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Finding Biomarkers for Transplantation

Finding Biomarkers for Transplantation. Raymond Ng (Computer Science & iCapture, UBC rng@cs.ubc.ca). Overview of Application. Focus on the Genome Canada project entitled “ Better Biomarkers Of Acute and Chronic Allograft Rejection” ( www.allomark.ubc.ca )

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Finding Biomarkers for Transplantation

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  1. Finding Biomarkers for Transplantation Raymond Ng (Computer Science & iCapture, UBC rng@cs.ubc.ca) Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  2. Overview of Application • Focus on the Genome Canada project entitled “Better Biomarkers Of Acute and Chronic Allograft Rejection” (www.allomark.ubc.ca) • Led by Drs. Paul Keown, Bruce McManus and Rob McMaster • 3-year project starting January 2005 • $9.1 million over 3 years including contributions from Novartis and IBM Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  3. Definitions • Acute Rejection • Injury that typically occurs within weeks or a few months after a solid organ is transplanted • Chronic Rejection • Injury that occurs over time to a transplanted organ • This injury occurs mostly in the blood vessels of the organ • Accommodation • Absence of either form of rejection • (current means of detecting rejection can be very invasive, e.g., frequent biopsies) Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  4. The Overall Goal To establish effective, minimally-invasive and affordable markers that reliably predict rejection of heart, liver, and kidney allografts Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  5. How do we accomplish this goal? • Determine patterns of gene expression in white blood cells that react specifically to the transplanted organ • Identify protein biomarkers in the plasma • Put the identified gene and protein markers together, and use new mathematical tools to determine the best predictors of and diagnostics for rejection Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  6. Milestones • Year 1 • To find possible biomarkers in blood that predict rejection • Year 2 • To evaluate how well the biomarkers found in Year 1 predict rejection in a separate set of patients • Years 3-5 • To use the biomarkers in clinical trials to further test their ability to predict rejection • To also use these biomarkers to personalize existing immunosuppressive treatment • To identify novel targets for new drug development Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  7. Discovery Strategy Biopsy Tissue Alloreactive T cells DE NOVO Patients <=1 year post-transplant CURRENT Patients 1-5 years post-transplant Acute Rejection Accommodation Chronic Rejection Blood, Urine, Tissue Immunology Laboratory BioLibrary Dr. Paul Keown Anonymized Data Biomarker Database Dr. Raymond Ng RNA Extraction from Blood, Alloreactive T Cells and Biopsy Tissue Jack Bell Research Centre Dr. Alice Mui Plasma Depletion Jack Bell Research Centre Dr. Robert McMaster Pax-gene Blood RNA Amplification and Affymetrix GeneChip Analysis Microarray Core Laboratory, Children’s Hospital, LA Dr. Tim Triche ITRAQ Analysis UVic Genome BC Proteomics Platform Victoria, BC Dr. Christoph Borcher Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  8. Proteomics Team Rob McMaster Lead Ross MacGillivray Co-Lead Janet Wilson-McManus & Martha Casey-Knight Novartis Andreas Scherer, Georges Imbert, Nelson Guerreiro, Stephan Gatzek Jack Bell Axel Bergman UVic Christoph Borcher Derek Smith Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  9. Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  10. 8 7 6 Non-rejection 5 Rejection log2(expression) 4 3 2 bl TPr-2 TPr-1 TPr TPr+1 TPr+2 TPr+3 TPr+4 TPr+5 TPr+6 TPr+7 Time An Example Protein Biomarker Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  11. The Major Plasma Proteins Leigh Anderson Leigh Anderson Dynamic range of plasma proteins: 1 pg/ml to 50 mg/ml (1010 range) Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  12. Depleted Plasma Column 1: albumin, fibrinogen, IgG, IgA, IgM, a1-antitrypsin, transferrin, haptoglobin, a1-acid glycoprotein , HDL Apolipoprotein A-I, HDL Apolipoprotein A-II, a2-macroglobulin Column 2: Apolipoprotein B, Complement C3 Biomarkers in TransplantationDiscovery Strategy: Proteomics Analysis Plasma Jack Bell Research Centre, Dr. Robert McMaster BioLibrary ITRAQ Analysis UVic Genome BC Proteomics Platform Victoria, BC Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  13. Peptide + iTRAQ Labeling (Applied Biosystems) 12 12 14 16 14 114 13 13 14 16 14 115 13 13 15 17 14 116 13 13 15 18 15 117 Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  14. Plasma Biomarker Discovery iTRAQ Experimental Design Each depleted plasma sample is digested with trypsin All 4 samples are pooled Quantitative 2DLC / MS/MS analysis Protein identification and differential expression analysis Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  15. 5 4 3 2 1 2 3 4 5 Plasma Biomarker Identification by iTRAQ Technology Heart acute rejection plasma : normal plasma number of proteins decrease increase iTRAQ ratio (117:114) Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  16. Informatics Team Raymond Ng Robert Balshaw Co-Leads Janet Wilson-McManus & Martha Casey-Knight Data Management Data Analysis iCAPTURE Mark Wilkinson Nina Opushneva, Wendy Alexander, Joe Comeau, Andrew Ferris iCAPTURE Bruce McManus, Mark Wilkinson, Zsuzsanna Hollander, Andrew Ferris Trainees: Benjamin Good, Gabriella Cohen Freue, Jon Carthy UBC Wyeth Wasserman Epicenter Tim Triche, Jonathan Buckley Novartis Andreas Scherer, Peter Grass Novartis Andreas Scherer, Mischa Reinhardt IBM Paul Moody, Tony Li Mahendran Maliapen, Agata Szewczyk IBM Jeff Betts, O.K. Baek, Kareem Saad, Usha Reddy, Prasanna Athma Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  17. 50,000 Genes, ESTs, Proteins Preliminary gene / protein selection 10,000 Genes, Proteins Secondary gene / protein selection Biological Knowledge Analytical Strategy Clinical Data 60-120 Genes / Proteins Statistical model building Biological Knowledge Clinical Data <10 Predictive Markers Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  18. Filtering Methods Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  19. Questions & Methods Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  20. Data Validation Checks • Out of range values • Data inconsistencies • performed within and across visits for an individual patient, as well as across all patients in the study • Warnings (flags) generated when manual verification is required • More to discuss later in the “Frontiers” session about QC issues Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  21. Concluding Remarks • Consistency of our sample handling is vital for this project • Depletion of plasma is key to our workflow. The more depleted the plasma is, the more sensitive our method becomes in order to identify a potential Biomarker in pg/mL range • A time-course analysis adequately supported by the four channels of iTRAQ Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  22. Plasma Biomarker Discovery Using iTRAQ Technology MS/MS spectra 114 115 Peptide quantitation reporter tags 117 Protein identification Plasminogen peptide FVTWIEGVMR 116 Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

  23. Plasma Protein Identification by iTRAQ Dynamic Range C4A C3A HGF mg per mL pg per mL That’s 100pg/mL Range with MALDI Apo E Specific to ESI Specific to MALDI Common to both methods Leak from column Better Biomarkers in Transplantation. A Genome Canada Initiative for Human Health

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