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Liquid Biopsy Research Applications Solutions

2014 thermo fisher scientific . All rights reserved. 1 For Research Use only . Not for use in diagnostic procedures. The world leader in serving science Liquid Biopsy Research Applications Solutions Rosella Petraroli Senior Manager Global Market Development 2014 thermo fisher scientific . All rights reserved. 2 For Research Use only . Not for use in diagnostic procedures. Evolution of Cancer Classification Cancer classification has changed from anatomical to molecular classification Molecular subtype based stratification is a promising new strategy in clinical Research From anatomical to molecular approach Breast Cancer Cervical Cancer Colorectal Cancer Liver Cancer Lung Cancer Ovarian Cancer Pancreatic Cancer Prostate Cancer Skin Cancer Stomach Cancer Thyroid Cancer ALK AKT1 BRAF EGFR ERBB2 KRAS NRAS MAP2K1 PIK3CA RET ROS1 Undefined 2014 thermo fisher scientific .

For Research Use Only. Not for use in diagnostic procedures. The world leader in serving science ©2014 Thermo Fisher Scientific. All rights reserved.

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Transcription of Liquid Biopsy Research Applications Solutions

1 2014 thermo fisher scientific . All rights reserved. 1 For Research Use only . Not for use in diagnostic procedures. The world leader in serving science Liquid Biopsy Research Applications Solutions Rosella Petraroli Senior Manager Global Market Development 2014 thermo fisher scientific . All rights reserved. 2 For Research Use only . Not for use in diagnostic procedures. Evolution of Cancer Classification Cancer classification has changed from anatomical to molecular classification Molecular subtype based stratification is a promising new strategy in clinical Research From anatomical to molecular approach Breast Cancer Cervical Cancer Colorectal Cancer Liver Cancer Lung Cancer Ovarian Cancer Pancreatic Cancer Prostate Cancer Skin Cancer Stomach Cancer Thyroid Cancer ALK AKT1 BRAF EGFR ERBB2 KRAS NRAS MAP2K1 PIK3CA RET ROS1 Undefined 2014 thermo fisher scientific .

2 All rights reserved. 4 For Research Use only . Not for use in diagnostic procedures. Overview of Tumor Molecular Evolution Over Time Primary tumor may not reflect current tumor molecular profile Treatment options may cause changes in tumor cells * * MRCA = Most recent common ancestor Source: Yates LR, et al. (2012) Evolution of the cancer genome. Nat Rev Gen 13: 795 806. 2014 thermo fisher scientific . All rights reserved. 5 For Research Use only . Not for use in diagnostic procedures. Tissue Sampling versus Liquid Biopsy Challenges Tissue sampling is invasive Tissue sampling is limited Advantages Archive serial samples Cost Information on tumor heterogeneity Tissue Sample Liquid Sample 2014 thermo fisher scientific .

3 All rights reserved. 7 For Research Use only . Not for use in diagnostic procedures. Multiple Sources of Tumor DNA in Blood Circulating Tumor Cell (CTC) 2 Main DNA Targets: CTCs & cfDNA Cell-free DNA (cfDNA) Exosomes Crowley E, et al. (2013) Liquid Biopsy : monitoring cancer genetics in the blood. Nat Rev Clin Onc 10: 472 484. 2014 thermo fisher scientific . All rights reserved. 8 For Research Use only . Not for use in diagnostic procedures. Liquid Biopsy Applications in Cancer Research Research Studies Review of past therapeutic decision Correlation with tumor stage Tumor evolution at different time points Review of tumor recurrence Circulating Tumor Cells and Circulating Cell-free DNA Research Applications Modified source: Haber, DA, and Velculescu, VE.

4 (2014). Blood-Based Analyses of Cancer: Circulating Tumor Cells and Circulating Tumor DNA. Cancer Discovery 4:650 661. 2014 thermo fisher scientific . All rights reserved. 9 For Research Use only . Not for use in diagnostic procedures. Liquid Biopsy Research Applications Sample stability Standardization Contamination between samples Limited material for molecular analysis Limit of detection below 1% Increase number of target mutations Today s Challenges 2014 thermo fisher scientific . All rights reserved. 10 For Research Use only . Not for use in diagnostic procedures. Challenges Scalable Solutions for Liquid Biopsy Research thermo fisher scientific Solutions Sample Collection Template Isolation Variant Detection Analysis Ion Torrent LiquidBiopsy Blood Collection Kit Ion Torrent LiquidBiopsy Instrument Applied Biosystems MagMAX Cell-Free DNA Kits and thermo scientific KingFisher Flex Magnetic Particle Processor Sample stability Standardization Limited material for molecular analysis Standardization Contamination Limited material for molecular analysis Limit of detection <1% Increase number of target mutations Standardization Limit of detection <1% Increase number of target mutations False positive rate

5 Applied Biosystems QuantStudio 3D Digital PCR System Ion Torrent sequencing platforms Ion Reporter Software CTC Enrichment cfDNA Isolation 2014 thermo fisher scientific . All rights reserved. 11 For Research Use only . Not for use in diagnostic procedures. LiquidBiopsy Instrument Workflow Three Templates from a Single Blood Sample Enables CTC enumeration and molecular analysis of cfDNA, CTC and germline DNA from a single blood sample Automates flow cell priming, blood sample loading, target cell isolation and immunofluorescence labeling of captured cells Enables multiplexing: Up to 4 samples in ~3 hours 2014 thermo fisher scientific . All rights reserved.

6 12 For Research Use only . Not for use in diagnostic procedures. Comprehensive 2-day LiquidBiopsy Instrument Workflow Sample to Genomic Data NGS Analysis Purify cfDNA Germline WBC Extracted CTCs LiquidBiopsy Instrument Enrichment Sequencing Analysis with Ion Reporter Software Multiple template analysis of CTC, germline and cfDNA isolation from a single blood sample Multiplex up to 4 samples in ~3 hours CTC enumeration and genomic data Invitrogen EVOS Floid Cell Imaging Station Optional Enumeration Optional verification with dPCR 2014 thermo fisher scientific . All rights reserved. 13 For Research Use only . Not for use in diagnostic procedures. Research More Tumor Types with Multiple Supported Antibodies The ability to enrich CTCs enables researchers to ask which molecular profile should be serially analyzed Recovery and purity of target cells can be matched with different reagents Various cell populations can be recovered informative on molecular level Winer-Jones, JP, et al.

7 (2014) Circulating Tumor Cells: Clinically Relevant Molecular Access Based on a Novel CTC Flow Cell. PLoS ONE 9: e86717. 2014 thermo fisher scientific . All rights reserved. 14 For Research Use only . Not for use in diagnostic procedures. Consistent Number of Captured WBCs Enables More Accurate Variant Calls Performance enables detection of ~3 mL blood sample at >1% limit of detection A predictable WBC capture rate leads to more accurate variant calls Normal and diseased Research samples show the equivalent performance at cell capture 0125250375500 Non-target events/mL Non-target Cell Carryover CD45 cells captured/mL blood = 22 20 Mean non-target cell capture + 2 SD 198 + = Captured WBCs is 22 cells/mL At least 3 captured CTCs Verified 1% limit of detection using Ion Reporter Software* * 1% LOD at 2,000x read depth using Ion PGM System 2014 thermo fisher scientific .

8 All rights reserved. 15 For Research Use only . Not for use in diagnostic procedures. Excluded based on inclusion in germline PIK3ca PIK3ca PIK3ca PIK3ca PIK3ca TP53 PIK3ca TP53 PTPN11 TP53 KIT KDR KDR KIT BRAF KIT TP53 TP53 KRAS PTPN11 PTEN KRAS COSMIC mutations Uncategorized mutations CTC cfDNA PIK3ca TP53 TP53 KDR PIK3ca PIK3ca TP53 TP53 TP53 TP53 TP53 KRAS ERBB2 KRAS KRAS VHL PTEN PTEN BRAF STK11 SMO SMO SMO FGFR3 VHL KDR ATM RB1 RB1 FBXW7 FBXW7 FLT3 APC AKT1 AKT1 AKT1 HNF1A PIK3ca MET cfDNA and CTCs are Complementary Templates 2014 thermo fisher scientific . All rights reserved. 19 For Research Use only . Not for use in diagnostic procedures. Simple protocol 3 reagents + Applied Biosystems Dynabeads Magnetic Beads Ship and store at room temp Fast 30 to 45 min manual protocol Automated on KingFisher Flex (24 samples) Automated on thermo scientific KingFisher Duo Prime Purification System (6 samples) Scalable Flexible input volumes from mL to 10 mL plasma Specific Enrich for cfDNA fraction over gDNA or isolate all DNA KingFisher Flex MagMAX Cell-Free DNA Isolation MagMAX cfDNA Kit 2014 thermo fisher scientific .

9 All rights reserved. 20 For Research Use only . Not for use in diagnostic procedures. 4 ml plasma 4 ml Buffer ACL + carrier RNA ProK 60oC 30 min Add ml incubate on ice Load ml vacuum Wash x3 vacuum Elute nucleic acid (20-150 ul) 56oC 10 min drying Manual cfDNA Workflow Comparison 4 ml plasma +5 ml L/B solution +60 ul beads 10 min shaking room temp pellet beads on magnet Resuspend beads in wash solution Transfer to microfuge tube Wash 2x 1 ml wash solution Wash 2x 1 ml 80% ETOH Dry 3-5 minutes Elute DNA 50 ul QIAamp Circulating Nucleic Acid kit MagMAX Cell-Free DNA isolation kit ~15 minutes 10 minutes <10 minutes 35 minutes hours spin 2014 thermo fisher scientific .

10 All rights reserved. 21 For Research Use only . Not for use in diagnostic procedures. MagMAX Cell Free DNA Kit versus Competitor Equivalent short DNA yield Lower long DNA yield Enriched cfDNA --- MagMAX --- Q --- MagMAX --- Q --- MagMAX --- Q --- MagMAX --- Q 4 mL plasma Automated MagMAX cfDNA Kit versus Q Manual Procedure 2014 thermo fisher scientific . All rights reserved. 23 For Research Use only . Not for use in diagnostic procedures. Personalized Medicine: The Path Forward McKinsey & Company, 2013 Genomic Research Ever Expanding List of Genes and Variants 2014 thermo fisher scientific . All rights reserved. 24 For Research Use only . Not for use in diagnostic procedures.


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