BIOMARKERS

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BIOMARKERS

• Diagnostics and Prognostics

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OMICS
Molecular Diagnostics Promises and
Possibilities, p. 12 and 26
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• 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.

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• 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)
  .

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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

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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

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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

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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

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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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• 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

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BIOLOGICAL BASIS
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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)

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• 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

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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

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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

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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

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SEQUENCING HOW FAST

• 10 years to develop draft of 1st human genome
• Now it takes 10 days

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FLOW OF INFORMATION
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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

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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 ?

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

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END