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Sample to Insight
Liquid biopsy overview, challenges and new solutions
Wei Cao, Ph.D.
Wei.Cao@qiagen.com
1
Sample to Insight
Legal disclaimer
Liquid biopsy overview 2
• QIAGEN products shown here are intended for molecular biolog...
Sample to Insight
Agenda
Liquid biopsy overview 3
The main areas of liquid biopsy
• Circulating tumor cells
• Circulating ...
Sample to Insight
Agenda
Liquid biopsy overview 4
The main areas of liquid biopsy
• Circulating tumor cells
• Circulating ...
Sample to Insight
Why liquid biopsy?
Liquid biopsy overview 5
Biomarkers
Personalized
therapies
• Need to correlate mutati...
Sample to Insight
What is liquid biopsy?
A liquid biopsy is a liquid biomarker that can be isolated from body
fluids, such...
Sample to Insight
Liquid biopsy: the 3 main areas
7
Liquid biopsy
CTCs
(circulating
tumor cells)
ctNA
(circulating
tumor
n...
Sample to Insight
Area 1: circulating tumor cells (CTCs)
8
Rolfo, C. et al. (2014) Liquidbiopsiesin lungcancer: the new am...
Sample to Insight
CTCs: characterization and challenges
9
Molecular characterizationof CTCs:
Paterlini-Bréchot, P. (2014) ...
Sample to Insight
CTCs: detection/capture methods in clinical studies
10Liquid biopsy overview
Sample to Insight
CTCs: detection/capture methods in clinical studies
11Liquid biopsy overview
Sample to Insight
CTCs: detection/capture methods in clinical studies
12
Ignatiadis, M. et. al. (2015) “Circulating Tumor ...
Sample to Insight
Area 2: circulating tumor nucleic acids (ctNAs)
13
• Biology of cell-free NA and clinical utilities
• Re...
Sample to Insight
ctDNA: detection and challenges
14
Methods for detecting circulating tumor DNA
based on the differences ...
Sample to Insight
Area 3: exosomes in cancer progression
15
Exosomes: small membrane vesicles (30–100 nm), secreted by mos...
Sample to Insight
Exosomes: isolation methods and challenges
16
Rolfo, C. et al. (2014) Liquid biopsiesin lungcancer: the ...
Sample to Insight
Exosomal RNAs
17
mRNAs:
• mRNAs released into the circulation are stable because they are protected from...
Sample to Insight
Agenda
Liquid biopsy overview 18
The main areas of Liquid Biopsy
▪ Circulating tumor cells
▪ Circulating...
Sample to Insight
Liquid biopsy solutions at QIAGEN – Sample to Insight
Liquid biopsy overview 19
For non-invasive biomark...
Sample to Insight
Solutions for circulating tumor cells
20
REPLI-g® Single Cell technology to analyze circulating tumor ce...
Sample to Insight
Solutions for CTCs
21
Learn about the technology: http://www.qiagen.com/us/resources/technologies/wga
Re...
Sample to Insight
REPLI-g Single Cell technology
22
Easy-to-use single-cell WGAand WTAmethod
• Offers complete genome/tran...
Sample to Insight
REPLI-g Single Cell technology
23
Unbiased amplification of DNA and RNA!
Complete genome coverage Maximi...
Sample to Insight
Applications of REPLI-g Single Cell Kits
24
WGA
or
WTA
Whole Genome Sequencing
• Detect variability in g...
Sample to Insight
AdnaTest: the CTC revolution
Blood sampling Determination of
prognostic or
predictive
biomarkers (liquid...
Sample to Insight
Solutions for circulating nucleic acid (ctNA)
26
Isolation of free-circulating DNA and RNA
Liquid biopsy...
Sample to Insight
Circulating nucleic acids: analysis workflow
Sample
Separate plasma
Extract circulating nucleic acids:
•...
Sample to Insight
QIAamp Circulating Nucleic Acid Kit
28
Simple procedure and high performance:
• 4 steps: lyse, bind, was...
Sample to Insight
QIAamp Circulating Nucleic Acid Kit
29
http://www.qiagen.com/search/qiaamp-circulating-nucleic-acid-kit/...
Sample to Insight
Circulating nucleic acids: analysis workflow
miRNeasy Serum/Plasma Kit:
For purification of circulating ...
Sample to Insight
miRNA & mRNA isolation from blood
31
• Purifying RNA and miRNA from 200 µl serum or plasma
• Minimal elu...
Sample to Insight
Solutions for exosomes
32
Isolation of exosomes and exoRNAs
Liquid biopsy overview
Sample to Insight
Isolation of exosomes and exoRNAs
33
exoRNeasy Serum/Plasma Kits
• Isolate RNA from exosomes and other e...
Sample to Insight
exoEasy Maxi Kit: principle and protocol
34
Starting material: serum, plasma or cell culture supernatant...
Sample to Insight
exoEasy Maxi Kit: elute intact vesicles
35
Highest specificity through affinity membrane-binding technol...
Sample to Insight
exoRNeasy Serum/Plasma Kits: principle and protocol
36
Pre-filter
plasma
to remove
particles
larger than...
Sample to Insight
*Arroyo, J.D. et al. (2011) Argonaute2complexescarry a population of circulatingmicroRNAsindependent of ...
Sample to Insight
Circulating nucleic acids: analysis workflow
38Liquid biopsy overview
Sample
Separate plasma
Extract cir...
Sample to Insight
Solutions for circulating miRNA biomarkers
39
miScript miRNA PCR Arrays
Liquid biopsy overview
Sample to Insight
miScript miRNA PCR system
40
• miRNome
• Human: miRBase v21, covers 2402 primer assays
• Mouse: miRBase ...
Sample to Insight
miScript miRNA PCR Array workflow
41
Workflow
1. Isolate total RNA
2. Performreverse transcription
3. Pr...
Sample to Insight
miScript miRNA PCR Array applications
42
miScript Serum & Plasma 384HC PCR Array profiled 381 miRNAs iso...
Sample to Insight
Solutions for lncRNAs: novel biomarkers in cancer
43
RT2 lncRNA PCR Arrays and Assays
Liquid biopsy over...
Sample to Insight
RT2 lncRNA qPCR system
44
• lncRNAdatabases: in-house database at QIAGEN GeneGlobe provides
cover > 40,0...
Sample to Insight
RT2 lncRNA qPCR Array: applications
45
Flexible layout and patented controls
Each 96-well plate has 84 l...
Sample to Insight
Solutions for next-generation sequencing
46
NGS portfolio: complete solutions for NGS
Liquid biopsy over...
Sample to Insight
Complete NGS solutions from “Sample to Insight”
47
• Streamlined, standardized and automated sample-to-i...
Sample to Insight
We provide service – send samples to us & receive results
48
. Whole genome
• Illumina gene expression p...
Sample to Insight
Summary of products offered at QIAGEN
49
1 Single-cell technology – REPLI-g WGASingle Cell Kit
2 CTC enr...
Sample to Insight
Agenda
Liquid biopsy overview 50
The main areas of liquid biopsy
• Circulating tumor cells
• Circulating...
Sample to Insight
51
Thank you for attending today’s webinar!
Contact QIAGEN
Call: 1-800-426-8157
Email: BRCsupport@QIAGEN...
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Liquid Biopsy Overview, Challenges and New Solutions: Liquid Biopsy Series Part 1

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A liquid biopsy is often described as a sensitive and specific blood test to detect circulating tumor cells (CTCs). CTCs, shed by both the primary and metastasized tumors, carry specific information about their origins and markers that will enable us to discover new diagnosis, prognosis and therapeutic targets. This slidedeck gives an overview of the recent progress in exploring the predictive potential of circulating biomarkers, including circulating tumor cells, circulating tumor DNA, microRNAs, long non-coding RNAs (lncRNAs) and exosomes. Addressing both biological and technical aspects, we detail the isolation and characterization of circulating biomarkers. Challenges and solutions are also featured.

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Liquid Biopsy Overview, Challenges and New Solutions: Liquid Biopsy Series Part 1

  1. 1. Sample to Insight Liquid biopsy overview, challenges and new solutions Wei Cao, Ph.D. Wei.Cao@qiagen.com 1
  2. 2. Sample to Insight Legal disclaimer Liquid biopsy overview 2 • QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease. • For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor.
  3. 3. Sample to Insight Agenda Liquid biopsy overview 3 The main areas of liquid biopsy • Circulating tumor cells • Circulating tumor nucleic acid • Exosomes, mRNA, miRNA and lncRNA Solutions provided by QIAGEN Questions 1 2 3
  4. 4. Sample to Insight Agenda Liquid biopsy overview 4 The main areas of liquid biopsy • Circulating tumor cells • Circulating tumor nucleic acid • Exosomes, mRNA, miRNA and lncRNA Solutions provided by QIAGEN Questions 1 2 3
  5. 5. Sample to Insight Why liquid biopsy? Liquid biopsy overview 5 Biomarkers Personalized therapies • Need to correlate mutation profiles with sensitivity or resistance to specific therapies • Molecular characterization of tumors allows progression from “one-size-fits-all” strategy to “personalized medicine” Monitoring of a disease is fundamental for successful treatment Prognostic markersDiagnostic markers Need novel non-invasive biomarkers – liquid biopsy as a game changer Cancer diagnosis Cancer genome analysis Clinical decision Monitor outcome Dancey, J.E. (2012) The genetic basisfor cancer treatment decisions. Cell 148,409. Personalized therapies for cancer patients
  6. 6. Sample to Insight What is liquid biopsy? A liquid biopsy is a liquid biomarker that can be isolated from body fluids, such as blood, saliva, urine, ascites or pleural effusion. Like a tissue biopsy, a liquid biopsy is representative of the tissue from which it has spread. Diaz, Jr., L.A. and Bardelli, A. (2014) Liquid biopsies: genotyping circulating tumor DNA.”Am. Soc. Clin. Oncol. 32,579. 6 • Cancer is a heterogeneous disease • Molecular properties differ within a tumor • Primary tumor biopsy may not reflect current disease condition • Therapy causes changes in tumor cells • Biopsy is invasive • May not be feasible based on patient condition or tumor accessibility • Impractical for periodic monitoring for progression/ recurrence • Biopsy tissue is limited • Greater demand due to molecular profiling • Surgery is costly Liquid biopsy overview • Allows early disease detection • Allows evaluation of metastasis in real-time and monitoring of the actual treatment response • Enables investigation of primary tumors and metastases through simple, non-invasive blood tests • Enables assessment of tumor heterogeneity and monitoring of tumor dynamics • Enables study of the “tumor dormancy” phenomenon • Is much faster than classical biopsy testing • Can be cheaper than classical biopsy testing Liquid biopsy addresses all these limitations
  7. 7. Sample to Insight Liquid biopsy: the 3 main areas 7 Liquid biopsy CTCs (circulating tumor cells) ctNA (circulating tumor nucleic acids) Exosomes Tumors shed both intact cells (resulting in circulating tumor cells) as well as cellular components, such as nucleic acids (resulting in cell-free DNA or RNA). Diaz, Jr., L.A. and Bardelli, A. (2014) Liquid biopsies: genotyping circulating tumor DNA. Am. Soc. Clin. Oncol. 32, 579. Cancer cells released from primary tumor into the bloodstream ctDNA (circulating tumor DNA), miRNAs, mRNA & long non-coding RNA Small membrane-derived vesicles (40–100 nm) that contain various molecules such as signal protein, miRNA, mRNA, lipid and exoDNA CTCs ctNA, mainly ctDNA Exosomes, exoDNA, exoRNA including miRNA and lncRNA Samples: blood, serum/plasma, urine, CSF saliva 1 2 3 Liquid biopsy overview
  8. 8. Sample to Insight Area 1: circulating tumor cells (CTCs) 8 Rolfo, C. et al. (2014) Liquidbiopsiesin lungcancer: the new ambrosiaof researchers. Biochimica et Biophysica Acta 1846, 539. • CTC enumeration: serves as a marker for tumor growth – higher CTC counts mean negative cancer prognosis • CTC characterization: protein and RNA expression, DNA aberrations and propensity for metastatic colonization Technologies to detect and capture CTCs: • EpCAM-affinity based: CellSearch® system, AdnaTest BreastCancerDetect, CTC-Chip, Dynal® , MACS® (magnetic- activated cell sorting system), MagSweeper, On-Q-Ity, CTC-ETI • Physical properties-based: ISET (isolation by size of epithelial tumor cells), ScreenCell® , ApoStream™, density gradient centrifugation • Other methods: FAST (fiber-optic array scanning technology), EPISPOT (epithelial immunospot), FACS (flow cytometry), PRO Onc Assay Limitations: • EpCAM-affinity based methods: low sensitivity, selection bias and poor specificity (false negatives and positives) • Physical properties-based methods: elementary and imprecise, with low specificity CTCs are extremely rare (~1 per 1 ml of blood), so optimization of CTC isolation and enrichment steps is essential. Liquid biopsy overview
  9. 9. Sample to Insight CTCs: characterization and challenges 9 Molecular characterizationof CTCs: Paterlini-Bréchot, P. (2014) Circulatingtumor cells: who isthe killer?”Cancer Microenviron. 7, 161. • Based on antibodies: EpCAM, cytokeratins (CK8, CK18, CK19), CD45-negative. Examples include CellSearch • Based on transcripts: rely on transcripts, performed on the total RNA extracted from blood by RT-PCR. Examples include AdnaTest BreastCancerDetect • Based on functions: protein-based assays such as the EPISPOT (Epithelial ImmunoSPOT) assay • Whole-genome amplifications: single-cell studies targeting CTC heterogeneity, or pooling CTCs to study the whole tumor cell population The clinical validity of CTCs as a modern, personalized, non-invasive and predictive test has been debated. Much effort is still needed to determine whether they represent a potential surrogate for clinical endpoints. Current clinical utility of CTCs • CTCs are extremely rare • CTCs are fragile • CTCs are heterogeneous Challenges Sensitivity is directly linked to clinical benefit at early stages of the disease Can easily vanish or morphology can be damaged during extraction from blood Require the isolation of all types of CTC without any loss Require broad-spectrum, specific cocktails of cell surface epithelial and mesenchymal markers covering all potential CTC phenotypes Liquid biopsy overview
  10. 10. Sample to Insight CTCs: detection/capture methods in clinical studies 10Liquid biopsy overview
  11. 11. Sample to Insight CTCs: detection/capture methods in clinical studies 11Liquid biopsy overview
  12. 12. Sample to Insight CTCs: detection/capture methods in clinical studies 12 Ignatiadis, M. et. al. (2015) “Circulating Tumor Cellsand Circulating Tumor DNA: Challengesand Opportunitieson the Path to Clinical Utility.” Clinical Cancer Research, 21(21): 4786 Updated list of CTC assays that have been used to test patient samples within the past 5 years Liquid biopsy overview
  13. 13. Sample to Insight Area 2: circulating tumor nucleic acids (ctNAs) 13 • Biology of cell-free NA and clinical utilities • Released from both healthy and tumor cells into circulation through various physiological events, such as apoptosis, necrosis and secretion • ctNAs reflect disease progression and treatment responses • The clinical utility of ctDNA depends on reliable detection of ctDNA when it is present • Cell-free vs. CTCs • Shorter half-life (CTCs: 1–2.4 h vs. ctNAs: <1.5 h) • Highly fragmented Types of cell-free nucleic acids ▪ DNA (defined as cell-free DNA, cfDNA) ▪ miRNA (microRNA) ▪ mRNA (messenger RNA) ▪ Long non-coding RNAs (lncRNAs) Schwarzenbach, H. et. al. (2011) Cell-freenucleic acidsas biomarkersin cancer patients. Nat. Rev. Cancer 11, 426. Diaz, Jr., L.A. and Bardelli, A. (2014) Liquid biopsies: genotyping circulating tumor DNA. Am. Soc. Clin. Oncol. 32, 579 Tumor cells Apoptosis Necrosis/secretion Exosome free-circulating DNA Normal circulatingNA Small RNA Liquid biopsy overview
  14. 14. Sample to Insight ctDNA: detection and challenges 14 Methods for detecting circulating tumor DNA based on the differences of ctDNAvs normal cfDNA by cancer-associatedgenetic mutations: • point mutations • copy number variations • chromosomal rearrangements • methylation patterns Challenges of current ctDNAassays • Sensitivity: sometimes extremely low levels of ctDNA (1.0%) are present • DNA extraction: the pre-analytical phases of cfDNA need to be better defined • Assay standardization: procedures need to be standardized • Quantification: accurate quantification is needed • Variability of assay platform: different platform assays have different assay sensitivity and specificity and analytical approach Diaz, Jr., L.A. and Bardelli, A. (2014) Liquid biopsies: genotyping circulating tumor DNA. Am. Soc. Clin. Oncol. 32, 579. Liquid biopsy overview
  15. 15. Sample to Insight Area 3: exosomes in cancer progression 15 Exosomes: small membrane vesicles (30–100 nm), secreted by most cell types into the bloodstream. • Exosomes play a central role in cell-to-cell communication. • The majority of DNA associated with tumor exosomes is double-stranded, representing whole genomic DNA. • Biological molecules (protein, RNA and miRNA) contained in exosomes are well protected by a lipid bilayer membrane that confers a high degree of stability. • Exosomal RNAs contain fingerprints for various diseases, so have potential as liquid biopsy Functional biomolecules: • DNA fragments (exosomal DNA, exoDNA) • Proteins and/or peptides • Lipids • mRNA (exoRNA) • microRNA (miRNA) • Long non-coding RNA (lncRNA) Rolfo, C. et al. (2014) Liquid biopsiesin lungcancer: the new ambrosiaof researchers. Biochimica et Biophysica Acta 1846, 539. Klevebring, D. et. al. (2014) Evaluation of exome sequencing to estimate tumor burden inplasma. PLOS One 9, e104417. Liquid biopsy overview
  16. 16. Sample to Insight Exosomes: isolation methods and challenges 16 Rolfo, C. et al. (2014) Liquid biopsiesin lungcancer: the new ambrosiaof researchers. Biochimica et Biophysica Acta 1846, 539. Challenges: improve and standardize methods for exosomes isolation • Many current protocols to isolate exosomes use ultracentrifugation based on size • Long processing time • Irreproducible and not selective for tumor exosomes • Different RNA isolation methods give extensive variation in exosomal RNA yield and patterns • Often fail to distinguish between differently-sized exosomes and membrane-free macromolecular aggregates, so should be taken cautiously Current methods • Differential centrifugation • Size exclusion • Immunoaffinity isolation • Microfluidic devices • Polymeric precipitation (ExoQuick) Issues • Long processing time • Cannot achieve absolute separation • Low reproducibility • High contamination • Low yields • Low purity Needs • Rapid, exosome- specific extraction • Simple, effective release from exosomes • Highly sensitive, specific detection Exosomal RNAs characterization methods: RNA-seq, microarray and real-time PCR based approaches. Liquid biopsy overview
  17. 17. Sample to Insight Exosomal RNAs 17 mRNAs: • mRNAs released into the circulation are stable because they are protected from degradation by being packaged into exosomes • The level of exosomal RNA implies genetic information miRNAs: • miRNAs are ~21 nt and have the potential to serve as diagnostic markers • miRNA expression is frequently deregulated in cancer • In blood, miRNAs are highly stable because most of them are included in apoptotic bodies, microvesicles or exosomes and can withstand known mRNA degradation factors Long non-coding RNAs (lncRNAs): • Play regulatory roles in cancer progression and metastasis • The long non-coding PCA3 RNA-based urine test is the first FDA-approved test for the diagnosis of prostate cancer patients Schw arzenbach, H. et. al. (2013) Cell-free nucleic acids as biomarkers in cancer patients. Nat. Rev. Cancer 11, 426. Rönnau, C.G.H. (2014) Noncoding RNAs as novel biomarkers in prostate cancer. Biomed. Res. Int. 2014; 591703: 17 Liquid biopsy overview
  18. 18. Sample to Insight Agenda Liquid biopsy overview 18 The main areas of Liquid Biopsy ▪ Circulating tumor cells ▪ Circulating tumor nucleic acid ▪ Exosomes, miRNA, and lncRNA Solutions provided by QIAGEN Questions 1 2 3
  19. 19. Sample to Insight Liquid biopsy solutions at QIAGEN – Sample to Insight Liquid biopsy overview 19 For non-invasive biomarker discovery: Sample to Insight Free-circulating nucleic acids • QIAamp Circulating NA Kit • QIAsymphony Circulating NA Kit • miRNeasy Serum/Plasma Kit Exosomes • exoEasy Maxi Kit for intact exosome isolation • exoRNeasy Serum/Plasma Kits for exoRNA isolation . Circulating tumor cells • Single cell analysis for amplification • The AdnaTest for CTC detection Sample Separate plasma Isolate analytes Real-time PCR NGS technology Analytical Blood draw (venipuncture) Pre-analytical Insight
  20. 20. Sample to Insight Solutions for circulating tumor cells 20 REPLI-g® Single Cell technology to analyze circulating tumor cells Liquid biopsy overview
  21. 21. Sample to Insight Solutions for CTCs 21 Learn about the technology: http://www.qiagen.com/us/resources/technologies/wga Read a white paper: “Genomic analysis of individual cells by NGS and real-time PCR” Problem • Incomplete or biased genome amplification with missing or underestimated sequence information Solutions • REPLI-g Single Cell Kit • Optimized Multiple Displacement Amplification (MDA) process with optimized form of Phi 29 DNA polymerase Results • High yield of high-molecular-weight DNA • High fidelity amplification – minimal error rate • Amplification of both DNA and RNA from a single sample for direct analysis with high accuracy and minimal amplification bias. QIAGEN’s REPLI-g Single Cell technology: • For fast and accurate genomic analysis of CTCs and single cells • DNA or RNA can be successfully amplified from just a single cell Liquid biopsy overview
  22. 22. Sample to Insight REPLI-g Single Cell technology 22 Easy-to-use single-cell WGAand WTAmethod • Offers complete genome/transcriptome coverage • Minimizes sequence bias, allowing for discovery of cell heterogeneity • Consistent yields of up to 40 μg from 1–1000 cells (average product length >10 kb) 15 min Whole genome amplification workflowThe REPLI-g WGA Single Cell Kit: for whole genome amplification (WGA) from samples as small as a single cell (1 tumor cell). The REPLI-g WTA Single Cell Kit: for transcriptome amplification (WTA) from samples as small as a single cell (1 tumor cell). The REPLI-g Cell WGA & WTA Kit: enables uniform WGA and WTA in parallel from a single sample. Start with the lysis of 25–1000 cells and easily get up to 40 µg of gDNA and cDNA, which can be used for comparative genome and transcriptome analysis via NGS, qPCR or microarray analysis. REPLI-g Single Cell WGA REPLI-g Single Cell WTA REPLI-g Single Cell WGA & WTA Liquid biopsy overview Learn about the technology: http://www.qiagen.com/us/resources/technologies/wga Read a white paper: “Genomic analysis of individual cells by NGS and real-time PCR”
  23. 23. Sample to Insight REPLI-g Single Cell technology 23 Unbiased amplification of DNA and RNA! Complete genome coverage Maximized genome coverage • Analysis of 267 loci spread out over different chromosomes across the entire human genome • Performed using RT2 qPCR Primer Assays (QIAGEN) • Low and consistent CT values, with no dropout from any marker, indicating a successful DNA amplification from all areas of the genome Maximized transcript coverage • Comparative genome and transcriptome analysis after parallel WGA and WTA from the same limited cell sample • An RT2 Profiler PCR Array was used to analyze over 60 genes and their corresponding transcripts • Link the genome to the corresponding gene expression profile and to its phenotype Liquid biopsy overview Learn about the technology: http://www.qiagen.com/us/resources/technologies/wga Read a white paper: “Genomic analysis of individual cells by NGS and real-time PCR”
  24. 24. Sample to Insight Applications of REPLI-g Single Cell Kits 24 WGA or WTA Whole Genome Sequencing • Detect variability in genome sequence (SNV, microsatellites, etc.) • Variability in genome structure (CNV, structural rearrangements, aneuploidy) • De novo sequencing of new, unidentified, and unculturable organisms TargetedDNASequencing • Detect variability in a target set of genes or region of the genome Microarrays • Use SNP-chips to genotype thousands of loci RNA-seq • Detect variability in transcript abundance for all expressed genes • Detect variability in isoform structure and abundance qRT-PCR profiling • Profile gene expression for a targeted set of transcripts • Accurately quantify specific splice-junctions, isoforms or other structural features Liquid biopsy overview
  25. 25. Sample to Insight AdnaTest: the CTC revolution Blood sampling Determination of prognostic or predictive biomarkers (liquid biopsy) Turn around time: 5 h CTC enrichment using multi AB labelled magnetic beads Lysis of the enriched cells RT and multiplex PCR Two-step procedure for the enrichment of CTCs from blood samples 1. Immunomagnetic capturing using a multi-antibody mix 2. Multiplex RT-PCR to determine a number of tumor associated transcripts • Every AdnaTest uses different antibody mixtures optimized for each tumor entity • This method is an easy and straightforwardmanual assay Type AdnaTestinto the questions box to request further information 25Liquid biopsy overview
  26. 26. Sample to Insight Solutions for circulating nucleic acid (ctNA) 26 Isolation of free-circulating DNA and RNA Liquid biopsy overview
  27. 27. Sample to Insight Circulating nucleic acids: analysis workflow Sample Separate plasma Extract circulating nucleic acids: • QIAamp Circulating NAKit • QIAsymphony Circulating NA Kit • miRNeasy Serum/Plasma Kit • Real-time PCR • NGS • Digital PCR therascreen assays Analytical Blood draw (venipuncture) Pre-analytical Insight QIAamp Circulating Nucleic Acid Kit: For isolating free-circulating DNA and RNA from human plasma or serum and other cell-free body fluids 27Liquid biopsy overview
  28. 28. Sample to Insight QIAamp Circulating Nucleic Acid Kit 28 Simple procedure and high performance: • 4 steps: lyse, bind, wash and elute • Allows simultaneous processing of multiple samples • Provides nucleic acids in less than 2 hours per 24 samples • No organic extraction or ethanol precipitation • Removal of contaminants and inhibitors • Suitable for human plasma, serum and urine Applications: Purified and concentrated circulating DNAand RNA is free of proteins, nucleases and other impurities, and is ready to use in wide range of downstreamapplications, including: • Biomarker research and verification for blood-based cancer detection • PCR and quantitative real-time PCR and RT-PCR • Viral nucleic acid detection http://www.qiagen.com/search/qiaamp-circulating-nucleic-acid-kit/#orderinginformation Purify & concentrate: Concentration of nucleic acids, with high input (5 ml) and low elution volumes (20–150 µl). Liquid biopsy overview
  29. 29. Sample to Insight QIAamp Circulating Nucleic Acid Kit 29 http://www.qiagen.com/search/qiaamp-circulating-nucleic-acid-kit/#orderinginformation Improved recovery of fragmentedDNA Reproducible recovery of circulating DNA Efficient purification with reproducible yields provides a representative population of the circulating nucleic acids in blood. Advanced technology involving selective binding to a silica-based membrane enables improved recovery of fragmented nucleic acids. Simplify your analysis of tumor-specific extracellular DNA fragments and mRNAs in the blood! Liquid biopsy overview
  30. 30. Sample to Insight Circulating nucleic acids: analysis workflow miRNeasy Serum/Plasma Kit: For purification of circulating RNA – primarily miRNAs and other small RNAs – from plasma, serum, CSF, urine, saliva, etc. 30Liquid biopsy overview Sample Separate plasma Extract circulating nucleic acids: • QIAamp Circulating NA Kit • QIAsymphony Circulating NA Kit • miRNeasy Serum/Plasma Kit • Real-time PCR • NGS • Digital PCR therascreen assays Analytical Blood draw (venipuncture) Pre-analytical Insight
  31. 31. Sample to Insight miRNA & mRNA isolation from blood 31 • Purifying RNA and miRNA from 200 µl serum or plasma • Minimal elution volume (14 µl) • Easy procedures and automatable protocol • High-purity RNA suitable for a variety of downstream applications: • Biomarker discovery • Northern blot analysis • Quantitative, real-time RT-PCRusing the miScript PCR system • Microarray analysis http://www.qiagen.com/search/mirneasy-serumplasma-kit/#orderinginformation Liquid biopsy overview
  32. 32. Sample to Insight Solutions for exosomes 32 Isolation of exosomes and exoRNAs Liquid biopsy overview
  33. 33. Sample to Insight Isolation of exosomes and exoRNAs 33 exoRNeasy Serum/Plasma Kits • Isolate RNA from exosomes and other extracellular vesicles from serum or plasma samples • Novel and efficient workflow: from sample to extracellular vesicle RNA in just 1 h https://www.qiagen.com/us/shop/sample-technologies/rna/rna-preparation/exorneasy-serum-plasma-kits/ exoEasy Maxi Kit Isolate intact exosomes and other extracellular vesicles from: • Plasma • Serum • Cell culture supernatant https://www.qiagen.com/shop-old/sample-technologies/exoeasy-maxi-kit/ Liquid biopsy overview
  34. 34. Sample to Insight exoEasy Maxi Kit: principle and protocol 34 Starting material: serum, plasma or cell culture supernatant • Isolation of bioactive exosomes and extracellular vesicles • Can be used for cell culture work • Speed: 25 minutes – no time-consuming procedure • Allows much shorter time-to-analysis • Ease of use allows processing of multiple samples in parallel • Standardization • Highest specificity for vesicle isolation through affinity membrane-binding technology • Excludes non-vesicular protein complexes and results in cleaner preparations Liquid biopsy overview
  35. 35. Sample to Insight exoEasy Maxi Kit: elute intact vesicles 35 Highest specificity through affinity membrane-binding technology to ensure delivery of intact vesicles of the expected size • Scanning electron microscopy images of isolated vesicles from ultracentrifugation and eluate from exoEasy column • exoRNeasy Serum/Plasma Kits yield a cleaner preparation Liquid biopsy overview
  36. 36. Sample to Insight exoRNeasy Serum/Plasma Kits: principle and protocol 36 Pre-filter plasma to remove particles larger than 0.8 μm Microvesicle isolation: 20 minutes exoRNA isolation: 35 minutes Liquid biopsy overview
  37. 37. Sample to Insight *Arroyo, J.D. et al. (2011) Argonaute2complexescarry a population of circulatingmicroRNAsindependent of vesiclesin human plasma. Proc. Natl. Acad. Sci. USA 108, 5003. mRNAs exclusively within vesicles – near 100% bound miRNA in vesicular and non-vesicular fractions (e.g., free Ago2 complexes) exoRNeasy: captures all mRNAs and vesicle-specific miRNAs 37Liquid biopsy overview
  38. 38. Sample to Insight Circulating nucleic acids: analysis workflow 38Liquid biopsy overview Sample Separate plasma Extract circulating nucleic acids: • QIAamp Circulating NA Kit • QIAsymphony Circulating NA Kit • miRNeasy Serum/Plasma Kit • Real-time PCR • NGS • Digital PCR therascreen assays Analytical Blood draw (venipuncture) Pre-analytical Insight
  39. 39. Sample to Insight Solutions for circulating miRNA biomarkers 39 miScript miRNA PCR Arrays Liquid biopsy overview
  40. 40. Sample to Insight miScript miRNA PCR system 40 • miRNome • Human: miRBase v21, covers 2402 primer assays • Mouse: miRBase v21, covers 1765 primer assays • Rat: 653 primer assays • Dog: 277 primer assays • Rhesus macaque: 469 primer assays • Cow: 744 primer assays • Pathway-focused arrays (>20 arrays) • Serum and plasma • miFinder • Cancer PathwayFinder • Liver miFinder • Brain cancer • Breast cancer • Ovarian cancer • Prostate cancer • Cancer stem cells • Apoptosis miScript PreAMP Kit • Optional step for small or precious samples • Full miRNome profiling from as little as 1 ng RNA Pre-formatted, single-use PCR arrays with wet-lab verified assays Liquid biopsy overview
  41. 41. Sample to Insight miScript miRNA PCR Array workflow 41 Workflow 1. Isolate total RNA 2. Performreverse transcription 3. Prepare PCR pre-mix 4. Load PCR arrays and perform real-time PCR 5. Analyze data  1 hour  2 minutes  2 hours  15 minutes 1. 2. 3. 4. 5. Request webinar slides for miRNA webinar series to learn more  type “miRNA webinar” in questions box. Liquid biopsy overview
  42. 42. Sample to Insight miScript miRNA PCR Array applications 42 miScript Serum & Plasma 384HC PCR Array profiled 381 miRNAs isolated using exoRNeasy Kits miRNA distribution Abundance of miRNAs inside and outside extracellular vesicles Liquid biopsy overview
  43. 43. Sample to Insight Solutions for lncRNAs: novel biomarkers in cancer 43 RT2 lncRNA PCR Arrays and Assays Liquid biopsy overview
  44. 44. Sample to Insight RT2 lncRNA qPCR system 44 • lncRNAdatabases: in-house database at QIAGEN GeneGlobe provides cover > 40,000 human and 27,000 mouse lncRNAtargets. • RT2 lncRNAassays: laboratory-verifiedfor optimal qPCR performance – high specificity, amplification efficiencyand sensitivity. • RT2 lncRNAqPCR Arrays: pathway or disease relevant lncRNA assays • RT2 lncRNA Cancer PathwayFinder Array (human and mouse) • RT2 lncFinder PCR Array (human and mouse) • Custom option: flexible custom design from the lncRNAand qPCR databases to profile mRNA and lncRNA simultanously. • lncRNAisolation: exoRNeasy or miRNeasy Kits • Data analysis: free online data analysis tool http://www.qiagen.com/us/landing-pages/lncrna/ Liquid biopsy overview
  45. 45. Sample to Insight RT2 lncRNA qPCR Array: applications 45 Flexible layout and patented controls Each 96-well plate has 84 lncRNA-specific assays, 5 reference genes and genomic DNA, reverse transcription and PCR controls. Arrays are also available in 384-well plates and 100-well ring discs for the Rotor-Gene Q. http://www.qiagen.com/us/landing-pages/lncrna/ Volcano plot of lncRNA gene expression changes in stage II prostate cancer tissue compared with normal tissue. Liquid biopsy overview
  46. 46. Sample to Insight Solutions for next-generation sequencing 46 NGS portfolio: complete solutions for NGS Liquid biopsy overview
  47. 47. Sample to Insight Complete NGS solutions from “Sample to Insight” 47 • Streamlined, standardized and automated sample-to-insight workflow • Overcome challenges at every step in your DNA-NGS workflow Isolate high- quality DNA Amount of DNA Long turnaround time Panel contents Panel performance Number of libraries per sample Platform dependence Massive amounts of data require expertise to get biological insight REPLI-g Single Cell Kit REPLI-g Cell WGA/WTA Kit GeneRead DNAseq Targeted Panel V2 DNA Amp Kit GeneRead Size Selection Kit Compatible with major sequencing platforms GeneRead data analysis portal (free) CLCbio Cancer Genomics Workbench Ingenuity® Variant Analysis™ Challenges Solutions NGS webinar series: learn more  type “NGS webinar” in the questions box to request webinar slides. Sample isolation Targeted enrichment Library construction NGS run Data analysis InterpretationSample Insight Sample QC Library QC Variant confirmation qBiomarker Somatic Mutation AssaysGeneRead DNAseq Library Quant KitGeneRead DNA QuantiMIZE System Liquid biopsy overview
  48. 48. Sample to Insight We provide service – send samples to us & receive results 48 . Whole genome • Illumina gene expression profiling • Illumina genotyping . Pathway/focused panels • Mutation profiling • Methylation • PCR arrays • miRNA PCR arrays • NGS . Individual gene/locus • Mutation detection • Methylation • qPCR • NGS . Sample preparation – DNA, RNA extraction and purification • Cells, tissues or biofluids • Fixed tissue • Small samples . http://www.qiagen.com/products/catalog/services/ Liquid biopsy overview
  49. 49. Sample to Insight Summary of products offered at QIAGEN 49 1 Single-cell technology – REPLI-g WGASingle Cell Kit 2 CTC enrichment and detection – AdnaTest and AdnaPanel 3 Sample isolation technology 1. QIAamp Circulating NA Kit 2. miRNeasy Serum/Plasma Kit 3. exoRNeasy Serum/Plasma Kit 4 Assay technology 1. miScript miRNA PCR Arrays for circulating miRNAbiomarker discovery 2. RT2 Profiler lncRNAPCR Arrays for lncRNA biomarker discovery 3. Complete NGS workflow to allow Sample to Insight 5 Service – We provide expert service Liquid biopsy overview
  50. 50. Sample to Insight Agenda Liquid biopsy overview 50 The main areas of liquid biopsy • Circulating tumor cells • Circulating tumor nucleic acid • Exosomes, mRNA, miRNA and lncRNA Solutions provided by QIAGEN Questions 1 2 3
  51. 51. Sample to Insight 51 Thank you for attending today’s webinar! Contact QIAGEN Call: 1-800-426-8157 Email: BRCsupport@QIAGEN.com Wei Cao, PhD Wei.Cao@QIAGEN.com QIAwebinars@QIAGEN.com Questions? Thank you for attending Liquid biopsy overview

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