• Biomarker discovery platforms and development
• Types of biomarkers
• Phases of evaluation of biomarkers
• Validation of biomarkers
• Study designs for biomarker studies
• Uses of biomarkers
• Limitations of biomarker in clinical trials
• The role of biomarkers has been exponentially increasing in
guiding decisions in every phase of drug development, from
drug discovery and preclinical evaluations through each phase
of clinical trials and into post-marketing studies.
• The high costs incurred when drugs fail during clinical trials
has prompted interest in biomarkers as biological indicators
for progress of disease, effect of therapeutic interventions,
and drug-induced toxicity.
• Biomarkers, thus also reduce attrition of drugs during the
preclinical and clinical phases of drug development, and
hence, the overall cost of drug development.
TYPES OF BIOMARKERS
TYPE 0: Natural History Marker
TYPE 1: Biological Activity Marker
TYPE 2: Single Or Multiple Markers Of Therapeutic Efficacy
TYPES OF BIOMARKERS
•Type 0 biomarkers are markers of the natural history of a
disease & correlate longitudinally with known clinical indices,
such as symptoms over the full range of disease states. Ex: CRP
•Type I biomarkers capture the effects of an intervention in
accordance with the MOA of the drug: HbA1C
•Type 2 biomarkers are considered to be surrogate endpoints as
a change in that marker predicts clinical benefit . Ex: LDL-C
CLASSES OF BIOMARKERS IN CLINICAL TRIALS
• Safety biomarkers
• Efficacy biomarkers
• Biomarkers include imaging (CT, MRI, PET, x-ray) or clinical
• Constantly monitored safety lab biomarkers can act as common
vital organ function tests applied across different therapeutic
areas or as specialized testing applied to detect unique
• Thus, at phases 1 and 2, careful selection of the correct tests is
essential, and the selection of those tests should be based on
the compound profile and pre-clinical toxicology data.
• Safety testing can be classified as follows:
1) Liver safety tests: AST , ALT , ALP, GGT, Bilirubin
2) Renal safety tests: BUN, Sr Creatinine, GFR
3) Hematology safety biomarkers: Complete blood count
4) Bone safety biomarkers: Calcium, Inorganic phosphates
5) Basic metabolic safety biomarkers: Glucose , Cholesterol, Uric
• Efficacy biomarkers are used to demonstrate a change in all,
or at least a good proportion of treated subjects.
• The more positive the biomarker, the higher the efficacy of a
• Efficacy biomarkers can be classified into the following groups:
1) Surrogate Biomarkers/Endpoints
2) Predictive Biomarkers
3) Pharmacodynamic Biomarkers
4) Prognostic Biomarkers
• A surrogate endpoint is a laboratory or physical measurement
used in clinical trials to indicate a drug’s response and can be
used in place of a clinical endpoint, which is usually
acceptable as evidence of efficacy for regulatory purposes.
• It can be used to assess the benefit of or harm from a
• Even if evidence for surrogacy is not enough, such types of
biomarkers are useful in proving the concept for which a
candidate drug is to be used.
• Examples of surrogate biomarkers are
Disease Surrogate Endpoints Clinical Endpoints
Hypertension Blood pressure Stroke
Dyslipidemia Cholesterol, LDL Coronary artery disease
Diabetes Glycosylated hemoglobin
neuropathy, heart disease
Glaucoma Intraocular pressure Loss of vision
Cancer Biomarkers Tumor
shrinkage, Response rate
• Predictive biomarkers can:
Stratify patient populations into responders and non-
Predict whether or not a drug will have the intended effect
Forecast the extent to which a drug can be effective and/or
toxic in different patient populations
DRUG INDICATION BIOMAKER
Imatinib CML BCR-ABL (PCR), c-KIT
Erlotinib NSCLC, pancreatic EGFR and KRAS mutation
Gefitinib NSCLC EGFR and KRAS mutation
Trastuzumab Breast cancer HER2
EXAMPLES OF PREDICTIVE BIOMARKERS
PHARMACODYNAMIC (PD) BIOMARKERS
• These are the biomarkers which demonstrate that a drug hits its
target and impacts its biochemical pathway.
• Such types of biomarkers are necessary to demonstrate proof of
the drug’s mechanism of action.
• This class of biomarkers:
Constitute the majority of biomarkers in early phases of drug
discovery (preclinical, phase I, and phase II).
Can help to determine effective dose and dose schedule.
• The non-imaging biomarkers include proteins, cytokines, and
enzyme activity in serum, CSF, or tissue lysates, proteins by
immunohistochemistry (IHC), and DNA and RNA gene
expression. Ex: Ki67 in Ca Prostate
• Prognostic biomarkers can predict the risk or outcome of a
disease in patient population without the involvement of
• In addition to its predictive power, prognostic biomarkers may
help enrich a clinical trial by choosing people more likely to
respond to treatment.
• Examples include prostatic specific antigen to predict survival
in prostatic cancer patients and CRP as a risk factor in
PHASES OF EVALUATION OF BIOMARKERS
• The identiﬁcation of biomarkers should proceed in a
• In 2002, the National Cancer Institute’s ‘Early Detection
Research Network’ developed a ﬁve-phase approach to
systematic discovery and evaluation of biomarkers.
• In Phase 1, the identified markers are prioritized based on
their diagnostic/prognostic/therapeutic value that could
suggest their evolution into routine clinical use.
• Phase II involves establishing an assay with a clear intended
clinical use. The assays need to be validated for reproducibility
and shown to be portable among diﬀerent laboratories.
• During Phase III, an investigator evaluates the sensitivity and
speciﬁcity of the test for the detection of diseases that have
yet to be detected clinically.
• Phase IV evaluates the sensitivity and speciﬁcity of the test on
a prospective cohort. An investigator can estimate the false
referral rate based on tested biomarkers and describe the
extent and characteristics of the disease detected.
• Phase V evaluates the overall beneﬁts and risks of the new
diagnostic test on the screened population.
BIOMARKERS IN PHASE I TRIALS
• In phase I studies, pharmacodynamic biomarkers are often of
interest based on assumptions that modulation of these
markers may provide proof of drug target inhibition and
support the selection of drug and dose for further evaluation.
• These are almost always exploratory biomarkers.
• The goal in these pharmacodynamic marker studies in phase I
trials is to provide evidence that the agent reaches or
modulates the putative target.
• These studies can be conducted by analysis of samples and/or
images obtained prior to and after treatment, or by
comparison to an untreated control.
BIOMARKERS IN PHASE II TRIALS
• In phase II trials biomarkers can be used to:
Provide evidence that the agent modulates the putative target
or pathway in a pharmacodynamic assessment similar to the
phase I setting
Evaluate the association between the biomarker and clinical
Determine patient eligibility (for example, HER2 status for
Determine the dose-response relationship of a
pharmacodynamic marker across a narrow set of dose cohorts
(generally one or two) and more homogenous patient
VALIDATION OF BIOMARKERS
• Once a biomarker candidate has been selected on the basis of
biological plausibility and technical feasibility, statistical
validation is required to justify its use in a clinical trial.
• Validation of a biomarker begins with an initial demonstration
that a correlation exists between the marker and the clinical
endpoint of interest, followed by independent statistical
validation of this relationship.
• Type 0 markers can be characterized in phase 0 clinical
studies, in which a reliable assay is used in a well defined
patient population for a specified period of time.
• Ideally, a linear (+ve or -ve) relationship is established with the
gold standard clinical assessor.
VALIDATION OF BIOMARKERS
• A priori validation of type I biomarkers is impossible for truly
novel targets without an effective positive control treatment. So,
for novel targets the biomarker will be validated in parellel with the
Ex:- A battery of cognition markers validated with the scopalamine
were used to confirm the pharmacological effect of a novel agonist
directed against the alfa 7 nicotinic acetylcholine receptor.
•Type 2 biomarkers (or surrogate endpoints) must be relevant both
to the MOA of the drug & to the pathphysiology of the disease.
•Changes in these biomarkers should reflect treatment benefit & so
effective therapy is necessary for their validation.
•Hence, a phase-3 study supporting claims of effectiveness for the
innovator drug is sufficient to validate type 3 biomarkers.
FIT-FOR-PURPOSE METHOD VALIDATION
Practical approach of validating biomarkers.
Fit-for-purpose method validation provides for efficient drug
development by conserving resources in the exploratory stages of
It is used to describe distinct stages of the validation process
including pre-validation, exploratory and advanced method
validation, and in-study method validation
FIT-FOR-PURPOSE METHOD VALIDATION
Practical approach of validating biomarkers.
Biomarker data must be reliable and accurate.
Decision making during drug development.
Analytical validation requirements are specific to the stage of
Consideration is given to
the intended use of the biomarker data
the regulatory requirements associated with that use
BIOANALYTICAL VALIDATION Vs. BIOMARKER VALIDATION
PARAMETER BIOANALYTICAL ASSAY BIOMARKER ASSAY
ASSAY METHOD Quantitative Quasi-quantitative
GLP No specific guidelines
NATURE OF ANALYTE Exogenous Endogenous
PRECISION/ACCURACY Robust with acceptance
SENSITIVITY LLOQ defined by
Limited sensitivity with
SPECIFICITY Drugs are not present in
Samples are subject to
clean-up and analyte
Biomarkers present in
Samples not subject to
QC Certified standard and
blank patient sample
Certified standard and
blank patient sample
matrix usually not available
DESIGN CONSIDERATIONS FOR BIOMARKER
• The choice of an appropriate design for a trial will largely
The strength of existing evidence for a biomarker
The nature of conclusions to be drawn
The strength of evidence desired at the trial’s conclusion
The available resources.
• For prognostic biomarkers, which offer information about the
likely course of a disease if there is no change in treatment of
an individual, retrospective studies using data from well-
conducted clinical trials will be sufficient.
• In the case of predictive biomarkers, more rigorous standards
must be met to justify their use in a clinical setting.
• Since predictive biomarkers seek to prospectively identify
patients likely to have a favourable clinical outcome in
response to targeted therapies, validation often may require
comparing outcomes between biomarker-positive and
• Thus, prospective, randomized controlled trials (RCTs) remain
the best approach for establishing the clinical utility of
• Yet traditional RCTs only allow for the estimation of the average
treatment effect in the overall study population rather than in
• Therefore, alternative trial designs like adaptive clinical trials
need to be considered for the evaluation and application of
• If evidence suggests that the benefits of a treatment are limited to
the biomarker-positive subpopulation, an enrichment design
strategy, in which only biomarker-positive patients are enrolled,
may be the appropriate choice.
• Because they require relatively small sample sizes to demonstrate
safety and efficacy, enrichment strategies may improve trial
• However, such designs only allow for partial evaluation of the
clinical validity of biomarkers since they do not provide
information on the effects of treatment in biomarker-negative
• If there is sufficient reason to suggest that a biomarker can
predict that a therapy will be more effective in biomarker-
positive patients, but the evidence is not compelling enough to
rule out clinical efficacy in biomarker-negative patients, a
biomarker-stratified trial design or an adaptive enrichment
trial design may be more appropriate.
• In the biomarker-stratified trial design, biomarkers are used
to guide analysis but not treatment assignment.
• In the adaptive enrichment trial design, biomarkers are used
to guide the enrollment and not treatment assignment.
• By assigning biomarker-positive and - negative patients to
both experimental and control groups, the biomarker-
stratified trial design provides more information on the effects
of treatment in both subpopulations, as well as more
definitive evidence for the clinical utility of the biomarker.
BIOMARKERS AS SURROGATE ENDPOPINTS
• Evaluation of biomarkers as surrogate endpoints is a
• It has been proposed that for a biomarker to be considered a
surrogate, it must be
(1) a correlate of the true clinical outcome and that
(2) the treatment effect on the surrogate should capture the full
effect of treatment on the clinical endpoint.
While the first criterion is relatively simple to demonstrate, the
second is not.
• For example, although the risk that human immunodeficiency
virus (HIV)-infected pregnant women will transmit the
infection to their infants is strongly correlated with maternal
CD4 counts, the provision of therapy to increase maternal CD4
counts has not been found to impact transmission risk
because the CD4 count is not in the causal pathway of the
disease processes that are responsible for transmission.
• Reasons why surrogates may fail to capture the effect of an
intervention on clinical outcomes include cases:
where a disease has multiple pathways and the intervention
affects only one pathway mediated through the surrogate
when a surrogate is not affected by the intervention’s effects
when the intervention has mechanisms of action independent
of the disease process.
• Hence, a particular biomarker’s status as a surrogate is
context-specific and cannot be assumed to be a general
surrogate endpoint separate from its designated use.
POTENTIAL USES OF BIOMARKERS IN DRUG
PHASE POTENTIAL USE
TARGET DISCOVERY &
To identify & justify targets for therapy.
Ex: Use of Her 2 proto-oncogene as a marker of poor
prognosis in Ca Breast.
LEAD DISCOVERY &
To identify leads and evaluate the effects of molecular
targeted drugs in preclinical development.
PRECLINICAL STUDIES Development and validation of new animal disease models.
To assess the toxicity and safety of drug
CLINICAL TRIALS For early evaluation of success and failure of drugs.
Rational selection of drug combinations
Optimization of dose and schedule
To identify responders in subpopulations
Development of new surrogate endpoints of clinical benefit
To predict clinical outcomes.
USE OF BIOMARKERS IN POSTMARKETING
•Information obtained from postmarketing studies has resulted in
recent labeling changes.
Ex: Finding of strong association of HLA-B*5701 to abacavir-
induced hypersensitivity reaction in HIV-infected patients.
Carbamazepine-induced SJS & the presence of HLA-B*1502 allele.
Genetic variants of CYP2C9 & VKORC1 leading to PK/PD variations
in patients on warfarin therapy.
ROLE OF FDA IN PROMOTING USE OF
BIOMARKERS IN DRUG DEVELOPMENT
•To facilitate the use of biomarkers in drug development &
clinical practice, the FDA has
1) Established a voluntary submission process
2) Developed online educational tools
3) Strived to ensure the integration of genetic/genomic
biomarkers information into drug labels
Valid genomic biomarkers in the context of FDA-approved drug labels
Genomic biomarker Context in label for which biomarker is valid Other drugs associated
with this biomarker
Representative label Test* Drug
*Reference is made to the requirement of testing for the biomarker (1 = test required, 2 = test recommended, 3 = information
LIMITATIONS OF BIOMARKERS
• Expensive (cost for analyses)
• Storage (longevity of samples)
• Laboratory errors
• Normal range is difficult to establish
• The following are the major pitfalls in the translation from
biomarker discovery to clinical utility:
1. Lack of making different selections before initiating the
2. Lack in biomarker characterisation/validation strategies.
3. Robustness of analysis techniques used in clinical trials.