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RNA Biomarker Discovery in Exosomes and Liquid Biopsies by Sequencing and qPCR
- 1. RNA biomarker discovery in exosomes and liquid biopsies by sequencing and qPCR September 2017 Peter Mouritzen Vice President R&D
- 2. Agenda ● Analysis of RNA in biofluids ● Overcoming the challenges ● A comparison of qPCR and NGS in liquid biopsies ● for RNA Biomarker discovery ● Case study ● miRNA biomarkers for prostate cancer in urine 2
- 3. Exiqon Diagnostics – strong focus on RNA in biofluids Diagnostics Diseases ● Colorectal and prostate cancer Technologies ● RNA isolation biofluids ● Exosome isolation ● ISH ● qPCR ● NGS Services ● High throughput RNA isolation biofluids ● NGS instruments HiSeq, NextSeq, MiSeq ● qPCR – 30,000 PCRs per day ● Automation ● SOPs, QC checks and LIMS ● Industry Leading Data Analysis ● Highly skilled and experienced scientists and technicians 3 Profiled over 50,000 samples for over 1800 clients in Pharma/Biotech and Academia since 2006
- 4. 4 Broad product and service portfolio for RNA analysis
- 5. MicroRNAs in liquid biopsies make excellent biomarkers ● Liquid biopsies ● Easy and non-invasive sampling ● microRNAs are excellent diagnostic biomarkers ● Actively secreted from cells e.g. in exosomes ● High stability ● Wide-ranging biological potential ● Limited number 5
- 6. Exosomes stabilize microRNAs in a range of biofluids 1) Valadi et al., Nature Cell Biology 2007 (First demonstration that exosomes contain microRNA) ● Secreted actively by most cell types ● Present in a range of biofluids ● including serum/plasma, urine and CSF ● Carriers for proteins, DNA and RNA1) (microRNA, piRNA, lncRNA & mRNA) ● microRNAs are selectively sorted into exosomes 6
- 7. Experience and specialized protocols are required when working with cell free serum/plasma 7
- 8. Experience and specialized protocols are required when working with cell free serum/plasma 8 RNA Isolation kit + optimized protocol • Samples with low RNA content • qPCR and NGS LNA™ based qPCR Optimized NGS library prep
- 9. Experience and specialized protocols are required when working with cell free serum/plasma 9 Monitor RT and PCR inhibitors (add RNA spike-ins to cDNA synthesis) Monitor RNA isolation efficiency (add RNA spike-ins to lysis buffer) Monitor RNA isolation and NGS library prep efficiency (52 RNA spike-ins added to lysis buffer)
- 10. Experience and specialized protocols are required when working with cell free serum/plasma 10 Unique hemolysis indicator ∆Cq (miR-23a – miR-451) Detects hemolysis down to 0.001% PLoS One. 2016 Apr 7;11(4)
- 11. miRCURY™ Exosome Isolation Kit provides greater yield than ultracentrifugation Nanosight data kindly provided by Dr. Ken Howard Aarhus University miRCURY™ Exosome Isolation Kit Ultracentrifugation Yield comparison ● Similar size distribution of pelleted vesicles ● Greater yield of exosomes compared to ultracentrifugation 11
- 12. miRCURY™ Exosome Isolation Kit increases microRNA signals ● Easy 2 hour protocol ● Concentrate or increase starting volume of biofluid ● Without co-purification of inhibitors 12
- 13. miRCURY™ Exosome Isolation Kit enables detection of more microRNAs in urine 13
- 14. Agenda ● Analysis of RNA in biofluids ● Overcoming the challenges ● A comparison of qPCR and NGS in liquid biopsies ● for RNA Biomarker discovery ● Case study ● miRNA biomarkers for prostate cancer in urine 14
- 15. High mapping percentages from liquid biopsy and exosome samples ● > 80 % of reads can be mapped in a typical biofluids microRNA sequencing project Typical distribution of mappable reads obtained by Exiqon biofluids microRNA sequencing
- 16. Exiqon’s LNA™ microRNA PCR system demonstrates excellent concordance with NGS results 16 Adapted from Mestdagh et al. Nature Methods 2014 Further information: exiqon.com/miRQC The largest peer- reviewed microRNA benchmarking study to date 12 platforms 14 samples 8 parameters
- 17. miRNome panel I+II microRNAexpression microRNAs • qPCR is a hypothesis-driven experimental set-up • Requires previous knowledge • Very accurate detection of even low copy number microRNAs • Profiling or validation of a subset of microRNAs • Requires smaller amount of sample Novel and less abundant/described microRNAs Platform sensitivity Advantages and limitations and of qPCR Most sensitive, specific and accurate detection method 17
- 18. All microRNA microRNAexpression microRNAs • NGS is hypothesis-free • Possibility to detect “all” microRNAs • Discovery of novel microRNAs and isomiRs • Requires a larger amount of sample • Possible ligation bias in library prep • PCR amplification in library prep Platform sensitivity Advantages and limitations of NGS Detection and quantification of novel microRNAs and isomiRs 18
- 19. Click to add date microRNAs detected by qPCR and/or NGS • Majority not present on qPCR panels • IsomiRs Possible adaptor ligation bias in library prep 19
- 20. Exiqon’s LNA™ microRNA PCR system demonstrates excellent concordance with NGS results in serum 20 Blondal T et al 2017, Methods Mol Biol. University Hospital of Pisa Ferruccio Bonino Maurizia Rossana Brunetto Daniela Cavallone
- 21. Agenda ● Analysis of RNA in biofluids ● Overcoming the challenges ● A comparison of qPCR and NGS in liquid biopsies ● for RNA Biomarker discovery ● Case study ● miRNA biomarkers for prostate cancer in urine 21
- 22. Today: Too many unnecessary biopsies on healthy men PSA blood test is not reliable 7 in 10 men with raised PSA are CANCER FREE 1 in 10 men with normal PSA DO have cancer PSA grey-zone (3-10 ng/ml) • Very low predictive value (25%) • About 40% of performed tests falls into this category Biopsy carries a risk • Invasive procedure • Risk of infection/bleeding/sepsis Consequences • Cancer is not detected in some patients • Too many healthy men undergo invasive biopsies • About 2.3 million biopsies performed annually • About 1.5 million biopsies per year could be avoided with more reliable tests Better non-invasive tests for prostate cancer are needed 22 Loeb & Catalona, Ther Adv Urol 2011 Aarhus University Hospital Skejby Torben F. Ørntoft Karina D. Sørensen Jacob Fredsøe The University of Sheffield James Catto
- 23. Simple workflow of Exiqon’s urine test 23
- 24. Prostate cancer biomarker discovery and validation overview BPH: Benign ProstaticHyperplasia 24
- 25. A 3-microRNA signature supports diagnosis in all three cohorts The urine microRNA signature identifies individuals with prostate cancer in independent cohorts DISCOVERY VALIDATION DK – no prostate massage, exosomes isolated UK, prostate massaged No exosome isolation 25 Fredsøe et al. 2017; Eur Urol Focus.
- 26. The 3-microRNA signature identifies individuals with prostate cancer in the PSA grey-zone group PPV ACC SENS SPEC Applied cut-off 0.98 0.91 0.92 0.89 (Prevalence 0.78) Cohort 2 sub-population (PSA< 10 ng/ml) 26 Fredsøe et al. 2017; Eur Urol Focus.
- 27. Summary ● Applying biofluids as clinical samples for RNA biomarker development requires ● Sensitive detection methods ● Adapted protocols ● RNA quality assessment ● The LNA™ based qPCR shows high concordance with NGS ● microRNA are promissing biomarkers ● A 3 microRNA signature for prostate cancer 27
- 28. Thank you for your attention