Variations in preanalytical steps at different testing sites may make it difficult to interpret
analytical performance studies. Thus, for all steps of preanalytical specimen handling and
preparation, sponsors should have a detailed standard operating procedure (SOP) or protocol
that is followed at each site that performs any of the preanalytical steps. The sponsor should
ensure that all sites handling the specimens are trained to use the specific method, follow the
SOPs, and record any deviations from the SOP.
For example, if the analyte is labile, a plan to collect several specimens from a small
number of subjects to assess lability to inform appropriate limitations on storage and
transport durations may be appropriate. Note that some analytical validation studies may
not require use of samples from therapeutic product clinical trial subjects, although the
studies should be conducted with samples from the same target population to ensure that
the variability parameters defined are relevant to the population to be tested.
In cases where multiple markers will be detected/measured by the test, analytical validation
of each reported marker may be required regardless of each marker’s prevalence. When it is
not possible for sponsors to obtain specimens containing a particular marker, validation
studies with contrived samples may be permitted.
Analytical validation studies may also be
complicated for IVDs that have the potential to detect a very large number of markers, in
which case it may be necessary for the study to use a representative sampling of markers.
For example, for next generation sequencing panels, the ability of the IVD to detect single-
nucleotide polymorphisms, copy-number variations, inversions or deletions, and other
relevant variant classes should be studied. Sponsors who are concerned about the feasibility
of conducting analytical validation studies for all markers detected by an investigational IVD
should consult with FDA before beginning sample collection and analytical validation
D. Therapeutic Product Clinical Trial Design Considerations
Understanding the population of subjects enrolled in a clinical trial is critical. It is
conceivable, for example, that assessment of preclinical or early clinical studies indicates a
Sponsors may find it helpful to consider resources on analytical validation studies, e.g., Mansfield, E., et al.
and harmful in a test-
negative subgroup. In such cases, subjects with false-positive results may be harmed by the
therapy, and subjects with false-negative results may be deprived of beneficial therapy.
Additionally, false-positive results could lead to underestimation of effect size, whereas
false-negative results could lead to underestimation of the proportion of subjects who are
more likely to respond. Therefore, the therapeutic product and IVD sponsors should work
closely to understand how the IVD’s analytical performance affects the selection of subjects
in the trial. To minimize the proportion of incorrect test results (i.e., false positives and false
negatives that would result in misclassification),
sponsors should ensure that the
appropriate analytical validation studies are carried out and that the level of analytical
validation of the proposed IVD(s), in relation to its specific role in the clinical trial, has been
adequately assessed. This is especially important when progressing from the versions of the
test used in a trial to the candidate IVD companion diagnostic (see Section III.E.3. of this
The following sections discuss considerations for the design of clinical trials for a
therapeutic product for use with a developmental IVD companion diagnostic.
This could result in the failure of a codevelopment program if, for
example, a late-phase clinical trial enrolls only “marker-positive” subjects, when positivity is
based on flawed exploratory programs. When using exploratory testing, it is advisable for
sponsors to establish procedures that specify the process for sample acquisition and handling
Note that the terms “test-positive” and “test-negative” are often used interchangeably with the term “marker-
positive” and “marker-negative;” however, it is important to be aware that tests for the same marker that have
different performance characteristics may identify different subpopulations of “marker-positive” patients.
For example, molecular tests that are intended to select for one target but have undetected cross-reactivity
not the target of interest.
Sponsors should consider principles laid out in the National Cancer Institute publication, “Criteria for the use
guidance for industry “Clinical Pharmacogenomics: Premarket Evaluation in Early-Phase Clinical Studies and
Recommendations for Labeling”
Some early therapeutic product clinical trial designs employ testing for multiple markers to
assign subjects to one of multiple different therapeutic arms with the goal of testing multiple
hypotheses under one study protocol. Sponsors of these clinical trials should consider the
pathway for continued development of selected therapeutic products with accompanying
IVDs in the event that such trials support further development of a candidate IVD companion
There are a variety of clinical trial designs that may be used to
study a developmental IVD companion diagnostic in combination with a therapeutic
product in premarket codevelopment programs. The appropriate clinical trial design to
support the diagnostic strategy depends on the proposed claim(s) for the IVD and what
has already been established about the predictive, prognostic, or other critical properties
of the marker.
The success of a clinical trial design strategy depends on many factors,
including but not limited to the following: a) the characteristics of the marker as applied
to the target population for whom the therapeutic product will be indicated, specifically
the mechanistic rationale for selecting the marker, its predictive/prognostic/other utility
and its intrinsic properties (e.g., variability and specificity with respect to the disease); b)
the nature of the disease; and c) the need to fully characterize the therapeutic product’s
benefits and risks, such as the safety profile (e.g., taking into account a possible lack of
benefit in the test-negative population), and the degree of observed benefit, if any, in the
population for whom the therapeutic product may not be indicated (e.g., test-negative
For IVDs, clinical validity typically refers to the accuracy with which the test identifies, measures, or predicts
diagnostic, clinical validity typically refers to the accuracy with which the test identifies the patients for whom
use of the therapeutic product is safe, effective, or both.
See Section III.D.3. and Section III.G.1 for additional discussion of predictive and prognostic markers.
For additional trial designs and further discussion, please also refer to FDA draft guidance “Enrichment
FDA draft guidance represents FDA’s proposed approach on this topic. When final, this guidance will
represent the FDA’s current thinking on this topic.
The clinical trial design depicted in
Figure 1A, in which both test-positive and at least some test-negative subjects are
enrolled and randomized, is the most informative design because treatment by marker
interaction, as well as the prognostic versus predictive value of the marker, can be
assessed. This approach may be particularly valuable when the biological plausibility or
medical relevance of the biomarker is not well understood (e.g., based on findings from
exploratory studies or post-hoc analyses in other trials). Other variations on this design
exist, such as those including interim futility analysis where, for example, further
enrollment could be limited to test-positive subjects if harm or lack of efficacy is
A purely predictive marker will predict that patients, given a particular marker status, will have better or
therapeutic product; that is, there is a clear therapy-marker interaction. A prognostic marker would suggest that
patients with the marker would, as a consequence of the natural history of the disease, have better or worse
outcomes even absent treatment with the investigational therapeutic product; that is, the marker has little or no
interaction with the therapy. Some markers may have both predictive and prognostic properties in a given
disease/therapy setting. For example, the presence of HER-2 protein overexpression indicates a poorer
prognosis in patients with breast cancer than in patients who do not overexpress HER-2, but the same marker
also predicts greater likelihood of response to the drug trastuzumab (Herceptin). Thus, it is important to
understand the role the marker is expected to play in the therapeutic product trial. The prognostic value of the
marker, if unknown at the time of the therapeutic product trial, should be assessed in clinical trials that are
stratified by marker status.
Sponsors planning to evaluate the safety and effectiveness of a therapeutic product only
in a subset of subjects identified by an IVD should consider whether there is persuasive
evidence (e.g., evidence from strong preclinical data, preliminary clinical data, or from
clinical trials with similar therapeutics) for the marker as a predictive measure of
response or non-response. Although the sponsor may select any cutoff , FDA
recommends that sponsors choosing a marker-positive only approach assure that the
chosen marker and assigned assay cutoff are relevant to the disease under study (i.e.,
known prevalence of marker positivity in the general patient population) within the
context of likelihood of a subpopulation’s response (e.g., biologic plausibility,
mechanism of action), and that sponsors make a persuasive case for use of the IVD to
identify patients who are to be treated.
See note 63. Sponsors may also find it helpful to consider resources on this topic, e.g., Wang SJ, O’Neill RT,
Fridlyand, J. et al. “Considerations for the successful co-development of targeted cancer therapies and
companion diagnostics.” Nat Rev Drug Discov. 2013. Vol. 10, pp. 743-55; Temple, R. “Enrichment of clinical
study populations.” Clin Pharmacol Ther. 2010. 88(6), pp. 774-8.