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BIOMARKERS

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Title: BIOMARKERS


1
BIOMARKERS
  • Diagnostics and Prognostics

2
OMICS
Molecular Diagnostics Promises and
Possibilities, p. 12 and 26
3
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4
  • Biomarkers are defined as endogenous or injected
    molecules whose presence or metabolism correlates
    with important disease related physiological
    processes and disease outcomes (Ferber, 2002).
    They should be identified or defined molecular
    entities to facilitate comparison across
    laboratories and technology platforms. A
    biomarker is a characteristic that is objectively
    measured and evaluated as an indicator of normal
    biologic processes, pathogenic processes, or
    pharmacologic responses to a therapeutic
    intervention.

5
  • According to the US National Institutes of
    Healths (NIH) Working Group and the Biomarkers
    Consortium, a biomarker is a characteristic that
    is objectively measured as an indicator of normal
    biological processes, pathogenic processes, or a
    pharmacological response to a therapeutic
    intervention (http//www.biomarkersconsortium.org)
    .

6
TOOLS FOR BIOMARKER DISCOVERY
  • Genomics
  • Ultra-fast DNA sequencing whole human genome or
    large number of samples
  • Connect to genome-wide associated studies (GWAS)
    for the discovery of disease-specific mutations

7
TOOLS FOR BIOMARKER DISCOVERY
  • Transcriptomics
  • Changes in gene expression levels in tissue and
    cells, i.e., microarrays
  • Using as many as 10,000 probes can compare
    numerous patient/normal populations, disease
    states and sample types

8
TOOLS FOR BIOMARKER DISCOVERY
  • Transcriptomics
  • identify genes differentially expressed
  • Oncotype DX breast cancer multigene (21 genes)
    expression evaluated in 14 clinical studies
    involving gt 4,000 breast cancer patients
    worldwide
  • RT PCR of RNA from tumor tissue 21 genes
  • Oncotype DX for colon cancer 12 genes
  • Both used to predict recurrence

9
Oncotype DX Assay
  • RNA extracted from piece of formalin fixed,
    paraffin embedded tissue, i.e., part of tissue
    used for pathology
  • Treated with Dnase 1
  • Measure total RNA concentration
  • Reverse transcription followed by quantitative
    TaqMan (Roche Molecular Systems, Inc.)
  • Each of 16 genes is measured in triplicate and
    then normalized relative to a set of five
    reference genes.
  • Real Time PCR to quantitate 21 genes in the
    sample

10
STEPS USED TO DEVELOP ASSAY
  • optimize RT-PCR technology
  • for high-throughput, real-time quantitation of
    specific RNA in FPET
  • to be reproducible regardless of the variability
    inherent in tumor block
  • 25,000 human genes - identify 250 candidate genes
    possibly associated with breast cancer tumor
    behavior

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12
  • Narrowing 250 candidate genes
  • Analyzed tissue from 447 patients ((3 independent
    clinical studies)
  • Identified panel of genes strongly correlated
    with distant recurrence-free survival
  • Selected 16 cancer genes and 5 reference genes to
    normalize amounts of cancer genes
  • Developed Recurrence Score result calculation to
    combine individual gene results into a single
    result

13
BIOLOGICAL BASIS
14
RECURRENCE SCORE
  • number between 0 and 100 that corresponds to a
    specific likelihood of breast cancer recurrence
    within 10 years of the initial diagnosis
  • lt 18 6.8 risk of recurrence
  • 18-30 14.3
  • gt 30 30.5
  • Guides treatment plan radiation, standard
    chemotherapy, monoclonal antibody therapy

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ONCOTYPE DX
  • Validated only for patients with
  • node-negative, estrogen-receptor-positive
    invasive breast cancer (spread beyond the ducts)

17
  • Validated Oncotype DX assay gene panel and
    Recurrence Score result calculation
  • large, independent, multicenter clinical trial
  • large population-based case-control study
  • Demonstrated consistent statistical link to
    distant breast cancer recurrence, as well as
    robust predictive power regarding chemotherapy
    benefit
  • www.oncotypedx.com

18
NARROWING AND INTERPRETATION REQUIRE
  • Robust bioinformatics and statistical
    capabilities
  • Appropriate data management, ability to extract
    knowledge from massive amounts of data, and
    availability of functional information for data
    interpretation
  • Data
  • Management
  • Analysis
  • Interpretation

19
GENOMICS APPLICATIONS
  • improve understanding of basic biological
    processes and their diversity
  • delineate mechanisms of efficacy and toxicity of
    xenobiotic compounds (e.g., drug, dietary
    supplements, and environmental agents)
  • understand disease processes
  • generate predictive models or molecular
    classifiers to provide better predictive and
    diagnostic accuracy

20
GENOMICS APPLICATIONS
  • animal husbandry, species, aging, and gender
    differences, e.g., genes expressed in liver
    distinguish genders, distinguish different albino
    rats
  • Drug metabolism, drug mechanism of action,
    pharmacotoxicity,
  • Differences/similarities of in vitro/in vivo
    cells, identify stem cell characteristics
  • Kidney disease, heart failure

21
SEQUENCING HOW FAST
  • 10 years to develop draft of 1st human genome
  • Now it takes 10 days

22
FLOW OF INFORMATION
23
PROTEOMICS
  • Biomarkers, proteome and protein-protein
    interaction data, pathways and 3-D structures
  • availability of gene and genome sequence
    databases and the discovery and development of
    protein ionization methods
  • Identify expression of proteins as a function of
    cell or tissue state
  • Widely applied
  • Mass spectrometry (MS) commonly used
  • designed to identify peptides not proteins
  • Identify thousands of proteins within complex
    samples (such as blood, urine, tissue, etc.)
  • identify and quantitate differences in proteins
    between comparative samples (e.g., healthy versus
    diseased)
  • Within hours, e.g., approximately 1000 peptides
    can be confidently identified in a single
    one-hour LC-MS2 experiment

24
MASS SPECTROSCOPY
  • Converts compounds into ions
  • move about and manipulate by external electric
    and magnetic fields
  • Parts
  • Ion source converts into (usually) cations by
    loss of electron
  • Mass analyzer sorts and separates ions
    according to mass and size operates in a near
    vacuum
  • Detector measures separated ions and displays
    in chart form ?

25
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26
  • Prior to MS other techniques used for preliminary
    separation
  • 2D gel electrophoresis
  • Liquid chromatography
  • .

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END
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