA – alternative methods delivering cell suspensions: àCell loss during staining in suspension too high (~ 50%) (confirmed by indpendent groups) àMethods preventing this cell loss are under development

✔

DLA - CellSearch – DEPArray isolation à validated by independent group

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Summary

Diagnostic Leukapheresis (DLA)

  • co-enrichment of CTCs with MNCs
  • improves significantly CTC detection vs PB sample

àComplimentary method in M1 patients àGuide adjuvant therapy in M0 patients àcomprehensive CTC analysis (liquid biopsy) à DLA might be further improved for CTC enrichment àBasis for more effective high-volume technologies

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  • Dept. for General, Visceral

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

  • Dept. for Pediatric Hematology,

Oncology, and Immunology University Hospital Düsseldorf Vera Binder Arndt Borkhardt