QIAGEN Complete Solutions for Liquid Biopsy Molecular Testing
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1 QIAGEN Complete Solutions for Liquid Biopsy Molecular Testing Christopher Swagell, PhD Market Development Manager, Advanced Molecular Pathology QIAGEN 1
2 Agenda QIAGEN Solid Tumor Testing and Liquid Biopsy Sample Collection PAXgene ccfdna Sample Extraction QIAamp Circulating NA Plasma EGFR Testing therascreen Next Generation Sequencing - GeneReader PowerPoint Style Guide,
3 Personalized Healthcare Portfolio - therascreen Brain MGMT IDH1 & IDH2 Melanoma BRAF NRAS Lung EGFR ALK KRAS Gastrointestinal KIT PDGFRA Colorectal KRAS NRAS BRAF PIK3CA UGT1A1 MLH1 Market leading portfolio for solid tumor testing RAS
4 QIAGEN solutions for Molecular Pathology by technology Mutation testing EGFR, KRAS, NRAS, BRAF etc. ARMS Scorpions qpcr High sensitivity Selective mutation detection Ease of use One-step procedure Pyrosequencing Sequence information Complex mutation testing with simple data interpretation Small amount of starting material Short TAT GeneReader NGS Workflow Sequence information Completely integrated sample to report workflow Highly powerful bioinformatics 4
5 Co-development projects with industry leaders Global pharma partnerships and selected co-development projects with a total of 15 master framework agreements signed 5
6 PHC/Oncology Development Pipeline Disease # Assays On-Market # Development Projects # Projects w/ Liquid Biopsy # Pharma Partner Lung NSCLC (9 x PMA, 1 x CE) 2 9 Colon/Gastric CRC/GIST 7 2 (2 x PMA) 1 2 Gynecological Breast/ Ovarian - 3 (2 x PMA, 1 x CE) 1 3 Brain Glioma 2 2 (1 x PMA, 1 x CE) - 2 Genitourinary Prostate/Bladder - 3 (2 x PMA, 1 x CE) 1 3 Skin Melanoma 2 1 (1 PMA) - 1 Blood Leukemia Lymphoma > (1 PMA, 1 CE) - in disc. > 30 >
7 QIAGEN offers Solutions for Liquid Biopsy Based on either NGS or (RT-)PCR PAXgene Blood ccfdna Tubes Manual Sample Extraction PCR Workflow (e.g. known somatic mutation or pathway analysis) PCR-Setup (Real Time) PCR Data Analysis & Interpretation Sample collection & Stabilization Sample Extraction & Isolation NGS GeneReader Workflow (e.g. identification of known / novel mutations) Pre- Amplification / Library Preparation NGS Run (Sequencing) Data Analysis & Interpretation Report Automated Sample Extraction Innovation is in the blood, 7
8 What is Liquid Biopsy? A minimal invasive technology for detecting signs of cancer and other diseases Free circulating nucleic acids RNA and DNA from dead cells shed into the bloodstream, can contain cancer-related mutations. Exosomes Tiny microvesicles found in body fluids that transport RNA between cells. Circulating tumor cells Tumor cells shed from a tumor into the bloodstream carrying genetic information. Tissue samples FFPE tissue samples of tumor extracted from the patient s body through an invasive procedure. Comprehensive portfolio offering proven ways to access nucleic acids Innovation is in the blood,
9 Critical Points Along the Workflow ccf DNA Blood draw (venipuncture) Separate plasma Extract circulating nucleic acids: QIAamp Circulating NA Kit, QIAsymphony Virus/Pathogen Kit Optional DNA modification (e.g., bisulfite treatment) Real-time PCR Sequencing library prep Next-generation sequencing hemolysis of blood cells increases Wildtype gdna background Sample requires rapid processing and/or stabilization Very low concentration of ccfdna(1-50 ng DNA/ml plasma) Efficient large volume extraction required Low concentration and highly fragmented DNA efficient and sensitive downstream processing From : QIAGEN solutions for Liquid Biopsy applications 9
10 Agenda Slide created with Agenda Assistant QIAGEN Solid Tumor Testing and Liquid Biopsy Sample Collection PAXgene ccfdna Sample Extraction QIAamp Circulating NA Plasma EGFR Testing therascreen Next Generation Sequencing - GeneReader PowerPoint Style Guide,
11 Blood sample processing - Workflow 1-2h Draw EDTA whole blood 1900g for plasma separation Draw whole blood in cfdna BCT (ccfdna PAXgene) 1900g for plasma separation 7-10 days Carefully save supernatant 16,000g Supernatant: plasma w/o cell debris and reduced gdna background Store at -80 C Thaw plasma Extraction of cfdna 11
12 Sample Collection and Handling PAXgene Blood ccfdna Tube (RUO) CE-IVD ccfdna PAXgene tubes expected in early 2017 Distributed by Disclaimer: For Research Use Only. Not for use in diagnostic procedures.
13 PAXgene Blood ccfdna Tube (RUO) - Features Stabilization of extracellular levels of ccfdna Non-crosslinking preservation Minimal hemolysis maximum yield from plasma BD Vacutainer Plastic Tube with Hemogard Safety Closure Minimized risk of tube breakage Enhanced safety for healthcare and laboratory personnel Consistent blood draw volume Integrated Preanalytical Workflow Seamless integration into manual or automated extraction technology QIAamp Circulating Nucleic Acid Kit QIAsymphony DSP Circulating DNA Kit 13
14 Prevention of Red Blood Cell Lysis During Transport and Storage PAXgene Blood ccfdna stabilization reagent prevents the hemolysis of erythrocytes 2,50 Hemolysis 2,00 1,50 EDTA Streck PAXccfDNA 1,00 0,50 0,00 t0 t1 t3 t6 t8 t10 Picture kindly provided by 14
15 Blood cell stabilization prevents apoptosis PAXgene Blood ccfdna stabilization reagent prevents the apoptosis of blood cells Reduces wildtype DNA background 15
16 Agenda Slide created with Agenda Assistant QIAGEN Solid Tumor Testing and Liquid Biopsy Sample Collection PAXgene ccfdna Sample Extraction QIAamp Circulating NA Plasma EGFR Testing therascreen Next Generation Sequencing - GeneReader PowerPoint Style Guide,
17 QIAGEN s solutions for cfdna extraction QIAGEN market leaders in sample extraction Current solution (manual): QIAamp Circulating NA Kit 5 ml plasma input 24 samples 3 hours for IVD use In development (automated): QIAsymphony Circulating DNA Kit Using new beads and chemistry 1-4 ml plasma input 96 samples 6 hours (hands-off) for IVD use 17
18 ccfdna isolation considerations Fragmentation QIAamp Circulating Nucleic Acid Kit: Cat. No genome e equiv./ sample x QIAamp circulating NA Kit 66 bp Standard blood kit 66 bp sample 1 sample 2 sample 3 Improved recovery of fragmented DNA Ultimately results in better sensitivity QIAamp Circulating Nucleic Acid Kit: Distributed by
19 Agenda Slide created with Agenda Assistant QIAGEN Solid Tumor Testing and Liquid Biopsy Sample Collection PAXgene ccfdna Sample Extraction QIAamp Circulating NA Plasma EGFR Testing therascreen Next Generation Sequencing - GeneReader
20 therascreen EGFR Plasma Testing - Assay Intended use The therascreen EGFR Plasma RGQ PCR Kit is a realtime PCR test for the qualitative detection of mutations of the EGFR gene derived from plasma from whole blood collected in EDTA tubes of NSCLC patients. Covers the most frequent and clinically validated mutations exon 19 deletions L858R point mutation T790M point mutation (resistance mutation) 20
21 therascreen EGFR Plasma Testing - Workflow 60 min 160 min 15 min Total < 4 hrs 21
22 Case Study 1 Title only (a) 74 yo, male, non-smoker Health degradation and dyspnea Tumor upper lobe right lung and plerual effusion L858R mutation detected in both lung biopsy and plasma After 2 month TKI treatment Health deterioration and weight loss No reduction in tumor size L858R remained stable in plasma T790M mutation detected plasma Identification of resistance mutation during patient progression Marcq et al, J Thorac Oncol. 9 e49-e50 (2014) Detection of EGFR Mutations in the Plasma of Patients with Lung Adenocarcinoma for Real-Time Monitoring of Therapeutic Response to Tyrosine Kinase Inhibitors? 22
23 Case Study 2 Title only (a) 63 yo, female, never-smoker Cough and weight loss Large tumor upper lobe right lung and single bone metastasis ex19del mutation detected in both lung biopsy and plasma After 2 month TKI treatment 50% reduction in lung tumor EGFR mutation became undetectable in plasma After 11 months TKI treatment Progression of tumor and appearance of ex19del mutation detected in plasma Dissapearance of EGFR mutation may be a marker of TKI response Marcq et al, J Thorac Oncol. 9 e49-e50 (2014) Detection of EGFR Mutations in the Plasma of Patients with Lung Adenocarcinoma for Real-Time Monitoring of Therapeutic Response to Tyrosine Kinase Inhibitors? 23
24 Agenda Slide created with Agenda Assistant QIAGEN Solid Tumor Testing and Liquid Biopsy Sample Collection PAXgene ccfdna Sample Extraction QIAamp Circulating NA Plasma EGFR Testing therascreen Next Generation Sequencing - GeneReader 24
25 QIAGEN NGS Workflow Solution - GeneReader QIAGEN GeneReader Launched December The first truly complete NGS workflow 2 Actionable Insights 3 Flexibility to fit your needs 4 Guaranteed results with predictable costs 5 Expertise and service you can count on 25
26 2016: GeneReader NGS System development program Solid Tumour Panel GeneReader NGS System development Expanding range of sample types Liquid biopsy, fresh and frozen tissue o Driving sensitivity down to <1% o Liquid Biopsy Summer launch A T G C A T A C G T Broadening test content to lung and breast Expand oncology panels, other applications o Enabling CNV & Fusion detection Expanding capabilities Expanding System capabilities Further laboratory platform integration and automation (LIMS capability) 26
27 Gracias Gracias por su atención ccfdna PAXgene tubes: QIAamp Circulating Nucleic Acid Kit: Distributed by Customer presentation:therascreen EGFR Plasma RGQ PCR Kit 27
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