Developing Targeted 
Therapies in Low-Frequency 
Molecular Subsets of a Disease 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
October 2018 
Clinical Pharmacology 
 
Developing Targeted Therapies in Low-Frequency Molecular Subsets of a Disease 
10/15/18   
Developing Targeted 
Therapies in Low-Frequency 
Molecular Subsets of a Disease 
Guidance for Industry 
 
 
Additional copies are available from: 
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Center for Drug Evaluation and Research 
Food and Drug Administration 
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Email: ocod@fda.hhs.gov 
https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/default.htm    
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
 October 2018 
Clinical Pharmacology 
 
 
Developing Targeted Therapies in Low-Frequency Molecular Subsets of a Disease 
10/15/18  Contains Nonbinding Recommendations 
 
TABLE OF CONTENTS 
 
 
 
I.  INTRODUCTION AND BACKGROUND ................................................................ 1 
II.  DEVELOPMENT AND REGULATORY CONSIDERATIONS............................... 2 
A.  Identification of Patients for Inclusion in Clinical Trials .................................................. 2 
B.  Generalizability of Findings ........................................................................................... 4 
C.  Benefit and Risk Determination and Labeling ................................................................. 4 
D.  Refining the Target Population/Indication After Initial Approval ..................................... 5 
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Developing Targeted Therapies in Low-Frequency Molecular Subsets of a Disease 
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Developing Targeted Therapies in Low-Frequency Molecular 
Subsets of a Disease 
Guidance for Industry1 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on 
this topic.  It does not establish any rights for any person and is not binding on FDA or the public.  You 
can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations.  
To discuss an alternative approach, contact the FDA office responsible for this guidance as listed on the 
title page.   
 
 
 
 
I.  INTRODUCTION AND BACKGROUND 
 
Insights into the molecular basis of disease have led to the development of targeted therapies.2  
Often, the pharmacological  effect of a targeted therapy is related to a particular molecular 
alteration.3  Many clinically  defined diseases are caused by a range of different molecular 
alterations, some of which may occur at low frequencies, that impact common proteins or 
pathways involved  in the pathogenesis of diseases.  In a population  of patients with the same 
clinical  disease, the heterogeneity in the molecular etiology  may influence responsiveness to a 
particular targeted therapy.  However, certain targeted therapies may be effective in multiple 
groups of patients who have different underlying  molecular alterations because the functional 
effect of the molecular alterations may be similar.  Therefore, the FDA is providing  guidance on 
the type and quantity of evidence that can demonstrate efficacy across molecular subsets within a 
disease, particularly  when one or more molecular subsets occur at a low frequency. 
 
The purpose of this guidance is to describe:  (1) the FDAs current recommendations on how to 
group patients with different molecular alterations for eligibility  in clinical  trials; and (2) general 
                                              
1 This guidance has been prepared by the Office of Translational Sciences in the Center for Drug Evaluation and 
Research in cooperation with the Center for Biologics Evaluation and Research at the Food and Drug 
Administration. 
 
2 For the purpose of this guidance, a targeted therapy is defined as a drug intended for populations that are subsets 
of clinically-defined diseases and that are identified by using diagnostic testing.  For the purposes of this guidance, 
all references to drugs include both human drugs and therapeutic biological products unless otherwise specified. 
 
3 For the purpose of this guidance, molecular alteration refers to a broad array of molecular changes in DNA, RNA, 
or proteins, including point mutations, gene fusions, mutational load, epigenetic changes, and over- or under-
expression.  Molecular subset refers to a subgroup of the clinically defined disease caused by a specific molecular 
alteration or group of molecular alterations that are observed in the clinical disease. 
 
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Developing Targeted Therapies in Low-Frequency Molecular Subsets of a Disease 
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approaches to evaluating the benefits and risks of targeted therapies within a clinically  defined 
disease where some molecular alterations may occur at low frequencies.4     
 
In general, FDAs guidance documents do not establish legally  enforceable responsibilities.   
Instead, guidances describe the Agencys current thinking  on a topic and should be viewed only 
as recommendations,  unless specific regulatory or statutory requirements are cited.  The use of 
the word should in Agency guidances means that something  is suggested or recommended, but 
not required.   
 
 
II.  DEVELOPMENT AND REGULATORY CONSIDERATIONS 
 
A.  Identification of Patients for Inclusion in Clinical Trials 
 
  The appropriateness of pursuing an indication  in a molecular subset of a disease depends 
on the strength of evidence supporting  the hypothesis that patients with the molecular 
alteration of interest will be more likely  to respond to the targeted therapy owing to some 
property of the drug (e.g., based on a putative mechanism of action or previous clinical 
experience with the drug) than patients without the molecular alteration.5  
 
  If a sponsor is pursuing  an enrichment strategy based on molecular criteria for the 
purposes of clinical  trial design and eligibility,  the FDA will accept grouping  patients 
with different molecular alterations if it is reasonable to expect that the grouped patients 
will have similar  pharmacological  responses based on a strong scientific rationale.  The 
rationale for grouping  patients can be based on computational  (e.g., in silico), 
experimental (e.g., in vitro or animal experiments), or clinical evidence.  The grouping  of 
molecular alterations may be defined in many ways, including  a list of individual 
alterations (within a single gene or multiple  genes) or a functionally  defined group of 
alterations (e.g., deleterious or sensitizing).   Sponsors should discuss any proposed 
grouping  strategy and plans for identifying the proposed group in the clinical setting with 
the FDA.   
 
  Types of evidence that could support a grouping  strategy are listed below, although other 
sources of evidence may also be appropriate.  In general, clinical  studies are considered 
                                              
4 For the purpose of this guidance, low-frequency molecular alterations are those that occur at frequencies low 
enough that enrolling a sufficient number of patients to conduct a clinical trial limited to the specific molecular 
alteration of interest is not feasible or practical.   
 
5 For further discussion on clinical trial enrichment strategies, refer to the draft guidance for industry entitled 
Enrichment Strategies for Clinical Trials to Support Approval of Human Drugs and Biological Products.  When 
final, this guidance will represent the FDAs current thinking on this topic.  For the most recent version of a 
guidance, check the FDA Drugs or Biologics guidance Web pages at 
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm or 
https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/default.htm.    
 
 
 
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Developing Targeted Therapies in Low-Frequency Molecular Subsets of a Disease 
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the strongest type of evidence; however, other types of evidence may also support a 
grouping  strategy (in combination  with or independent of clinical  studies).  When 
consistent effects are observed across more than one line of evidence, the strength of the 
evidence increases. 
 
  Clinical studies:  Preliminary studies demonstrate that patients with the proposed 

group of specific molecular alterations exhibit  similar responses to the investigational 
drug based on a clinical  efficacy endpoint or pharmacodynamic (PD) biomarker. 
 
  Nonclinical studies:  Similar drug effects are observed across a model system with 

the proposed group of molecular alterations in nonclinical  studies (e.g., similar 
activity demonstrated across molecular alterations at clinically  relevant 
concentrations or dosages). 
 
  In silico or mechanism-based evidence:  The investigational  drug is expected to 

have a similar  effect across patients with the proposed group of molecular alterations 
based on computational  studies or mechanistic rationale (e.g., all protein-truncating 
mutations or over-expression of the drug target in tumor tissue). 
 
  Evidence from other drugs in the same pharmacological class:  Drugs with the 

same mechanism of action as the drug under investigation  show similar  effects across 
the proposed group of molecular alterations in a nonclinical  model or clinical  trials. 
Note that confidence in the extrapolation  of findings  from one drug to another in the 
same class depends on their similarity  in structure, binding  sites, and other drug 
properties.   
 
  Phenotypic characterization of molecular alterations:  The proposed group of 

molecular alterations results in a similar baseline of disease-related nonclinical  or 
clinical  phenotypes (e.g., mutations that result in increased activity of the drug target).   
 
  In some situations,  it may be desirable to include patients who have molecular alterations 
that are less likely to be responsive (e.g., unmet medical need) or for which the likelihood 
of response is not known based on the available evidence.  In this setting, the sponsor has 
the option of either splitting  study alpha assessment of the primary efficacy endpoint 
between a subgroup of patients with specific molecular alterations of interest and a 
broader enrolled population  or using an adaptive design with interim assessments of 
efficacy.  Alternatively,  the sponsor may limit  the assessment of the primary efficacy 
endpoint to the subgroup of patients with the specific molecular alteration(s) of interest 
while enrolling  additional  subgroups to generate preliminary  clinical  data.  
 
 
 
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Developing Targeted Therapies in Low-Frequency Molecular Subsets of a Disease 
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  Sponsors should use analytically  validated assays for the identification  of molecular 
alterations used for enrollment of patients into clinical  trials.6  The FDA recognizes that 
the clinical  trial assay method may limit  who is eligible  for clinical  trials.  Ideally, 
clinical  trial assays should be designed to detect all possible molecular alterations that 
comprise the group that is expected to respond.  
 
B.  Generalizability of Findings 
 
  Targeted therapies may be effective in multiple  molecular subsets that make up a clinical 
disease.  However, the FDA anticipates that for certain subsets only a small number of 
patients (or even none despite eligibility  criteria that are inclusive  of such patients) will 
be enrolled into the trial.  The low numbers or the absence of such patients would in most 
settings preclude meaningful  empirical inferences about treatment benefits or risks in 
patients with those particular molecular alterations.  However, when sponsors follow the 
principles  for grouping  patients set out in section II.A., Identification  of Patients for 
Inclusion  in Clinical  Trials, extrapolation  of efficacy findings  across multiple  subsets 
may be possible despite the low frequency or absence of patients in some subsets. 
 
  Given the above considerations,  if the clinical trials are successful and other conditions 
for approval are met, the FDA will,  in most circumstances, approve the drug for all 
patients who meet the inclusion  criteria for the trial based on the prespecified criteria, 
irrespective of the extent to which patients with various molecular alterations were 
represented in the clinical  trial.  Substantial evidence of effectiveness for the drug in the 
indicated population  would be based on the strength of evidence (see section II.A., 
Identification  of Patients for Inclusion  in Clinical  Trials) demonstrating  that patients in 
the molecular grouping  defined by the inclusion  criteria will respond similarly  to the 
patients who participated in the study.   
 
  As with all new drug approvals, the FDA will consider the totality of evidence in 
weighing  the benefits and risks of the drug.  If after trial enrollment,  the FDA or sponsor 
newly identifies  a substantial scientific issue essential to determining  the safety or 
effectiveness of the drug in some molecular subset included  in the trial, the indicated 
patient population  may be narrower than the clinical  trial enrollment  criteria.    
 
C.  Benefit and Risk Determination and Labeling 
 
  Labeling will reflect the overall benefits and risks of the drug in the target population.   
When the drug is approved for all patients who meet the inclusion  criteria for the 
                                              
6 For additional information, see the draft guidance for industry and FDA staff entitled Principles for 
Codevelopment of an In Vitro Companion Diagnostic Device with a Therapeutic Product.  When final, this guidance 
will represent the FDAs current thinking on this topic.  For the most recent version of a guidance, check the FDA 
Drugs or Biologics guidance Web pages at 
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm or 
https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/default.htm.    
 
 
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Developing Targeted Therapies in Low-Frequency Molecular Subsets of a Disease 
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registration trials as discussed in section II.B., Generalizability  of Findings,  the indication 
as described in the INDICATIONS AND USAGE section of labeling  should be 
sufficiently  broad to include treatment of patients with low-frequency molecular 
alterations who would have been eligible  for the trial irrespective of the extent to which 
they were represented in clinical  trials.  The studies informing  the basis for grouping 
patients (e.g., cell or animal models, PD data) should be clearly specified (e.g., in the 
CLINICAL PHARMACOLOGY or CLINICAL STUDIES section of labeling). 
 
  Recognizing  the analyses may be exploratory, evidence supporting  the efficacy of the 
drug for each molecular subset should be transparently displayed  (e.g., tabulation  of the 
number of patients with specific molecular alterations that were enrolled in the clinical 
trial and the outcomes of the patients) in the CLINICAL STUDIES section of labeling. 
 
  When accurate testing for molecular alterations (whether as a class or as specific 
alterations) is essential for the safe and effective use of the drug, an FDA-cleared 
or -approved assay should be commercially  available at the time of drug approval to 
identify patients in the clinical  setting.  The FDA may grant exceptions when the drug is 
intended to treat a serious or life-threatening  condition  for which no satisfactory 
alternative treatment exists, and the FDA determines that the benefits from the use of the 
drug outweigh the risks from the lack of an approved or cleared in vitro companion 
diagnostic device.7 
 
D.  Refining the Target Population/Indication After Initial Approval 
 
  Incremental expansion of the population  eligible  for a drug may be part of a lifecycle 
strategy, and sponsors are encouraged to conduct additional studies in molecular subsets that 
may respond to the drug but were not eligible for inclusion in the original trials.  The amount 
and nature of clinical  efficacy data needed to expand a drugs indication  depends on the 
similarity  of pharmacologic  responses and the mechanistic rationale for the drugs effect 
in the population  for which efficacy was initially  established and in the population  to 
which the indication  is being expanded.  Similarly,  if substantive data emerge from 
studies (whether observational  or randomized  trials) indicating  lack of efficacy in certain 
molecular subgroups for which the drug was initially  indicated, the FDA will consider 
narrowing the intended population  as appropriate. 
 
  In some cases, generating data in the postmarket setting may be necessary to provide 
additional  information  regarding the risks and benefits of the drug in subsets of patients 
with limited  or no enrollment in clinical  trials.  Real-world evidence (e.g., from 
observational  studies or registry data), traditional  controlled trials, or data from other 
sources (including  trials already ongoing)  may be appropriate.  As with all drug 
development programs, if the FDA approves the drug under the accelerated approval 
                                              
7 For additional information, see the guidance for industry and FDA staff entitled In Vitro Companion Diagnostic 
Devices.   
 
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Developing Targeted Therapies in Low-Frequency Molecular Subsets of a Disease 
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program8 or identifies  safety concerns during the review process, the FDA may require 
postmarketing  studies.   
 
 
                                              
8 See the guidance for industry entitled Expedited Programs for Serious ConditionsDrugs and Biologics. 
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Developing Targeted Therapies in Low-Frequency Molecular Subsets of a Disease 
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