Biomarkers—A General Review UNIT 9.

23
Jeffrey K. Aronson1 and Robin E. Ferner2,3
1
Centre for Evidence Based Medicine, Nuffield Department of Primary Care Health
Sciences, Radcliffe Infirmary, Oxford, United Kingdom
2
West Midlands Centre for Adverse Drug Reactions, City Hospital, Birmingham, United
Kingdom
3
School of Medicine, University of Birmingham, Birmingham, United Kingdom

A biomarker is a biological observation that substitutes for and ideally predicts

a clinically relevant endpoint or intermediate outcome that is more difficult
to observe. The use of clinical biomarkers is easier and less expensive than
direct measurement of the final clinical endpoint, and biomarkers are usually
measured over a shorter time span. They can be used in disease screening,
diagnosis, characterization, and monitoring; as prognostic indicators; for de-
veloping individualized therapeutic interventions; for predicting and treating
adverse drug reactions; for identifying cell types; and for pharmacodynamic
and dose-response studies. To understand the value of a biomarker, it is nec-
essary to know the pathophysiological relationship between the biomarker and
the relevant clinical endpoint. Good biomarkers should be measurable with
little or no variability, should have a sizeable signal to noise ratio, and should
change promptly and reliably in response to changes in the condition or its
therapy.  C 2017 by John Wiley & Sons, Inc.

Keywords: adverse drug reactions r biomarkers r drug discovery r drug de-

velopment r diagnosis r monitoring drug therapy r screening r surrogate
endpoints r surrogate markers

How to cite this article:

Aronson, J.K., & Ferner, R.E. (2017). Biomarkers—A general review.
Current Protocols in Pharmacology, 76, 9.23.1-9.23.17.
doi: 10.1002/cpph.19

INTRODUCTION Here we discuss the history, advantages,

Although they have been used for decades,
the term “biomarker” and its synonyms, such
as “surrogate marker” and “surrogate end-
point,” are used much more widely today.
A search for these terms in papers indexed
in PubMed shows that interest in biomark-
ers began to increase dramatically in 2005
(Fig. 9.23.1).
The increase in interest since 2010 has
been particularly striking; about half of all
papers published on the topic have appeared
since 2014. Recent examples include system-
atic reviews of procalcitonin as a biomarker
for medullary thyroid cancer (Karagian-
nis, Girio-Fragkoulakis, and Nakouti, 2016),
KRAS mutations as prognostic biomarkers
in pancreatic cancer (Li, T. et al., 2016),
and serum microRNA-21 as a diagnostic
biomarker in breast cancer (Li, S. et al.,
2016).
uses, and taxonomies of biomarkers and
the pitfalls and challenges associated with
them. We also propose a new definition of
a biomarker. This paper builds upon and ex-
tends previous publications and replaces them
(Aronson, 2005, 2008, 2012).
HISTORY
The term “biomarker” dates from the
1970s, having been first used, as far as we
can discover, in an article title in 1973 (Rho
et al.). In that instance the term was used to
indicate the presence of material of biological
origin. It continues to be used in this way in
the geological and ecological literature. The
earliest clinical use dates from 1977 in a pub-
lication titled “Tumor biomarkers of value in
the management of gynecologic malignancy
Drug Discovery
will also be correlated with clinical course” Technologies

Current Protocols in Pharmacology 9.23.1–9.23.17, March 2017 9.23.1

Published online March 2017 in Wiley Online Library (wileyonlinelibrary.com).
doi: 10.1002/cpph.19
Copyright C 2017 John Wiley & Sons, Inc. Supplement 76
 Figure 9.23.1 Numbers of publications indexed under the terms “biomarker(s),” “surrogate
marker(s),” and “surrogate endpoint(s)” in PubMed. Top panel: Absolute numbers of reports;
bottom panel: Numbers of reports as percentages of the total numbers of publications appearing
that year.

(Order et al., 1977). However, the concept it-
self is much older, as evidenced by references
to “biochemical markers” in 1949 (Mundkur)
and to “biological ‘markers’” in 1957 (Porter).
The word surrogate comes from the Latin
surrogare (sub + rogare; supine surrogatus),
literally meaning ‘asked in place of.’ It is de-
fined as “a person or thing that acts for or
takes the place of another” (Oxford English
Dictionary online, 2016). Its use as a syn-
onym for a biomarker dates from the early
1980s. The earliest example of “surrogate end-
point” of which we are aware dates from
1983, but in the context of competitive strategy
(Shubik, 1983). In that case the author stated
that “...this means that at any point of time ... it
Biomarkers—A is necessary and desirable that a surrogate end-
General Review point be assigned together with a measurement
criterion which enables the planners to view
what they have been doing up to that point
in time and to be able to state whether or not
their long-term strategic plan met their criteria
of success.” The term was used in 1989 to refer
to clinical trials and was defined as “one that
we elect to measure as a substitute for some
other variable” (Wittes, Lakatos, and Probst-
field, 1989). The earliest use of “surrogate
marker” in a biomedical sense was in 1985.
The term appeared in the English language
abstract of a report written in German stating
that “Since AIDS-specific laboratory tests are
not yet commercially available, laboratory di-
agnoses of AIDS or of the AIDS-related com-
plex (ARC) are based on ‘surrogate markers.’”
(Joller-Jemelka, Vogt, and Joller, 1985). The
expression “surrogate response variable” was

9.23.2
Supplement 76 Current Protocols in Pharmacology
used in the same year in a textbook on clin-
ical trials, where it was noted that a change
in tumor size could be used as a surrogate for
mortality (Friedman, Furberg, and De Mets,
1985).
DEFINITIONS
The biomarker field is populated by a con-
fusion of terms, such as biological mark-
ers, surrogate markers, surrogate endpoints,
surrogate response variables, intermediate
endpoints, intermediate markers, biomarker
endpoints, and even intermediate marker end-
points. We suggest that the term “biomarker”
should replace all other terms.
In 2000 the U.S. National Institutes of
Health (NIH) convened a Biomarkers Def-
initions Working Group (NIH Definitions
Working Group, 2000; Biomarkers Defini-
tions Working Group, 2001) that defined a
biomarker as “a characteristic that is objec-
tively measured and evaluated as an indica-
tion of normal biologic processes, pathogenic
processes, or pharmacologic responses to a
therapeutic intervention.” There are two prob-
lems with this definition. First, evaluation of
a biomarker is not always completely ob-
jective (for example, histological and radio-
logical observations). Secondly, attempting to
enumerate the processes or responses that are
indicated by a biomarker results in omissions.
For example, non-pharmacological interven-
tions, such as surgical procedures and devices
should have been included. A more recent def-
inition characterizes a biomarker as “a func-
tional variant or quantitative index of a bi-
ological process that predicts or reflects the
evolution of or predisposition to a disease or a
response to therapy” (Fitzgerald, 2016). This
description is also problematic, in that it omits
both the possibility of structural, as opposed
to functional, variants and qualitative, as op-
posed to quantitative, indices. It also omits to
mention the use of biomarkers in verifying the
presence of a disease in addition to the evo-
lution of or predisposition to a clinical condi-
tion. Thus, both definitions represent incom-
plete attempts to enumerate specific aspects
of biomarkers rather than elaborating general
features.
The Biomarkers Definitions Working
Group defined a surrogate endpoint as “a
biomarker intended to substitute for a clinical
endpoint . . . expected to predict clinical bene-
fit (or harm or lack of benefit or harm) based on
epidemiologic, therapeutic, pathophysiologic,
or other scientific evidence” (Biomarkers
Current Protocols in Pharmacology
Definitions Working Group, 2001). While
this implies that all surrogate endpoints are
biomarkers, not all biomarkers are surrogate
endpoints, because they can substitute for non-
clinical intermediate outcomes or endpoints
(for example, breath alcohol concentration and
impaired driving or a risk of crashing). We
see no advantage in this distinction. Indeed,
an analysis of the use of these terms in re-
ports indexed in PubMed shows the dominance
of the term “biomarker(s)” over “surrogate
marker(s)”: “biomarker(s)” but not “surrogate
marker(s)” appears in 799,915 (98.8%) of
809,419 hits; only 6328 (0.78%) used “surro-
gate marker(s)” and not “biomarker(s)”; only
3176 (0.39%) used both.
We therefore suggest that the preferred term
for all such markers should be “biomarker,”
for which we propose a new definition
below.
ADVANTAGES OF BIOMARKERS
Clinical biomarkers have the advantages of
being simpler and less expensive to measure
than final clinical endpoints and they can be
analyzed repeatedly and over a shorter period
of time. For example, it is easier to measure
a patient’s blood pressure than to use echo-
cardiography to measure left ventricular func-
tion, and much easier to use echocardiography
than to measure morbidity and mortality from
hypertension in the long term. Whereas blood
pressure can be readily measured immediately
and repeatedly, it takes years to collect data on
morbidity and mortality.
The use of biomarkers allows clinical
trials to be performed with fewer subjects than
might otherwise be possible. For example, to
determine the effect of a drug on blood pres-
sure requires relatively few patients, say 100
to 200, particularly in a crossover design, and
the trial would be completed within a year or
two. To study the prevention of deaths from
strokes would require a much larger patient
population, crossover is not possible, and the
study would take many years.
Biomarkers are also useful for avoiding
ethical problems associated with the mea-
surement of clinical endpoints. For example,
when treating paracetamol (acetaminophen)
overdose it is unethical to wait for evidence
of liver damage before deciding whether or
not to institute therapy. Instead, the plasma
paracetamol concentration, a pharmacological
biomarker, is used to predict whether treat-
ment is required (Ferner, Dear, and Bateman, Drug Discovery
Technologies
2011).
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Supplement 76
 USES OF BIOMARKERS
Biomarkers can be used to:
r screen for diseases
r characterize diseases (e.g., trinucleotide
repeats; Pearson, 2011)
r rule out, diagnose, stage, and monitor
diseases
r inform prognosis
r individualize therapeutic interventions by
monitoring responses to therapies or pre-
dicting outcomes in response to them
(Table 9.23.1)
r predict adverse drug reactions
(Table 9.23.2)
r predict and guide treatment of drug toxi-
city (e.g., measurement of serum concen-
trations following medication overdose)
r identify cell types (e.g., histological
markers).
The use of genetic biomarkers for individ-
ualizing and optimizing dosage requirements
of certain drugs has so far been disappoint-
ing. For example, a complex model of the ef-
fects of warfarin on coagulation that included
two genetic biomarkers produced only a small
improvement in therapeutic anticoagulation
(Pirmohamed et al., 2013).
Biomarkers are also used at various stages
of drug discovery and development:
r as targets for screening compounds
during drug discovery, e.g., mea-
surement of cyclooxygenase activity
to identify potential anti-inflammatory
agents
r as endpoints for pharmacodynamic
studies, e.g., serum cholesterol as a
marker for the action of a drug in-
tended for prevention of cardiovascu-
lar disease, and in pharmacokinetic/
pharmacodynamic studies
r in studying the relationship between the
concentration or dose of a drug and its
effect
r to measure efficacy in clinical trials
r to help define the adverse effects of drug
candidates.

Table 9.23.1 Examples of Biomarkers Used in Individualizing Drug Therapy During Treatment

Level Biomarker Treatment/condition

Molecular Drug concentrations Aminoglycoside antibiotics,
digoxin, lithium, phenytoin
Cellular Serum electrolytes Diuretics
Thyroxine/TSH Thyroid disease
Blood glucose Diabetes mellitus
Serum lipids Hyperlipidemias
Tissue QT interval Antiarrhythmic drugs
HbA1c Diabetes mellitus
Organ International normalized ratio Warfarin
(INR)
Activated partial thromboplastin Heparin
time (APTT)
Peak expiratory flow rate (PEFR) Reversible airway obstruction
Echocardiography Cardiac failure
Blood pressure Hypertension; pre-eclampsia
Visual tests Macular degeneration
Intraocular pressure Glaucoma
Renal function tests Renal disease
Renal biopsy Renal transplantation
Radiology Arthritis; chest infections
Visual analogue scales Symptoms (e.g., pain)
Rating scales Depression
Biomarkers—A
General Review Whole body Body weight Congestive cardiac failure; obesity

9.23.4
Supplement 76 Current Protocols in Pharmacology
Table 9.23.2 Examples of Biomarkers That Can Be Used in Predicting Risks of Adverse Drug
Reactions Before Therapy
Biomarker Drug
Pseudocholinesterase activity Suxamethonium
(dibucaine number) (succinylcholine)
Thiopurine methyl Azathioprine
transferase (TPMT) activity
Glucose-6-phosphate Many antimalarial drugs
dehydrogenase (G6PD)
activity
HLA-B*5701 polymorphism Abacavir
HLA-B*5801 polymorphism Allopurinol
HLA-B*1502 polymorphism Carbamazepine
SLCO1B1 polymorphism Statins
Four uses of biomarkers have been sug-
gested for the management of cancers (Gion
and Daidone, 2004):
r to assess the risk of cancer
r to study tumor-host interactions
r to reflect tumor burden
r to reflect cellular function, such as path-
ways of apoptosis.
Examples of tumor markers that have been
categorized on the basis of pathology are
shown in Table 9.23.3 (Gargano et al., 1990).
Another application of biomarkers in the
treatment of cancers is the identification of
tumor markers that predict responses to par-
ticular medications (Table 9.23.4).
TAXONOMIES OF BIOMARKERS
Biomarkers can be classified in three ways.
Classification by the Pathophysiology
of the Disorder or Illness
Biomarkers can be classified in terms of the
chain of causative mechanisms, perhaps asso-
ciated with susceptibility factors that can trig-
ger a pathophysiological process (Fig. 9.23.2).
For each disorder there may be more than
one such causative mechanism, more than one
susceptibility factor, and more than one cor-
responding pathophysiological process. The
disorder or illness results directly or through
additive effects or a final common pathway.
Each primary pathophysiological process can
Current Protocols in Pharmacology
Relevance
Prolonged duration of effect
in pseudocholinesterase
deficiency
Increased risk of toxicity in
homozygotes for reduced
enzyme activity
Risk of hemolysis in G6PD
deficiency
Increased risk of skin
hypersensitivity reactions
Increased risk of rashes in
Han Chinese
Increased risk of skin
hypersensitivity reactions in
Han Chinese
Increased risk of
rhabdomyolysis
be considered to occur because of an effect at
the molecular level, which results, through a
chain of subsequent events at the cellular, tis-
sue, and organ levels, in the signs and symp-
toms of the disease. There may also be sec-
ondary pathology and complications. Each of
these can be analyzed analogously, as in the ex-
ample of asthma (Fig. 9.23.3), in which each
of the mechanisms in the chain of pathophys-
iological events is matched by a biomarker or
biomarkers that could be used to monitor dis-
ease progress or changes in response to thera-
peutic interventions.
Classification by the Mechanism of
Action of the Intervention
Biomarkers can be classified according to
the pharmacological level at which they occur
(Fig. 9.23.4). The closer the biomarker is to
the therapeutic or adverse effect, the better it
is likely to be as a measure of the clinical
endpoint.
Classification by the Nature
of the Measurement
A biomarker can be extrinsic to the in-
dividual, for example cigarette smoking as
a biomarker for lung cancer, or intrinsic
(Table 9.23.5). Intrinsic biomarkers can be
physical (symptoms and signs), psychologi-
cal, or based on laboratory tests. The cate- Drug Discovery
Technologies
gories shown in Table 9.23.5 can be further
9.23.5
Supplement 76
 Table 9.23.3 Examples of Biomarkers in Clinical Oncologya

Level Biomarker Examples

Molecular DNA repair MGMT and hMLH1 promoter
pathology hypermethylation
Cell pathology Immune responses Auto-antibodies against MUC1 protein, p53
mutated protein, overexpressed erbB2/neu
protein
Tumor burden Carcinoembryonic antigen, prostate-specific
antigen, mucin markers (CA15.3, CA19.9,
CA125), alpha-fetoprotein, human chorionic
gonadotropin, cell-free DNA
Oncogene or p53, soluble HER2neu, soluble epidermal
oncosuppressor gene growth factor receptor, APC, RAR, p73, FHIT,
deregulation RASSF1A, LKB1, VHL, BRCA1 promoter
hypermethylation
Cell-cycle regulation Cyclin D1 mRNA, p14, p15, p16 promoter
hypermethylation
Apoptosis Survivin, M30 antigen
Extracellular matrix TIMP2, MMP, uPA, DAPK1, E-cadherin,
modification TIMP3 promoter hypermethylation
Detoxification GSTP1 promoter hypermethylation
Tissue pathology Angiogenesis Vascular endothelial growth factor, fibroblast
growth factor
Organ pathology Organ damage Tissue-specific enzymes (e.g., creatine kinase
MB fraction) and other components (e.g.,
troponin, myogloblin), acute-phase proteins,
markers of inflammation, hemoglobin
Susceptibility Genetic BRCA1 and BRCA2, MSH, MLH1
factors susceptibility
Environmental Steroid hormones, insulin-like growth factors
susceptibility and their binding proteins, gene polymorphisms
(lactate dehydrogenase gene)
a Adapted from Gargano et al., 1990, with additions.

subdivided according to whether the markers
are used for diagnosis, staging, or monitor-
ing disease, or for determining the response
to a therapeutic intervention. They can also be
divided according to the level at which they
occur (molecular, cellular, tissue, organ) and
according to whether they relate to suscepti-
bility factors, primary or secondary pathology,
or complications of the disease.
CRITERIA FOR USEFUL
BIOMARKERS
There are many links in the chain of events
that leads from the pathogenesis of a disease to
its clinical manifestations (see Figures 9.23.3,
9.23.4, and 9.23.5). A biomarker may be iden-
Biomarkers—A tified at any point in the chain, at the molecu-
General Review
lar, cellular, tissue, organ, or whole organism
9.23.6
Supplement 76
levels. Likewise, an intervention that modi-
fies such a biomarker may prove to be a use-
ful therapy. Any observation short of the ac-
tual outcome measure could be regarded as a
biomarker.
However, as illustrated in Figure 9.23.6,
there are different ways in which a biomarker
can be linked to the disease and its outcome.
To understand fully the value of a biomarker it
is necessary to know which type of model best
fits the disease condition.
Bradford Hill’s Guidelines
The Austin Bradford Hill guidelines are
useful when looking for information that can
be used to establish a causal association be-
tween a biomarker and a clinical disorder
(Table 9.23.6; Howick, Glasziou, and
Current Protocols in Pharmacology
Table 9.23.4 Examples of Markers that Predict Tumor Responses to Medications

Markers of treatment
response
Enzyme and receptor
polymorphisms
Receptor-associated
drug sensitivities
Examples
Anaplastic lymphoma kinase (ALK) mutation associated with
non-small cell lung cancers, responsive to drugs such as
ceritinib, crizotinib, and nilotinib
Human epidermal growth factor receptors (HER2) associated
with breast cancer, responsive to trastuzumab
CD-117 associated with gastrointestinal stromal tumors;
responsive to imatinib, sunitinib
KRAS wild-type proto-oncogene associated with colorectal
tumors; responsive to cetuximab and panitumumab
BRAF V600E or V600K mutations associated with unresectable
or metastatic melanoma; responsive to trametinib
Estrogen-sensitive breast cancer
Androgen-sensitive prostate carcinoma

Figure 9.23.2 A representation of the pathways whereby causative mechanisms and sus-
ceptibility factors contribute to disease (adapted from Aronson, 2008, page 52; used with
permission).

Aronson, 2009). Biomarkers that conform to
these guidelines are more likely to be useful
than those that do not.
IDENTIFYING BIOMARKERS
The first step in identifying suitable
biomarkers is to understand the pathophysi-
ology of the disease and the factors associated
with it. Thus, understanding the pathophys-
iology of asthma makes it possible to iden-
Current Protocols in Pharmacology
tify factors that might be useful as biomark-
ers (Fig. 9.23.3). In a study of the use of
biomarkers in heart failure, biomarkers that
were linked to mechanisms involved in the
etiology seemed to be best suited to predicting
and diagnosing the disease, selecting therapy,
or assessing progression (Jortani, Prabhu, and
Valdes, 2004).
The next step in identifying potential Drug Discovery
biomarkers is based on the mechanism Technologies
9.23.7
Supplement 76
 Figure 9.23.3 Biomarkers in the pathophysiology of asthma (adapted from Aronson, 2008, page
53; used with permission).

Figure 9.23.4 Examples of biomarkers at different pharmacodynamic levels in the management

of diseases (adapted from Aronson, 2008, page 54; used with permission).

whereby the intervention affects the patho-
physiology of the condition (Fig. 9.23.5).
Finally, the putative biomarker should cor-
relate with the process. For example, it has
been suggested that the following require-
ments must be fulfilled when searching for
Biomarkers—A useful biomarkers of ageing (Ingram et al.,
General Review 2001):
9.23.8
Supplement 76
r the biomarker should have a signif-
icant cross-sectional correlation with
age
r there should be a significant longitudinal
change in the same direction as the cross-
sectional correlation
r there should be significant stability of in-
dividual differences over time
Current Protocols in Pharmacology
Table 9.23.5 Classifying Biomarkers by the Type of Measurement

Types of biomarker Examples Relevant clinical endpoints

A. Extrinsic markers
B. Intrinsic markers
1. Physical evaluation
a. Symptoms
b. Signs
2. Psychological evaluation
3. Laboratory evaluation
a. Physiological
b. Pharmacological
i. Exogenous
ii. Endogenous
c. Biochemical
d. Hematological
e. Immunological
f. Microbiological
g. Histological
h. Radiographic
i. Genetic
Cigarette consumption
Daily defined dose (e.g.,
antihypertensive drugs)
Breathlessness
Slow-relaxing ankle jerks
Likert scales/visual analog
scales
Questionnaires
Blood pressure
Thiopurine methyltransferase
(TPMT) phenotype
Docetaxel clearance
Blood glucose concentration
CD4 count
Autoantibodies
Clostridium difficile toxin
Jejunal biopsy
White dots on MRI scan
Hepatitis C virus genotypes
Lung cancer
Antihypertensive drug
consumption (Callisaya et al.,
2014)
Heart failure
Hypothyroidism
Pain
Self-harm
Heart attacks and strokes
Risk of neutropenia
Risk of neutropenia
Complications of diabetes
HIV/AIDS
Autoimmune diseases
Pseudomembranous colitis
Gluten-enteropathy
Multiple sclerosis
Hepatitis C infection

r the rate of change in a biomarker of aging
should be predictive of lifespan.
VALIDATION OF BIOMARKERS
The final test of a biomarker is that in
real-world conditions it faithfully predicts the
desired outcome. This is ideally tested in
well-designed randomized controlled clini-
cal trials, and there are cases in which such
trials have shown that a proposed biomarker
is not in fact valid, such as the example of
cardiac arrhythmias mentioned above and dis-
cussed below (Scenario e in Fig. 9.23.6; Fig.
9.23.7). As another example, Dr. Benjamin
Spock hypothesized that the position of a
sleeping baby was a biomarker for the risk
of cot death and that the risk was increased
when a baby lay on its back; this was dis-
proved by studies that showed that the risk of
cot death is increased by following this ad-
vice, and that babies should in fact be put
“back to sleep” (Gilbert et al., 2005). The
monoclonal antibody HA-1 A (Centoxin)
binds to the lipid A domain of endotoxin,
which was regarded as a biomarker that was
assumed to have a pathogenic role in sep-
ticemia; however, it did not alter mortality or
even increased it (National Committee for the
Evaluation of Centoxin, 1994).
PITFALLS AND PROBLEMS
ASSOCIATED WITH THE USE OF
BIOMARKERS
A major problem encountered when us-
ing biomarkers is failure to understand the
relationship between the pathophysiology of
the condition and the mechanism of action
of the intervention. For example, smoking
causes lung cancer, and a trial of the benefit
of education in preventing lung cancer might
use smoking as a biomarker rather than the
occurrence of the cancer itself. On the other
hand, if chemotherapy is used to treat lung
cancer, smoking is of no use as an indicator of
outcome, since smoking is irrelevant to the ac-
tion of the intervention. Other cases may be
less obvious.
Because ventricular arrhythmias cause sud-
den death and antiarrhythmic drugs prevent Drug Discovery
ventricular arrhythmias, it was assumed that Technologies

9.23.9
Current Protocols in Pharmacology Supplement 76
 Figure 9.23.5 The chain in the pharmacodynamic process for beta-adrenoceptor agonists in the
treatment of acute severe asthma and the potential monitoring measurements that might be made
at each pharmacodynamic level (adapted from Aronson, 2008, page 56; used with permission).
antiarrhythmic agents would prevent sudden
death. In fact, the assumption was wrong:
The results of the Cardiac Arrhythmia Sup-
pression Trial (Cardiac Arrhythmia Suppres-
sion Trial Investigators, 1989) showed that the
Class Ic antiarrhythmic drugs encainide and
flecainide increased sudden death significantly
in patients who developed asymptomatic ven-
tricular arrhythmias after a myocardial in-
farction. The mechanism remains undefined
(Fig. 9.23.7).
Another example is based on results ob-
tained with enalapril and vasodilators, such
as hydralazine and isosorbide, whose hemo-
dynamic actions and effects on mortality
associated with heart failure are dissociated.
Although vasodilators improved exercise ca-
pacity and left ventricular function to a greater
extent than enalapril, the latter reduced mor-
tality to a significantly greater extent than
vasodilators (Cohn, 1991). So in this case
hemodynamic effects are poor biomarkers.
Confounding factors, particularly the use
of drugs, can negate the value of biomark-
ers. Thus, the serum T3 concentration is used
as a marker of the tissue damage caused by
thyroid hormones in patients with hyperthy-
Biomarkers—A roidism. However, its utility as a biomarker is
General Review
9.23.10
Supplement 76
blunted in patients taking amiodarone, which
interferes with the peripheral conversion of
T4 to T3 without necessarily altering thy-
roid function. In a patient with gastrointestinal
bleeding whose heart rate does not increase
because of beta-blockade, the physician may
underestimate the seriousness of the bleeding.
Likewise, corticosteroids can mask the signs
of an infection or inflammation.
As a general principle, if the concentration-
effect (dose-response) curves for the effects of
an intervention on the primary outcome and
the biomarker differ, a change in the biomarker
may not accurately reflect the degree of change
in the outcome (Fig. 9.23.8). However, there
is little information about this.
Lack of reproducibility of the methods
used to measure biomarkers may impair
their use. For example, there are differ-
ences between ciclosporin concentrations in
serum and blood when they are measured by
radioimmunoassay and HPLC (Reynolds and
Aronson, 1992). While the association be-
tween thiopurine methyltransferase (TMPT)
activity and the risk of adverse reactions to
mercaptopurine was described some years
ago, methods for measuring the enzyme re-
main unstandardized, optimal treatment is
Current Protocols in Pharmacology
Figure 9.23.6 Five scenarios relating pathophysiology, biomarkers, and clinically relevant outcomes (from Aronson, 2008,
page 56; used with permission).
Drug Discovery
Technologies

9.23.11
Current Protocols in Pharmacology Supplement 76
Figure 9.23.6 Continued.

Table 9.23.6 Bradford Hill’s Guidelines Applied to Biomarkers

Guideline Characteristics of useful biomarkers

Strength Strong association between marker and outcome, or between
the effects of a treatment on each
Consistency The association persists in different individuals, in different
places, in different circumstances, and at different times
Specificity The marker is associated with a specific disease
Temporality The time-courses of changes in the marker and outcome occur
in parallel
Biological gradient Increasing exposure to an intervention produces increasing
(dose-responsiveness) effects on the marker and the disease
Plausibility Credible mechanisms connect the marker, the pathogenesis of
the disease, and the mode of action of the intervention
Coherence The association is consistent with the natural history of the
disease and the marker
Experimental evidence An intervention gives results consistent with the association
Analogy There is a similar result from which we can adduce a
relationship

often not achieved, and guidelines differ (Bur-
nett et al., 2014).
It is unusual for a single biomarker to pro-
vide all the information needed for monitor-
ing interventions. For example, patients with
asthma feel breathless if they have a low
peak expiratory flow rate (PEFR). However, in
one study different drugs produced different
relationships between PEFR and breathless-
Biomarkers—A ness (Higgs and Laszlo, 1996). Patients taking
General Review beclomethasone did not feel as breathless as
those taking theophylline for a given PEFR.
This finding raises the question about whether
the biomarker should be the objective mea-
sure of peak flow or the subjective evaluation
of how the patients feel. It is probably best to
use both.
The statistical properties of biomarkers
have been discussed (Buyse et al., 2016).
Statistical problems can arise when biomark-
ers are used as entry criteria for clinical
trials (Shubik, 1983). If a patient is randomized

9.23.12
Supplement 76 Current Protocols in Pharmacology
on the basis of an abnormal value of a
biomarker, repeat measurements are likely to
be closer to the mean as a consequence of re-
gression to the mean, whether or not an effec-
tive treatment was used, thereby reducing the
power of a study. There is also the likelihood of
missing or censored data when biomarkers are
used. Although a biomarker may allow the use
of a small sample size, the study may not then
be large enough to detect adverse drug effects
or adverse reactions to drug candidates.
CHOOSING THE BEST
BIOMARKER TEST
The following criteria can be used for de-
ciding among various biomarker tests (Irwig
and Glasziou, 2008):
(1) Clinical validity: The test should be either
a measure of the clinically relevant out-
come or a good predictor of the clinically
relevant outcome. It should be subject to
little or no systematic variability.
(2) Responsiveness: The biomarker results
should change promptly in response to
changes in therapy, improving when the
condition improves and worsening when
it deteriorates.
(3) Large signal to noise ratio: The biomarker
analysis should differentiate clinically im-
Figure 9.23.7 The model and hypothesis that drove the Cardiac Arrhythmia Suppression Trial
(CAST), which was based on the use of an inappropriate biomarker.
Current Protocols in Pharmacology
portant changes over time from back-
ground values, such as short-term biolog-
ical fluctuations and technical measure-
ment errors. Such background noise can
be reduced by identifying and reducing the
sources of biological and technical vari-
ability to achieve standardization and by
using multiple clustered measurements,
with repetition if an abnormal result
occurs.
If a biomarker analysis does not fulfill all
these criteria it might be better to use several
biomarkers, such as one for monitoring the
short-term response to therapy and another for
long-term effects, or one for monitoring bene-
fits and another for adverse effects. The mea-
surement of blood glucose is useful for day-to-
day monitoring of the management of diabetes
mellitus, while HbA1c (glycated hemoglobin)
is used to monitor progress over a longer pe-
riod of time. Even with these biomarkers it
is still necessary to monitor for the long-term
complications of the disorder, such as renal
impairment, neuropathy, and retinopathy.
PRACTICAL MATTERS
Practicability is important when biomark-
ers are used for monitoring therapy. The tests
should be non-invasive, inexpensive, and easy
Drug Discovery
Technologies
9.23.13
Supplement 76
 Figure 9.23.8 Theoretical concentration-effect curves displaying different sensitivities for a
biomarker and the primary outcome to a therapeutic intervention or pathology.
to execute, and the results should be rapidly
available. Ideally, the test should be suitable
for patient self-monitoring.
Point-of-care testing has increased in re-
cent years, making monitoring more practica-
ble (Drancourt et al., 2016; Tian et al., 2016).
Since 1986 over 3900 publications have ap-
peared with the term “point of care” in the
title, and near-patient testing has been studied
since the 1940s (Carr, 1946; Steinitz, 1947).
A committee of the U.S. Institute of
Medicine (IOM) has concluded that the
widespread implementation of biomarkers
into routine clinical practice has been
restrained by several interrelated factors
(Graig, Phillips, and Moses, 2016):
r lack of consensus over common standards
of evidence
r inefficient and inconsistent regulation
r the lack of an effective framework of stan-
dards for collecting patient data on tests,
treatments, and outcomes
r poor translation of such data into new
knowledge to improve patient care and
outcomes.
To this should be added the lack of agree-
ment on terminology.
The IOM’s committee made a range of
Biomarkers—A recommendations, including: establishment of
General Review common standards of evidence of clinical
9.23.14
Supplement 76
usefulness; coordinated regulatory processes;
enhanced communication with patients and
providers; accreditation of laboratories and
good laboratory practice; continual assess-
ment of clinical usefulness; development and
use of biomedical informatics tools; a database
of biomarkers; equity of access; and improved
clinical guidelines on the appropriate use of
biomarkers.
A PROPOSED DEFINITION
As defined in the Oxford English Dictio-
nary, a marker is an object or observation that
serves to label or distinguish something; an ob-
servation, in this context, is a measurement or
other piece of scientific information. Having
reviewed previous definitions of a biomarker
(Friedman et al., 1985; Department of Health
and Human Services, 1992; NIH Definitions
Working Group, 2000; Biomarkers Definitions
Working Group, 2001), and in the light of
the matters covered in this review, we believe
that the essential features of a biomarker that
should be encompassed in a definition are that
it is a biological observation of any kind that
is intended to substitute for some other obser-
vation that cannot be easily measured.
We therefore propose that a biomarker be
defined as follows:
Current Protocols in Pharmacology
 biomarker, n. /ˌbʌɪəˈmɑrkər/ A biolog-
ical observation that substitutes for and
ideally predicts a clinically relevant end-
point or intermediate outcome that is more
difficult to observe. [ancient Greek βίο- com-
bining form life + Old English (Mercian) merc
a boundary, a limit +-er suffix]
The phrase “difficult to observe” encom-
passes difficulties with intermediate outcomes
or endpoints that are, for example, hard to ac-
cess or temporally remote.
CONCLUSIONS
There are potential benefits in using
biomarkers in drug development, in studying
various aspects of diseases, and in monitor-
ing the beneficial effects of therapeutic inter-
ventions. However, because the events linking
disease pathogenesis to outcome are generally
complex, the more that is known about the
underlying abnormalities associated with the
condition and the mechanism of drug action
the easier it is to identify useful biomarkers
for diagnosing an illness, for monitoring the
response to a drug, and for studying disease
progression. Accumulation of this information
is a challenge for basic and clinical pharma-
cologists as well as others involved in the iden-
tification of biomarkers.
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