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Challenges in Clinical Trial Specimens Testing Laboratory and Importance of Establishing Specimen Biorepository
By Rishab K. Gupta, Ph. D., Professor Emeritus, Division of Oncology, Department of Surgery, and David Geffen School of Medicine at UCLA and Past Director of Research Core Services, and Immunodiagnosis & Protein Biochemistry, John Wayne Cancer Institute
The main goal in any medical investigation is to discover
effective therapeutic agents and validate their effectiveness
through randomized clinical trials. These trials involve a large
number of subjects in all arms of the trial to assess
statistical significance of clinical outcome in response to the
therapeutic agent. To achieve the clinical outcome as an
endpoint, the trials are lengthy and expensive. It is highly
appropriate to take advantage of these trials to incorporate
possible surrogate biomarkers to identify which markers are
reliable for early detection of disease, i.e., cancer, and
prediction of therapeutic response of the agent. Clearly, the
use of biomarkers in oncology or other diseases is not only to
address the therapeutic effect of an agent, but also to
determine the safety of the agent. Such biomarkers could also be
evaluated for appropriate diagnosis and prognosis for
appropriate stratification of patients who would otherwise not
benefit from the treatment, i.e., those patients who are already
cured or are at no risk of developing the recurrent disease.
Thus, selection of high-risk subjects not only spares no-risk
patients from unnecessary treatment, but allows randomization of
patients with uniform clinical status for an effective
assessment of the treatment effect. Here we outline the unique
methodological and logistical challenges faced by clinical
specimen testing laboratories, which attempt to conduct
controlled trials of therapeutic agents.
Randomized controlled trials for the efficacy of laboratory
tests with respect to efficacy of therapeutic agents are
generally designed and conducted based on preliminary or
background information. The vital role of clinical laboratory in
the delivery of safer and more effective healthcare requires a
careful evaluation. This includes not only the analytical
characteristics of the biomarker assay, but also any other
variable that may affect the clinical usefulness and diagnostic
performances of laboratory test. Therefore it would allow more
accurate interpretation and utilization of laboratory
information. Better understanding of the overall process of
biomarker discovery, and of the challenges inherent in each
phase, should thus improve experimental study design.
Development of strategies along these lines should lead to
increasing the efficiency of the biomarker development and
facilitate the delivery and deployment of novel clinical tests.
Role of Clinical Laboratory in Moving Biomarkers from Initial
Discovery into Clinical Practice
Clinical laboratories, particularly those that are research and
development (R&D) oriented, play a major role in moving
appropriate biomarkers from initial discovery to clinical
practice. This role, which is comprised of several aspects, is
one of the keys to the translational research. These critical
aspects include but are not limited to
(1) Providing expertise to validate analytic methods
(2) Ensuring accuracy and precision that are compliant with
respect to the Clinical Laboratory Improvement Amendments of
1988 (CLIA) and other agencies' regulatory requirements
(3) Offering a standardized infrastructure for collection and
processing of specimens
(4) Performing tests under strictest of laboratory regulations
with rigid quality controls
(5) Providing appropriate documentation required, complying with
regulatory agencies
(6) Offering professional staff for conducting laboratory and
clinical studies to validate clinical claims of new biomarker
(7) Developing interpretive criteria of results for use in
medical practice Biomarkers could be classified as
(a) Exploratory
(b) Demonstration
(c) Characterization
(d) Surrogate
This
classification is based on the purpose for which the biomarkers
will be used. Exploratory biomarkers require a minimum set of
assay validation experiments, but demonstration and
characterization biomarkers require more advanced assay
validation. This is especially true if they will be used as a
basis for drug development decisions, such as whether a drug is
effective, or at what dose the drug should be used. According to
the National Cancer Policy Form established in March 2006,
exploratory biomarkers should be for the generation of
hypotheses and should be on the R&D side. Demonstration
biomarkers should be considered better than probable or emerging
biomarkers. Characterization biomarkers should then be
considered as established biomarkers that often aid drug
development decision-making. The surrogacy biomarkers can
substitute for clinical endpoints in drug efficacy studies. All
biomarkers must undergo some degree of validation and
qualification.
By definition, qualification is an evidentiary process of
linking a biomarker with biology and clinical endpoints, leading
to data that are scientifically and clinically meaningful with
respect to intended use of the biomarker. Validation of a
biomarker assay on the other hand, is obtaining reliable
biomarker data that satisfy the experiment or study objective.
The degree of validation and qualification of biomarkers should
be within their purpose and should depend upon whether they are
target-engagement biomarkers or disease-related biomarkers.
A target-engagement biomarker that is used in drug development
decision making would need some advanced validation, but would
not be subject to qualification assessments, whereas a
disease-related biomarker that would be used for such decision
making should undergo qualification assessments. For certain
diagnostics, such as the immunohistochemistry (IHC) tests that
are already on the market, the costs and risks are low. These
diagnostics have allowed diagnostic companies to develop
marketable tests for a particular disease with ease. However,
the same companies would be less inclined to develop more
complicated diagnostics that might have to undergo an extensive
in vitro diagnostics (IVD) approval process with the Food and
Drug Administration (FDA) to reach the market. This is because
the IVD process has more extensive testing requirements than the
home-brew development process often used for diagnostic tests,
which only requires CLIA certification of the laboratory that is
performing the test.
With respect to the field of oncology, despite the promise of
many cancer biomarkers, only a few biomarker-based cancer tests
have entered the market. Clearly, translation of research
findings of cancer biomarkers into clinically useful tests
appears to be lagging. This is due to the technical, financial,
regulatory and social challenges linked to the discovery,
development, validation, and incorporation of biomarker tests
into clinical practice. Appropriate analysis and interpretation
of biomarker data presents enormous challenges, especially with
the advent of genomic and proteomic technologies that can
generate a tremendous amount of data on individual samples.
Challenges for Clinical Laboratories Involved in Clinical Trials
According to the FDA's analysis, the recent slowdown, instead of
the expected acceleration, in the development of biomarkers as
they relate to innovative medical therapies for the care of
patients, is due to the fact that the current medical product
development path is highly challenging. This is mainly because
the technologically applied sciences needed for biomarker and
its assay development have not kept pace with the advances in
the basic sciences research. Some of these challenges are:
1) Type of Biomarkers (Based on Application)
These could be:
(a) Diagnostic and prognostic to assess disease free survival or
overall survival or therapeutic assessment. Once evaluated and
validated, their importance is clear with respect to assessing
the disease outcome and therapeutic efficacy as surrogates
during early stages of treatment that would spare lengthy and
expensive treatment follow-up.
(b) Safety biomarkers to assess toxicity to vital organs
(cardiac, renal, hepatic, etc) and their management. Therefore,
clinical laboratory results, used as safety biomarkers, can
influence decision-making at many levels during the clinical
development and regulatory review of investigational therapies,
including (i) initial eligibility for protocol therapy; (ii)
analyses used to estimate and characterize the safety profile;
and (iii) treatment delivery, based on specific rules to modify
or discontinue protocol treatment. Safety biomarkers may be
appropriately used for decision-making by the application of
uniform criteria, especially in situations where there is a
degree of correlation between biomarker changes and
corresponding clinical outcomes.
2) Places where Biomarkers Fall in the Disease Process and
Outcome
Biomarkers could be target-engaged or disease related. It is
apparent that biomarkers fall at various intervals on the
pathophysiology path from the initial trigger or cause of a
disease to final disease outcome. Biomarkers that occur close to
the actions of the target are termed target-engagement
biomarkers. Those that are closer to the disease outcome are
called disease-related biomarkers. Target-engagement biomarkers
provide information about how well a drug is acting; on the
contrary, disease-related biomarkers are used to assess the
effect of a particular drug on a disease. Therefore, some
biomarkers may not be directly related to pathophysiology of the
disease; however, they are still useful. For example hemoglobin
A1c, which is a measure of glycated hemoglobin. When there are
higher than normal levels of blood glucose, as occurs with
diabetes, more hemoglobin becomes glycated. Thus, blood levels
of hemoglobin A1c serve as an excellent surrogate endpoint in
diabetes drug trials, yet this biomarker has nothing to do with
the diabetes disease process.
3) Outsourcing Clinical Laboratory Activities (Right
Opportunities vs. Risks - Patent and Intellectual Property
Protection)
Several problems are associated with outsourcing R&D clinical
laboratory operations. Only relatively routine
tasks are usually outsourced to benefit from cost and time
compression due to 24/7 activity and systematic management from
a distance. However, clinical activities requiring undeveloped
and unfamiliar technology are best done at the original
location. In addition to the time and cost benefits, other
advantages of outsourcing are: access to needed number of study
subjects, availability of trained personnel and labor pool, and
increased productivity. In order to reap all of the benefits, it
is necessary that an appropriate location be selected where
analytical activities are conducted in compliance as mandated by
the regulatory agencies for acceptance of the end results.
Regardless of close watch, there could always be issues about
safety of the subjects, analytical/technical aspects, strict
adherence to mandated guidelines, and long-term sustainability
of the outsourced site. Protection of intellectual property and
patent rights could be compromised. Management could be
problematic due to trained personnel turn-over rate, cultural
differences, language barrier, different regulatory environment,
and timely data acquisition. Despite these limitations,
practically all major US and European drug firms have the
clinical testing operations for new drugs in India performed by
Indian contract research firms that have proven to be highly
successful.
4) Human Subject Protection
It is apparent that laboratories performing tests on human
specimens and reporting patient-specific results must be
certified under the provisions of the CLIA. This agency makes an exception for research
laboratories that test human specimens but do not report patient
specific results for the diagnosis, prevention or treatment of
any disease or impairment of, or the assessment of the health of
individual patients. Therefore, if investigators provide
diagnostic test results to subjects or to physicians to alter
care, they should have laboratory tests performed under the
auspices of a clinical laboratory that has been certified in
accord with CLIA. However, it is not clear if the CLIA applies
to a variety of tests that are used solely for research
purposes. Therefore, caution should be exercised and attempts
should be made in complying with CLIA. The customary paradigm
has been that the Institutional Review Board (IRB) has acted as
both administrative and protective body for subjects from any
harm by the research investigation, including the clinical
laboratory. However, the new approach promotes integrated
accountability across all involved parties, which include, but
are not limited to IRB, subjects, sponsors, investigators,
administration and clinical R&D laboratories.
5) Specimen Collection and Processing
Patient biopsy tissue and other materials, i.e., blood, urine,
etc., collected during clinical trials are invaluable for
researchers trying to discover or develop biomarkers for
clinical application. However, there is a general lack of quality sample collection.
Furthermore, specimen collection, handling of collected
specimens, their processing and archiving for future use differ
from center to center as well as within a given center if the
guidelines are not followed strictly. This is especially the
case for patients having relapses of their disease. If the
standard operating procedures are not followed, the preserved
specimens should be precluded from inclusion in various
biomarker studies. In addition, annotating and archiving
clinical specimens can be costly. As a result, only a few
investigators or institutions are willing to undertake these
endeavors and then provide the specimens and data to others.
Variability in the way specimen associated data are entered and
categorized in a database can also make it difficult for
investigators to retrieve the information they need to include
the clinical laboratory results in final statistical analyses.
Another major challenge could be a lack of informed consent
forms that are broad enough to encompass new uses of the
specimens beyond the use for which they were initially
collected. Also, patient driven materials are often considered
of having some inherent intellectual property value. As
indicated in the previous section (4), the current paradigm of
the role of IRB is change that promotes an integrated
accountability across all involved parties. Specimen collection,
handling, processing and archiving must include a set path, and
must include a mandate on concomitant transmission of the
specimen-associated data to the specimen processing
laboratory/unit. The specimen receiving and processing unit must
operate under the direction of the Clinical Trial Office (CTO).
There should be a pathway to communicate any deficiency of the
specimen associated data or inadequacy of amount of the specimen
to the CTO, which in turn should rectify the problem immediately
with the center collecting and shipping the specimen. Feasible
and effective SOPs must be developed for the transport of the
specimens from collection point to the central processing
laboratory or facility. The processing laboratory should archive
and distribute the specimens to testing laboratory and maintain
appropriate records for audit trail and regulatory compliance.
6) Storage Conditions and Quality of Stored Specimen
These are major challenges, particularly for those biomarkers
for which there are no pre-existing data to base the selection
of storage conditions. For centralized processing of the specimens, the
specimens should be transported within specified time and
temperature. There should be proper SOPs, quality assurance (QA)
and quality control (QC) procedures in place in the
specimen-processing laboratory. The proper storage conditions of
the processed specimens depend on a variety of factors. These
factors include the intended use of the specimen, i.e., whether
the specimens will be used within a short period or need to be
stored for longer periods. As for all laboratory and biorepository procedures, blood collection, shipment, processing
and storage should be conducted under a strict quality assurance
program, including periodic review of SOPs and regular quality
control systems. Procedures should be in place to determine if
the storage conditions chosen will maintain integrity of the
stored specimens. Some biomarkers are stable whereas some are
highly labile. Thus selection of storage condition will depend
on the end use. Specimens collected during a clinical trial are
highly likely to be used in different research projects in the
future. In these situations, informed consent must be checked
and reviewed, and IRB approval must be re-obtained if needed.
The storage condition (temperature) must be low enough to
prevent degradation of the biomarker of interest. Thus selection
of storage temperature should be based on existing information.
The R&D clinical laboratory or associated biorepository must
therefore establish criteria for periodic evaluation of the
stored specimens for stability of the biomarker(s). The storage
containers should be able to withstand the storage temperature.
Temperature of storage freezers must be monitored continuously
24 hours and 7 days a week electronically if possible and
manually at each shift of staff, and must have provisions to
transfer contents of the malfunctioning freezer to another
functioning spare one. Temperature differential from bottom to
top of liquid nitrogen freezer could be significant. Thus, one
should pre-establish if the specimen will be in vapor-phase or
in liquid-phase of the nitrogen. This is particularly the case
to maintain viability of the program frozen cryopreserved cells.
Deviations to any of these issues will cause lost data points,
exclusion from final data analyses and inappropriate disease
management. Guidelines must be developed with respect to
biohazard, biosafety and final disposition of the stored
specimens in accordance with the subject's consent and
regulatory agency's mandates.
7) Specimen Bioinformatics (Tracking, Inventory Control and
Data Management)
Recent developments have led to technological advance that allow
genomic and proteomic analyses of specimens resulting in vast
amount of data. Therefore, a robust and
reliable bioinformatics system that is flexible for change must
be applied. It should be capable of supporting all aspects of
clinical laboratory/biorepository operations, including the fact
that a subject's confidentiality is ensured by assigning a
unique identification code through CTO in compliance with IRB
and Health Insurance Portability and Accountability Act of 1996
(HIPAA). It should encompass all aspects of the specimen
processing unit, including donor confidentiality, specimen
collection, processing, storage, distribution, QA/QC records,
collection associated data from patients, data security,
validation documentation, and management reporting functions.
Within certain limits and maintaining donor privacy under all
conditions, it should be searchable via varying levels of
Web-based access for different individuals and needs. The
bioinformatics system in itself must be highly secure and temper
proof. The National Cancer Institute (NCI) has developed a
number of initiatives to assist the biorepositories to implement
many of these guidelines.
Importance of Archived Specimens
It is apparent that availability of high-quality human specimens
that are appropriately annotated with essential clinical data
and collected with robust informed consent is essential to take
advantage of rapidly growing technological advances. This is
particularly true to explore the promise of proteomics and
genomics for preventing and curing human diseases. These
valuable specimens allow researchers to investigate
characteristics of disease at the molecular level by providing
information about the physiologic or pathologic condition of the
donor subject. It expands collaborative opportunities with
investigators and research institutions that have advanced the
biomedical technology. Availability of associated clinical
information with each specimen in the well-designed
biorepository allows testing of a new hypothesis and/or to
discover new biomarkers in a short time using the specimens that
have been collected retrospectively over a period of many years.
Such hypothesis testing would otherwise take a long time if it
has to wait for prospective collection of the specimens in the
years to come. Similarly, it is an excellent resource to
prospectively validate clinical biomarkers using the
prospectively collected retrospective specimens, and for
assessment of sensitivity and specificity of biomarkers by
inclusion of clinically defined disease and control specimens.
Several positive correlations between a biomarker and clinical
outcome of the disease have already been established using
specimens from well-maintained biorepositories.
Summary
A well-established and well-managed clinical laboratory with
research and development insight is crucial in moving biomarkers
from their initial discoveries to clinical practice. This is
because they are in compliance with the regulatory agency's
requirements and offer a standardized infrastructure for
collection, processing and analyses of the specimens according
to established and functionality proven SOPs. The biomarker
assays are performed under the strictest possible regulations
with rigid quality control and appropriate documentation. The
results obtained in such laboratories are the best for
bio-statistical analyses for clinical correlative studies. These
laboratories offer professionally trained staff for developing
basic science and clinical studies to validate clinical claims
of a new biomarker assay. These laboratories are in ideal
situation to establish authenticated specimen biorepositories
under the auspices of or in concert with federal agencies, i.e.,
NCI, FDA, etc; thus, creating an invaluable resource for basic
scientists, applied investigators, the biotechnology industry
and collaborative research.
Rishab K. Gupta, Ph. D., M.S. (Microbiology and Biochemistry,
Rutgers University, 1968), M.Sc. (Microbiology, GB Pant
University, 1965) is a Professor Emeritus at Geffen School of
Medicine at UCLA. He has completed his post-doctoral training at
Yale University before joining at UCLA research/faculty. Rishab
has significant research experience in tumor immunology and has
taught both undergraduate and graduate students, and
post-doctoral fellows at Rutgers, California State Colleges,
UCLA and John Wayne Cancer Institute (JWCI). He is one of the
leaders in the area of biomarkers assay development and
application. Rishab identified and characterized three
tumor-associated macromolecular antigens. One of these antigens,
TA90 glycoprotein, has been studied extensively for its
relevance in prognostic utility with respect to tumor metastasis
and for its clinical application for patients receiving various
treatment modalities. For article feedback contact Rishab at rgupta@ucla.edu
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