BIOMARKERS

Diagnostics and Prognostics

OMICS

Molecular Diagnostics:

Promises and Possibilities, p. 12

and 26

• 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.

• According to the US National Institutes of Health’s (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).

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

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

TOOLS FOR BIOMARKER DISCOVERY

• Transcriptomics– identify genes differentially expressed • Oncotype DX breast cancer multigene (21 genes)

expression evaluated in 14 clinical studies involving > 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

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

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

• 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

BIOLOGICAL BASIS

RECURRENCE SCORE

• = number between 0 and 100 that corresponds to a specific likelihood of breast cancer recurrence within 10 years of the initial diagnosis

• < 18 6.8% risk of recurrence• 18-30 14.3%• > 30 30.5%

• Guides treatment plan: radiation, standard chemotherapy, monoclonal antibody therapy

ONCOTYPE DX

• Validated only for patients with– node-negative, estrogen-receptor-positive

invasive breast cancer (spread beyond the ducts)

• 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

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

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

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

SEQUENCING – HOW FAST

• 10 years to develop draft of 1st human genome

• Now it takes 10 days

FLOW OF INFORMATION

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

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

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

END