BSR|ProfMa.
Biomarker Technology
Luisa G. Daroy || August 11, 2017
TOPIC OUTLINE
I. History
A. Early biomarkers
B. Era of Molecular Biology
C. 1980s 1990s: Molecular Biomarkers
D. Scientific &Technological Advances in Biomarker
Research
E. Biomarkers Consortium in 2006
II. Definition of Terms
III. Biomarker Development Pathway
A. Pathway
B. Advantages of Biomarker Development
C. Genetic Biomarkers
D. Protein Biomarkers
E. Proteome and Proteomics
IV. Bioinformatics
V. References
VI. Quiz
PPT Audio Book
TRANSERS NOTE: The part IV Bioinformatics of this trans is
in book trans format since it was skipped by the lecturer.
I. HISTORY OF BIOMARKERS
Term introduced by Karpetsky, Humphrey and Levy in April
1977 edition of J National Cancer Institute
7th C, B.C.: Diabetes first associated with sweetness of
urine
A. Early Biomarkers
Uroscopy
Blood pressure
Imaging
Electrocardiography
Hematology
Blood and urine chemistry
B. Era of Molecular Biology
26 February 1953, Cambridge Watson and Crick model of
DNA structure
o 1972: 1st gene sequence (of a bacterium)
o 1976: 1st complete RNA genome sequence
o 1977: 1st complete DNA genome sequence
Genomics, Proteomics, Metabolomics foundations for
the molecular basis of disease
With the advent of molecular technology, the discovery of
DNA molecule in 1953 by Watson and Crick spawned the
revolution in biology and medicine in which you have
complete sequences of the genes which encode for life
processes coming one after the other, laid the foundations in
genomics, proteomics, metabolomics for the molecular basis
of the disease.
Major Scientific Contributions and Research
Infrastructure Supporting Biomarker Discovery: These
are the technologies that gave rise to new novel biomarkers
as we now analyze it the medical practice
o Human Genome Project
o Mouse models of disease (recombinant DNA technology)
o Information management (informatics tools, open-source
databases, open-source publishing, biomarker reference
services.
o Population-based studies and gene-environment
interaction studies
o Computational biology and biophysics
o Medical imaging: structural and functional
o High-throughput technologies - where you have massive
volumes of data being produced in vitro cell-based
screening, nanotechnology platforms, molecular
separation techniques, robotics, automated microassays,
high-resolution optics
o Proteomics, metabolomics, epigenomics
o Pharmacogenomics
o Molecular toxicology
o Genome-wide association studies
o Molecular pathways systems biology and systems
engineering
C. 1980s-1990s: Molecular Biomarkers
Viral and Immune Indicators of disease
o CD4+ T-lymphocyte counts in HIV cases were used as
surrogate endpoints
Target-designed therapies
o Companion diagnostics for cancer therapeutics
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o Clinical cancer therapeutics led to the use of lab tests to
distinguish responders from non-responders to targeted
therapies
o Ex. Philadelphia chromosome in imatinib response in
CML(Chronic Myelogenous Leukemia) cases
o Ex. Her2/neu tyrosine kinase receptor for Herceptin
response in breast cancer patients
D. Scientific&Technological Advances in Biomarker
Research
Completion of the Human Genome Project provided
reference data of human DNA sequences and of other
organisms from which were derived certain biomarkers of
disease.
E. Biomarkers Consortium (2006)
Public-private initiative with industry and government to spur
biomarkers development and validation projects in cancer,
CNS, and metabolic disorders.
II. Definition of Terms
A. Biomarker
A biologic indicator of health or disease.
A characteristic that is measured and evaluated objectively
as an indicator of normal biologic processes, pathogenic
processes, or pharmacologic response to therapeutic
intervention.
B. Surrogate Endpoint Marker
A biomarker that can substitute for a clinical endpoint and is
expected to predict clinical benefit or harm, or lack of benefit
or harm, based on epidemiologic, therapeutic,
pathophysiologic, or scientific evidence.
(2020) something you can observe clinically such as
cardiac troponins, CK-MB for myocardial infarction
C. Uses of Biomarkers
Diagnosing, classifying or grading the severity of disease in
both clinical and laboratory settings
Provide efficacy, toxicity, and mechanistic info for the
preclinical and clinical phases of drug discovery
Together with therapeutics, may be applied to produce
commercial tests that aid patient selection or drug dosing
(personalized medicine)
D. Biomarker Tests
The best biomarkers are:
o Accurate
o Relatively non-invasive
o Easy-to-perform tests that can be done at the bedside or
in an outpatient setting
Examples:
o Blood or spot urine specimens that can be measured
serially and have a fast turnaround
o (2020) rapid PCR test instead of TB culture
E. Biomarkers
Any parameter of a patient that can be measured:
o Genetic mutations
o Gene polymorphisms
o mRNA expression profiles
o Epigenetic markers
o Imaging methods
o Electrical signals
o Lipids
o Proteomic patterns
o Proteins
F. Requirements for Biomarker Research
Innovations in technology applications
New statistical methods and clinical research designs
Data management and informatics
Clinical registries
Repositories of biological specimens
Imaging files
Common reagents
G. Requirements for Successful Biomarker Development
Access to sufficient, well-characterized sample biobanks
(repositories of sufficient and well-characterized samples of
biological specimens)
Understanding of the entire complex biomarker development
process.
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Use of multi-disciplinary team approach.
Validation of every step of both assay performance and
diagnostic utility.
III. BIOMARKER DEVELOPMENT PATHWAY
A. PATHWAY
Step 1. Understand and Define the Disease
Detailed knowledge of the disease: definition, differential
diagnosis, disease subsets, and the local or systemic
responses.
Reliable case definition is very valuable: may be extremely
specific or based on a constellation of symptoms and signs
(systems-based), should include a temporal element.
If the disease is poorly defined, then the newly discovered
biomarker may ultimately change the definition of the
disease.
Step 2. Frame the question: What critical information will
the biomarker provide?
The best biomarkers serve a basic science, translation or
clinical need that advances the diagnosis, prevention or
treatment of the disease.
A biomarker may be targeted at early detection of disease or
to monitor the stage, severity, progression or regression of
disease after diagnosis.
A biomarker may be targeted to predict drug response or
follow the effect of an intervention. (the best question may
not be the most obvious question).
Step 3. Desired site of clinical measurement
Biomarkers can be assayed in easily obtainable fluids;
serum, plasma or urine, or other sites, such as saliva, sweat,
hair and biopsy material. Sometimes a little bit more
invasive: aspirates, CSF, synovial fluid, bone marrow.
Choice is driven by a balance between clinical relevance,
ease of collection and stability versus disease specificity and
analytical simplicity of the discovery step.
B. Advantages of Biomarker Development
Compared with the cost and risk of drug development,
biomarkers offer the opportunity to have an impact on patient
health in a more economical manner and may provide an
opportunity to speed up the drug development process.
Biomarker development leads to earlier disease detection
and prediction of which patients will respond to which
therapies.
C. Genetic Biomarkers
Genetic variation gives rise to unique DNA profiles of each
individual
o Mutations
o Single nucleotide polymorphisms (SNPs)
o Copy number variants (CNV)
o Microsatellites
o mRNA expression profiles
o Epigenetic markers, i.e. DNA methylation patterns
o Micro RNA (miRNA)
o Circulating tumor cells
D. Protein Biomarkers
Protein alterations in disease may occur in many different
ways that are not predictable from genomic analysis; a better
understanding of these alterations in a protein level will have
substantial impact in medicine
Benefits
o Better understanding of disease process
o Development of new biomarkers for diagnosis and early
detection of disease
o Accelerated development of new drugs
E. Proteome and Proteomics

Step 4. Devise a strategy for the discovery process

The biomarker discovery strategy is influenced by the
diagnostic difficulty, disease variability, subclasses,
biomarker goals, ultimate site of measurement, and
discovery platform.
Questions that you have to answer:
o Should the discovery process start on the diseased tissue
or the material that will make up the final clinical assay?
So for example in cancer, should it start in a tumor, which
is highly invasive to get or should it start with the blood?
Should human samples or samples from animal models
be used?
o Should subtractive comparison between normal and
diseased or before and after samples be used, or should
comparison of several groups of samples be made in the
discovery step?

Step 5. Which samples should be used for the discovery
phase?
1. PROTEOME
Samples should match the clinical question as closely as
possible.
Samples should mirror the disease process being
2. PROTEOMICS
investigated.
Samples should come from carefully defined sources, be of
high quality, sufficient in quantity and carefully preserved.
Step 6. Determine which discovery method to use
Measurement of mRNA expression.
2D differential in-gel electrophoresis (proteins)
Surface-enhanced laser desorption ionization (SELDI) mass
spectrometry.
Liquid Chromatography-Tandem mass spectrometry
Tissue microarray
Single nucleotide polymorphisms (SNPs) detection
Step 7. Review the significance and feasibility
Step 8. Perform the experiments and prioritize the hit list
Step 9. Develop a robust clinical assay and initial clinical
evaluation to detect existing disease
Step 10. Evaluate the clinical utility
3. GOALS OF PROTEOMICS
Step 11. Combine biomarker with clinical data and other
biomarkers.
Figure #1. Steps in Protein Analysis
The entire protein complement in a given cell, tissue or
organism.
A global approach to the molecular biology study of the
overall distribution of proteins in cells, identification and
characterization of individual proteins of interest, and the
elucidation of their relationships and functional roles
(example: biomarkers for heart disease: apolipoprotein,
cytokine protein, interleukin, cystatin C).
Characterize the information flow through the protein
circuitry that interconnects the extracellular
microenvironment to the serum or plasma macro-
environment thru intracellular signaling systems and their
control of gene transcription.
Functional Proteomics function of a particular protein
Differential Proteomics differentiates between diseased
and normal or before and after
By studying global patterns of protein content and activity,
and how these change during development or in response
to disease, proteomics research is poised to boost our
understanding of systems-level cellular behavior.
Clinical research also hopes to benefit from proteomics by
both the identification of new drug targets and the
development of new diagnostic markers.
To define the identities, quantities, structures and
functions of complete complements of proteins.
To characterize how these properties vary in different
cellular contexts.

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Such studies enable genetic, biochemical and cell
biological technologies to be applied on a systemic level,
leading to the assignment of biochemical activities, the
construction of protein arrays, the identification of
interactions, and the localization of proteins within cellular
compartments.
IV. BIOINFORMATICS
All in all, biomarkers will be composed of these different
signals and integrated data base. Medicine is becoming
evidence-based and at a molecular level.
A. Applications
PROFILING OF DISEASE TISSUES
o Classification of leukemia into different subtypes
o Changes in myocardial proteins associated with heart
failure in humans and anima l models (rat, monkeys)
SERUM BIOMARKERS
o Detection of low-abundance and potentially new
circulating disease marker proteins
DIFFERENTIAL IN-GEL ELECTROPHORESIS
(DIGE)
o Quantification of protein expression between esophageal
CA cells and normal epithelial cells
DISEASE BIOMARKER IDENTIFICATION
o Comparative analysis of protein expression in normal
anddisease tissues to identify aberrantly expressed
proteins thatmay represent new markers
o Analysis of secreted proteins in cell lines and
primarycultures
o Direct serum protein profiling by mass spectrometry
(ex.Auto-antibodies against tumor proteins)
o Identificationoftumorantigens
BIOMARKER VALIDATION
o Prospective, well controlled clinical studies of
diversepatients across multiple institutions, with well-
established standards for all steps in the process
Tissue collection
Purification
Amplification
Hybridization or ligand-binding
Data capture
Normalization
Statistical analysis
Scoring
o Reproducibility within and among laboratories
B. Chronology of Biomarker Development
A biomarker is first identified then evaluated for a particular
clinical indication.
Analytical and clinical validations must be performed
before submission for US FDA approval.
Alternatively, the marker might bypass the FDA approval
process if it is to be used for research purposes only.
Once approved, the Centre for Medicaid and Medicare
Services (CMS) might determine that it is reasonable and
necessary for improved patient care and therefore,
reimbursable.
Because CMS decisions indirectly influence coverage by
private insurance carriers, a marker is not widely used in the
clinic unless all of the steps in the process have been
completed.
C. Regulatory Bodies
FDA: in vitro diagnostics (IVD) safety and effectiveness
of an IVD refers to the consequences expected from reliance
on it to make clinically significant diagnostic or treatment
decisions. i.e. tumor markers.
Center for Medicaid and Medicare Services: Analyte-
specific reagents (ASRs) biomarkers used in in-house
clinical laboratory for restricted for research only purposes
D. Challenges
Biological variability among patient samples as well as the
huge dynamic range of biomarker concentrations is the main
challenge to deduce diagnostic patterns unique to specific
cancer states.
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Reproducibility and validation of tumor biomarkers should
be addressed carefully.
Standardization of sampling and analytical techniques
Integration of proteomic, genomic, and metabolomics-level
data.
Cost: R&D expenditure was at $5.3B (2009).
Need to develop multiplex assay systems.
E. Biomarkers in the future
Understanding the molecular basis of tumor characteristics
such as neoplasia development, metastases, invasion and
resistance to therapy will play an important role in the
development of personalized cancer therapy.
New biomarker technologies combined with advanced
bioinformatics is being used to identify molecular signatures
of individual tumors based on protein pathways and
signaling cascades.
The discovery of new highly sensitive and specific
biomarkers for early disease detection and risk stratification
coupled with personalized therapies holds the key to future
cancer treatment.
V. REFERENCES
Lecturers PPT
2020 Trans
VI. QUIZ
1. The following are the foundations for the molecular basis of
disease except:
A. Genomics
B. Metabolomics
C. Epigenomics
D. Proteomics
2. Which of the following is the 5th step of biomarker
development pathway?
A. Review the significance and feasibility
B. Develop a robust clinical assay and initial clinical
evaluation to detect existing disease
C. Understand and Define the Disease
D. Determine which samples should be used for the
discovery phase
3. Which of the following is not a requirement for a successful
biomarker development?
A. Repositories of biological specimens
B. Understanding of the entire complex biomarker
development process.
C. Use of multi-disciplinary team approach.
D. Validation of every step of both assay performance and
diagnostic utility.
4. Which biomarker is considered a companion diagnostics for
cancer therapeutics?
A. Target-designed therapies
B. Single nucleotide polymorphisms (SNPs)
C. CD4+ T-lymphocyte
D. DNA methylation patterns
5. What describes a proteome?
A. A collection of cells used for genetic tests
B. A biologic indicator of health or disease
C. Protein alterations in disease
D. The entire protein complement in a given cell, tissue or
organism
Answers: 1.C, 2.D, 3.A. 4.B, 5. D
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