Webinar, February 23rd , 2012, Adam Baker, PhD. adam@exiqon.commicroRNA discovery and biomarker   development in clinical ...
Agenda• How to study microRNAs – introduction to LNA™• Detection of microRNAs in clinical samples and diagnostic developme...
Introduction to microRNAsSix Facts about microRNAs:      1.   Short non-coding RNA molecules of 19-22 nucleotides      2. ...
MicroRNA biogenesis and mode of action • Imperfect complementarity              • Perfect complementarity         → transl...
Reasons to use microRNAs as Biomarkers (1)● Key roles in pathway regulation and tissue differentiation   ● Important regul...
Reasons to use microRNAs as Biomarkers (2)● Highly stable in clinical sample preparations   ● Archival FFPE material   ● S...
Analyzing microRNAs –challenges using traditional DNA technologyFeature                               ChallengeShort seque...
LNA™ technology overviewLNA™ technology increases binding affinity•   LNA is a bicyclic high affinity RNA mimic with the  ...
Analyzing microRNAs –overcoming challenges by using LNA™Feature                      Challenge                LNA™ allowsS...
Variation in full length DNA capture probe Tm due to variationsin microRNA sequences
DNA capture probes have decreased sensitivity for lowTm targets and decreased specificity for high Tm targets
Optimally designed LNA™ capture probes result inunmatched sensitivity and specificity
What are your challenges of microRNA research?Please reply to the survey, we will display the results at the end of the we...
Agenda• How to study microRNAs – introduction to LNA™• Detection of microRNAs in clinical samples and diagnostic developme...
Clinical application of the miRCURY LNA™ISH and Universal RT microRNA PCR System          Clinical tissue samples   Bio-fl...
LNA™ based high Resolution ISH
Exiqon has developed a robust ONE DAY microRNAISH protocol for manual and automated hybridization                         ...
LNA™ ISH technology: miR-21 targets tumor suppressor& Fibroblast activation
LNA™ ISH technology: High resolution miR 126 ISH.Tumor biology of microRNAs in action
Clinical application of themiRCURY LNA™ Universal RT microRNA PCR System
LNA™ Universal RT microRNA PCR System                           Advantages:                           ● Universal RT → no ...
microRNA PCR profiling from as little as 0,18mm2 ofFFPE tissue or 20 µl of plasma                                  ● 20 µl...
Extreme sensitivity allows microRNA PCR profilingfrom clinical samples
Extensive QC and data analysis pipeline • Automated, user-guided QC     pipeline • Data flagging and cleanup     customize...
Normalization of serum/plasma microRNA qPCR datais challenging●   Normalization adjusts for technical biases (RNA amount, ...
Application of the miRCURY LNA™ microRNAPCR System in biofluids          Clinical tissue samples   Bio-fluids
You have to be able to detect the microRNAs present invery limited clinical samples  Exiqon Diagnostics optimized RNA isol...
Blood collection tubes and subsequenteffect on microRNA qPCR               Cq-values
Near perfect linearity at very low concentrations asfound in plasma and serum        Plasma or serum                      ...
Circulating microRNAs are promisingbiomarkersSerum miR-21 associated with relapse-free survival in diffuse large cell B   ...
microRNAs are actively secreted into thecirculation and are stable in serum / plasma                              ● Three ...
microRNAs are actively secreted into thecirculation and are stable in serum / plasmaMicroRNAs in body fluids--the mix of h...
microRNAs in serum have been identified to bepotential biomarkers in many diverse diseases
Normal reference range – Human serum1400+ human samples with over 1 million PCR data points make up the referencerange    ...
Normal reference range – Human serum1400+ human samples with over 1 million PCR data points make up the referencerange    ...
Normal reference range for other biofluidsHuman urine Profile                                             Dynamic         ...
Which sample types do you work with?Please reply to the survey, we will display the results at the end of the webinar.
Agenda• How to study microRNAs – introduction to LNA™• Detection of microRNAs in clinical samples and diagnostic developme...
Unmet need for minimally invasive earlydetection test of Colorectal Cancer • Estimated new cases in 2010 in the USA: 142,5...
Exiqon’s discovery process and PCR platformare compatible with clinical procedures
Development of microRNA Early Detection      Test of CRC in blood plasma                              DISCOVERY PHASE     ...
Focused panel of microRNAs in plasmamay be used as biomarker for CRC   All Samples (n=227)                  Hospital 1 (n=...
microRNA Early Detection Biomarker fromDiscovery to Development          Development of sensitive quantitative PCR platfor...
microRNA Biomarker discovery workflowand panel selection options                     DISCOVERY PHASE                      ...
Service Programs at Exiqon• Customized Assay development, screening, biomarker  discovery or companion diagnostic project ...
Pre clinical Animal Models • Customized sample prep, biomarker isolation, Assay   development, biomarker screening      Cu...
Conclusions● microRNAs are promising biomarkers● microRNAs can be sensitively and reproducibly detected in clinical    sam...
Acknowledgements
THANK YOU FOR YOUR ATTENTION  Webinar, February 23rd , 2012, Adam Baker, PhD. adam@exiqon.com
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microRNA discovery and biomarker development in clinical samples

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microRNA discovery and biomarker development in clinical samples

  1. 1. Webinar, February 23rd , 2012, Adam Baker, PhD. adam@exiqon.commicroRNA discovery and biomarker development in clinical samples
  2. 2. Agenda• How to study microRNAs – introduction to LNA™• Detection of microRNAs in clinical samples and diagnostic development using LNA™ Universal RT microRNA PCR• Case Study and Services – Early detection of colorectal cancer from human patient blood plasma 2
  3. 3. Introduction to microRNAsSix Facts about microRNAs: 1. Short non-coding RNA molecules of 19-22 nucleotides 2. Post-transcriptional regulators of mRNA 3. 1921 annotated human microRNAs* 4. Regulate at least one third of all human genes 5. Phylogenetically well conserved 6. Altered microRNA expression profiles are associated with a variety of diseases (cancer, diabetes, neurological disease, viral infection) *Sanger miRBase release 18.0, November 2011 http://www.mirbase.org
  4. 4. MicroRNA biogenesis and mode of action • Imperfect complementarity • Perfect complementarity → translational repression and → mRNA cleavage mRNA decay
  5. 5. Reasons to use microRNAs as Biomarkers (1)● Key roles in pathway regulation and tissue differentiation ● Important regulatory roles in many diseases● Regulate expression of key proteins in drug response pathways ● Predict response to targeted therapies● Actively secreted into the circulation ● Act as signalling molecules● Integrate biology from entire organism including diseased tissue and host response
  6. 6. Reasons to use microRNAs as Biomarkers (2)● Highly stable in clinical sample preparations ● Archival FFPE material ● Serum and plasma (routinely collected, minimally invasive) ● Other body fluids● Relatively small number of genes to profile● Huge dynamic range (0 to 40,000 molecules per cell)
  7. 7. Analyzing microRNAs –challenges using traditional DNA technologyFeature ChallengeShort sequences (19-22 nt) Hard to achieve high sensitivityHighly homologous families Hard to achieve sufficient specificity(single base differences)Large variation in base composition Hard to design good multiplex assays(GC content varies between 5-95%)
  8. 8. LNA™ technology overviewLNA™ technology increases binding affinity• LNA is a bicyclic high affinity RNA mimic with the sugar ring locked in the 3’-endo conformation• Obeys Watson-Crick base-pairing rules• Stable A-helix with good base-stacking• Increased Tm (Tm increases by 2 - 8ºC per base)• Tm normalization (adjust oligos to the same Tm) K. Bondensgaard et al., Chem. Eur. J. 2000, 6, 2687• Improved mismatch discrimination M. Petersen et al., J. Am. Chem. Soc. 2002, 124, 5974• High sensitivity and specificity in hybridization assays
  9. 9. Analyzing microRNAs –overcoming challenges by using LNA™Feature Challenge LNA™ allowsShort sequences (19-22 nt) Hard to achieve high Increased affinity sensitivityHighly homologous families Hard to achieve Single nucleotide(single base differences) sufficient specificity discriminationLarge variation in base Hard to design good Tm normalizationcomposition (GC content multiplex assaysvaries between 5-95%)
  10. 10. Variation in full length DNA capture probe Tm due to variationsin microRNA sequences
  11. 11. DNA capture probes have decreased sensitivity for lowTm targets and decreased specificity for high Tm targets
  12. 12. Optimally designed LNA™ capture probes result inunmatched sensitivity and specificity
  13. 13. What are your challenges of microRNA research?Please reply to the survey, we will display the results at the end of the webinar.
  14. 14. Agenda• How to study microRNAs – introduction to LNA™• Detection of microRNAs in clinical samples and diagnostic development using LNA™ Universal RT microRNA PCR• Case Study and Services – Early detection of colorectal cancer from human patient blood plasma 14
  15. 15. Clinical application of the miRCURY LNA™ISH and Universal RT microRNA PCR System Clinical tissue samples Bio-fluids
  16. 16. LNA™ based high Resolution ISH
  17. 17. Exiqon has developed a robust ONE DAY microRNAISH protocol for manual and automated hybridization One day Protocol
  18. 18. LNA™ ISH technology: miR-21 targets tumor suppressor& Fibroblast activation
  19. 19. LNA™ ISH technology: High resolution miR 126 ISH.Tumor biology of microRNAs in action
  20. 20. Clinical application of themiRCURY LNA™ Universal RT microRNA PCR System
  21. 21. LNA™ Universal RT microRNA PCR System Advantages: ● Universal RT → no bias, no pre-amplification ● LNA™ in two specific primers → sensitivity and specificity
  22. 22. microRNA PCR profiling from as little as 0,18mm2 ofFFPE tissue or 20 µl of plasma ● 20 µl plasma/serum or 0,18mm2 tissue / One RT reaction ● 96 or 384-well PCR including calibrators / controls ● QC / normalization and data analysis
  23. 23. Extreme sensitivity allows microRNA PCR profilingfrom clinical samples
  24. 24. Extensive QC and data analysis pipeline • Automated, user-guided QC pipeline • Data flagging and cleanup customized for qPCR platform • Diverse normalization protocols implemented • Comprehensive QC report • Visualization of potential technical and sample biases • Identify samples that may be affected by contamination from blood cells
  25. 25. Normalization of serum/plasma microRNA qPCR datais challenging● Normalization adjusts for technical biases (RNA amount, quality etc)● No housekeeping genes are stably expressed in all situations● U6 and 5S are not applicable to serum/plasma samples● With qPCR panels, no prior assumptions about housekeeping genes● Select a normalization method appropriate for the dataset Raw data values Mean centered Mean centered Quantile normalization (top 50 expressed) normalization normalization
  26. 26. Application of the miRCURY LNA™ microRNAPCR System in biofluids Clinical tissue samples Bio-fluids
  27. 27. You have to be able to detect the microRNAs present invery limited clinical samples Exiqon Diagnostics optimized RNA isolation protocol from serum / plasma (Andreasen et al., Methods 50 (2010) S6–S9)
  28. 28. Blood collection tubes and subsequenteffect on microRNA qPCR Cq-values
  29. 29. Near perfect linearity at very low concentrations asfound in plasma and serum Plasma or serum ● Serial dilution of a pool of 647 synthetic microRNAs spiked into plasma
  30. 30. Circulating microRNAs are promisingbiomarkersSerum miR-21 associated with relapse-free survival in diffuse large cell B Lawrie et al., BJH, 2008lymphomahuman prostate cell line implanted into mice and tumor-derived miRNAs Mitchell et al., 2008, PNAS,identified in blood 105(30):10513.lung, colon cancer and diabetes have specific serum-miRNA profiles Chen et al., 2008, Cell Res, 1-10microRNAs differentially expressed in serum of ovarian cancer patients Resnick et al., 2009 Gyn Oncology 112: 55-59microRNAs differentially expressed in circulating tumor-derived exosomes of Taylor et al., 2008, Gynovarian cancer patients Oncology, 110:13Plasma miR-208 as a Biomarker of Myocardial Injury Ji et al., 2009, Clin. Chem. 55: 11Circulating microRNAs, potential biomarkers for Wang et al., 2009, PNASdrug-induced liver injury
  31. 31. microRNAs are actively secreted into thecirculation and are stable in serum / plasma ● Three mechanisms of export: ● Exosomes ● RNA binding proteins ● HDL ● Packaging stabilizes the endogenous microRNAs in serum/plasma Cortez, M. A. et al. Nat. Rev. Clin. Oncol. 8, 467–477 (2011)
  32. 32. microRNAs are actively secreted into thecirculation and are stable in serum / plasmaMicroRNAs in body fluids--the mix of hormones and biomarkers. Cortez et al., 2011, Nat Rev Clin Oncol, 8(8):467-77.Export of microRNAs and microRNA-protective protein by mammalian Wang et al., 2010, Nucleic Acidscells. (nucleophosmin 1, export is energy dependent) Res. 38(20):7248-59.Argonaute2 complexes carry a population of circulating microRNAs Arroyo et al., 2011, PNAS,independent of vesicles in human plasma. 108(12):5003-8.MicroRNAs are transported in plasma and delivered to recipient cells Vickers et al., 2011, Nat Cell Biol.,by high-density lipoproteins. 13(4):423-33.Exosome-mediated transfer of mRNAs and microRNAs is a novel Valadi et al., 2007, Nat Cell Biol.,mechanism of genetic exchange between cells. 9(6):654-9.Glioblastoma microvesicles transport RNA and proteins that promote Skog et al., 2008, Nat Cell Biol.,tumour growth and provide diagnostic biomarkers. 10(12):1470-6.Secretory mechanisms and intercellular transfer of microRNAs in Kosaka et al., 2010, J Biol Chem.,living cells. 285(23):17442-52.Selective release of microRNA species from normal and malignant Pigati et al., 2010, PLoS One,mammary epithelial cells. 5(10):e13515.
  33. 33. microRNAs in serum have been identified to bepotential biomarkers in many diverse diseases
  34. 34. Normal reference range – Human serum1400+ human samples with over 1 million PCR data points make up the referencerange Dynamic Window Of miRNA Expression In serum
  35. 35. Normal reference range – Human serum1400+ human samples with over 1 million PCR data points make up the referencerange Dynamic Window Of miRNA Expression In serum
  36. 36. Normal reference range for other biofluidsHuman urine Profile Dynamic Window Of miRNA Expression In serum
  37. 37. Which sample types do you work with?Please reply to the survey, we will display the results at the end of the webinar.
  38. 38. Agenda• How to study microRNAs – introduction to LNA™• Detection of microRNAs in clinical samples and diagnostic development using LNA™ Universal RT microRNA PCR• Case Study and Services – Early detection of colorectal cancer from human patient blood plasma 38
  39. 39. Unmet need for minimally invasive earlydetection test of Colorectal Cancer • Estimated new cases in 2010 in the USA: 142,570 • Estimated deaths in 2010 in the USA: 51,370 • Early diagnosis results in resectable cancer with much improved prognosis • BUT around 50% of patients are diagnosed at Stage III / IV Stage 5 yr relative Treatment survival 0-I 93% Surgery II 80% Surgery/discretionary adjuvant chemotherapy III 58% Surgery/adjuvant chemotherapy IV 6.9% Chemotherapy
  40. 40. Exiqon’s discovery process and PCR platformare compatible with clinical procedures
  41. 41. Development of microRNA Early Detection Test of CRC in blood plasma DISCOVERY PHASE VALIDATION PHASE Genome wide Normalization, QC, Candidate miRNA Bioinformatics, data Validation Set screening processing discovery screen analysis miRNA signature• 50 controls • Multiple QC • 76 controls • Data analysis • 3000 patients• 50 CRC patients check • 151 CRC patients • Quality control • Defined miRNA • Focused• 742 miRNA • Data flagging Serum/Plasma • ROC curve signature screen • Normalization panel • miRNA selection • Pick & Mix panel• Genome wide • Multiple controls • Multiple controls
  42. 42. Focused panel of microRNAs in plasmamay be used as biomarker for CRC All Samples (n=227) Hospital 1 (n=85) Hospitals 2-5 (n=142) All Samples Hospital 1 Hospitals 2-5 Sensitivity * 75% 80% 73% Specificity * 80% 78% 82% (n) Cancer 151 49 102 (n) Control 76 36 40 * The same cutoff score was applied on all samples in the study
  43. 43. microRNA Early Detection Biomarker fromDiscovery to Development Development of sensitive quantitative PCR platform2009 Optimized for clincial serological samples Conventional PCR. Directly implement in clinical labs Biomarker pre-discovery • 50 stage II CRC + 50 matched healthy (colonoscopy-verified)2010 • Multi-centre, prospective protocol Biomarker complete discovery • 151 stage II/III,76 healthy sex age matched (colonoscopy-verified) • Data Analysis Signature development.2011 Assay development and retrospective validation • 3000 symptomatic individuals • Prospective protocol, multi-centre trial, blood drawn pre-endoscopy • Complete clinical information (other diseases, medicine usage) Prospective validation2012 • 5000 symptomatic individuals • Prospective protocol, multi-centre trial, blood drawn pre-endoscopy • Complete clinical information (other diseases, medicine usage)
  44. 44. microRNA Biomarker discovery workflowand panel selection options DISCOVERY PHASE VALIDATION PHASEGenome wide Normalization, Candidate miRNA Bioinformatics, Validation Set screening QC, processing discovery screen data analysis miRNA signature
  45. 45. Service Programs at Exiqon• Customized Assay development, screening, biomarker discovery or companion diagnostic project development Customized Sample preparation method development Custom discovery,development and validation projects• Full Experimental Design Guidance Consultation on experimental setup for best data quality Broad experience in biomarker discovery, development and validation projects• GLP processes Standard operating procedures Process control and QC checks to demonstrate performance• Industry Leading Data analysis Fully customized data analysis package to fit customers needs. Comprehensive QC processing and statistics. Webex, teleconference and on site support augment data analysis and provide industry leading project feedback.
  46. 46. Pre clinical Animal Models • Customized sample prep, biomarker isolation, Assay development, biomarker screening Customized Sample preparation method devlopment Toxicology studies, xenograph, dose response studies • Full animal Experimental Design Guidance Consultation on specific experimental setup for best data quality Broad experience in biomarker animal model studies, pre clinical and IND /NDA applications. • miRCURY LNA™ Universal RT MicroRNA PCR Standard operating procedures for animal work Process control and QC checks to demonstrate performance • Industry Leading Data analysis Fully customized data analysis package to fit customers needs. Comprehensive QC processing and statistics. Webex, teleconference and on site support augment data analysis and provide industry leading project feedback.
  47. 47. Conclusions● microRNAs are promising biomarkers● microRNAs can be sensitively and reproducibly detected in clinical samples.● Exiqon has developed a one day protocol that allows for sensitive detection of microRNAs in clinical tissue samples.● Without pre-amplification, the miRCURY LNA™ Universal RT microRNA PCR system provides unrivalled sensitivity and specificity in clinical tissue and biofluid samples.● Flexible platform enables whole genome profiling and focused / customizable panels● A plasma microRNA signature for colorectal cancer was developed and performs well in samples from independent hospitals
  48. 48. Acknowledgements
  49. 49. THANK YOU FOR YOUR ATTENTION Webinar, February 23rd , 2012, Adam Baker, PhD. adam@exiqon.com

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