Use of Circulating 
Tumor DNA for Early-
Stage Solid Tumor Drug 
Development 
Guidance for Industry 
 
 
 
DRAFT GUIDANCE
 
 
  This guidance document is being distributed for comment purposes only. 
 
Comments and suggestions regarding this draft document should be submitted within 60 days of 
publication in the Federal Register of the notice announcing the availability of the draft 
guidance.  Submit electronic comments to https://www.regulations.gov.  Submit written 
comments to the Dockets Management Staff (HFA-305), Food and Drug Administration, 5630 
Fishers Lane, Rm. 1061, Rockville, MD  20852.  All comments should be identified with the 
docket number listed in the notice of availability that publishes in the Federal Register. 
 
For questions regarding this draft document, contact Julia Beaver (OCE) at 240-402-0489.  
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Oncology Center of Excellence (OCE) 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
Center for Devices and Radiological Health (CDRH) 
 
May 2022 
Clinical/Medical 
 
 
     
   
Use of Circulating 
Tumor DNA for Early-
Stage Solid Tumor Drug 
Development  
Guidance for Industry 
 
 
Additional copies are available from: 
 
Office of Communications, Division of Drug Information  Office of Communication, Outreach and Development 
Center for Drug Evaluation and Research  Center for Biologics Evaluation and Research 
Food and Drug Administration  Food and Drug Administration 
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Silver Spring, MD 20993-0002  Silver Spring, MD 20993-0002 
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353  Phone: 800-835-4709 or 240-402-8010 
Email: druginfo@fda.hhs.gov  Email: ocod@fda.hhs.gov 
https://www.fda.gov/drugs/guidance-compliance-regulatory- https://www.fda.gov/vaccines-blood-biologics/guidance-
information/guidances-drugs  compliance-regulatory-information-biologics/biologics-
  guidances 
 
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Food and Drug Administration 
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Tel: 301-796-5900 
E-mail: CDRH-Guidance@fda.hhs.gov 
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/default.htm 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Oncology Center of Excellence (OCE) 
Center for Devices and Radiologic Health (CDRH) 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
May 2022 
Clinical/Medical 
   
 
Table of Contents 
I.  INTRODUCTION ........................................................................................... 1 
II.  BACKGROUND.............................................................................................. 2 
III.  DEVELOPMENT OF CTDNA AS A BIOMARKER FOR REGULATORY 
USE IN EARLY-STAGE SOLID TUMOR CLINICAL TRIALS ............................. 2 
A.  ctDNA for Patient Selection based on Molecular Alteration:.................................. 2 
B.  ctDNA Molecular Residual Disease for Patient Enrichment: ................................. 3 
C.  ctDNA as a Measure of Response......................................................................... 4 
IV.   ASSAY CONSIDERATIONS.......................................................................... 5 
A.  Types of Molecular Residual Disease Panels ......................................................... 5 
B.  Sampling Considerations .................................................................................... 5 
C.  Assay analytical validation considerations for marketing applications.................... 6 
V.  INVESTIGATIONAL DEVICE CONSIDERATIONS................................... 7 
 
 
 
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
1  Use of Circulating Tumor DNA for Early-Stage Solid Tumor Drug 
2  Development  
3  Guidance for Industry1 
4   
5   
6  This draft guidance, when finalized, will represent the current thinking of the Food and Drug 
7  Administration (FDA or Agency) on this topic.  It does not establish any rights for any person and is not 
8  binding on FDA or the public.  You can use an alternative approach if it satisfies the requirements of the 
9  applicable statutes and regulations.  To discuss an alternative approach, contact the FDA staff responsible 
10  for this guidance as listed on the title page. 
11   
12   
13   
14  I.  INTRODUCTION  
15   
16  This guidance is intended to help sponsors planning to use circulating cell-free plasma derived 
17  tumor DNA (ctDNA) as a biomarker in cancer clinical trials conducted under an investigational 
18  new drug application (IND) and/or to support marketing approval of drugs and biological 
19  products2 for treating solid tumor malignancies in the early-stage setting.  This guidance reflects 
20  FDAs current thinking regarding drug2 development and clinical trial design issues related to 
21  the use of ctDNA as a biomarker in clinical trials for solid tumor malignancies in the early stage 
22  (curative intent) setting.  This guidance does not address the use of ctDNA for the early detection 
23  of cancer or cancer screening (e.g. situations where cancer has not yet been diagnosed), or in the 
24  metastatic cancer setting.  Additional information on the related topic on use of minimal residual 
25  disease in hematologic malignancies can be found in guidance for industry Hematologic 
26  Malignancies: Regulatory Considerations for Use of Minimal Residual Disease in Development 
27  of Drug and Biological Products for Treatment (December 2020).3 
28   
29   The contents of this document do not have the force and effect of law and are not meant to bind 
30  the public in any way, unless specifically incorporated into a contract.  This document is 
31  intended only to provide clarity to the public regarding existing requirements under the law.  
32  FDA guidance documents, including this guidance, should be viewed only as recommendations, 
33  unless specific regulatory or statutory requirements are cited.  The use of the word should in 
34  Agency guidances means that something is suggested or recommended, but not required.  
35   
36   
 
1 This guidance has been prepared by the Oncology Center of Excellence in collaboration with the Center for Drug 
Evaluation and Research (CDER), the Center for Biologics Evaluation and Research (CBER), and the Center for 
Devices and Radiological Health (CDRH) at the Food and Drug Administration.  
 
2 For the purposes of this guidance, all references to drugs include both human drugs and therapeutic biological 
products unless otherwise specified. 
 
3 We update guidances periodically.  For the most recent version of a guidance, check the FDA guidance web page 
at https://www.fda.gov/RegulatoryInformation/Guidances/default.htm. 
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
37  II.  BACKGROUND 
38   
39  Drug development for solid tumors in the early stage, non-metastatic setting, typically involves 
40  large trials and multiple years of conduct and follow-up with time-to-event endpoints.  Certain 
41  patients with early-stage solid tumors can be cured with local therapy alone (e.g., surgery, 
42  radiation or chemoradiation), other patients require (neo)adjuvant systemic therapy in order to be 
43  cured, and others may progress to metastatic disease despite surgery and/or systemic therapy.  
44  ctDNA is tumor-derived fragmented DNA shed into a patients bloodstream that is not 
45  associated with cells.  ctDNA quantity can vary among individuals and depends on the type of 
46  tumor, location, stage, tumor burden, and response to therapy.  ctDNA as a biomarker has a 
47  number of potential regulatory and clinical uses in the early stage setting that may assist and 
48  expedite drug development.  In the early-stage cancer setting, ctDNA may be used to detect a 
49  certain targetable alteration, to enrich a high- or low-risk population for study in a trial, to reflect 
50  a patients response to treatment, or potentially as an early marker of efficacy.  We will discuss 
51  each of these potential uses below.  
52   
53  The evidence to support the clinical validity or clinical utility of ctDNA varies across solid tumor 
54  malignancies, patient populations, and testing modalities.  However, multiple small studies have 
55  suggested that residual ctDNA detecting molecular residual disease (MRD) after surgery or 
56  completion of standard systemic therapy confers a poor prognosis and selects a population at 
57  high risk of relapse.4  
58   
59  ctDNA assessments can vary among laboratories and technologies used to detect ctDNA which 
60  can result in discrepant results.  Many clinical laboratories develop their own protocols that can 
61  impact ctDNA measurements and detection.  Further standardization of assays will allow for 
62  better use of ctDNA in a regulatory setting and will allow for analyses across studies to validate 
63  the use of ctDNA.  
64   
65   
66  III.  DEVELOPMENT OF CTDNA AS A BIOMARKER FOR REGULATORY USE IN 
67  EARLY-STAGE SOLID TUMOR CLINICAL TRIALS 
68   
69  Sponsors should consult the FDA if they plan to incorporate ctDNA for patient selection or as an 
70  endpoint in early-stage solid tumor clinical trials.  The following are potential uses for ctDNA:  
71   
72  A.  ctDNA for Patient Selection based on Molecular Alteration:  
73  In the adjuvant treatment setting, patients typically receive curative local therapy 
74  followed by systemic treatment to prevent disease recurrence.  In this situation, 
75  sampling a patients plasma can allow for detection of ctDNA and for potential 
 
4 Powles, T., Assaf, Z.J., Davarpanah, N. et al. ctDNA guiding adjuvant immunotherapy in urothelial carcinoma. 
Nature (2021); Tie J, et al. Sci Transl Med. 2016; 8(346); Garcia-Murillas et al. JAMA Oncol. 2019; 5(10): 1473-
1478; Chaudhuri et al. Cancer Discovery 2017; 7: 1394  1403; Christensen et al. J Clin Oncol 2019; 37: 1547  
1557; Reinert, Henriksen et al. Analysis of Plasma Cell-Free DNA by Ultradeep Sequencing in Patients with Stages 
1 to III Colorectal Cancer. JAMA Oncol. 20195(8): 1124-1131; Coombes, Page et al. Personalized Detection of 
Circulating Tumor DNA Antedates Breast Cancer Metastatic Recurrence. Clin Cancer Res 2019. Jul 15; 25(14): 
4255-4263; Abbosh Birkbak et al. Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution. Nature 
2017. 
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
76  selection of a patient population harboring genetic or epigenetic alterations that 
77  could be targetable by a given drug under study.  
78   
79    ctDNA can be used as patient selection for detection of alterations for 
80  eligibility criteria for a clinical trial. 
81    ctDNA can also be used as a stratification factor if a trial enrolls both a 
82  marker-positive and marker-negative population.  Hierarchical testing 
83  procedures with the control of Type-I error rate may allow testing of 
84  multiple ordered endpoints in both the intent-to-treat population and 
85  biomarker-selected (ctDNA-positive) subgroup.  
86    The sensitivity of the ctDNA assay for detecting all variants of clinical 
87  interest contained within tumor tissue (i.e. discordance between 
88  ctDNA and tumor assays) should be evaluated.  If no variants are 
89  detected in ctDNA, tumor testing may need to be performed to confirm 
90  the negative result.  
91   
92  B.  ctDNA Molecular Residual Disease for Patient Enrichment:  
93  ctDNA can be used as a marker of MRD after definitive surgery and/or after 
94  (neo)adjuvant therapy to enrich a trial for patients with higher risk disease and 
95  increased events of disease recurrence or death.  
96   
97    ctDNA testing after surgery or (neo)adjuvant therapy could determine 
98  study eligibility of a biomarker positive population.  
99    ctDNA status at baseline could alternatively be used as a stratification 
100  factor in a study enrolling both ctDNA negative and positive patients.  
101  Hierarchical testing procedures could be performed to test both the 
102  intent-to-treat population (including both the ctDNA positive and 
103  negative group) as well as just the ctDNA positive group. 
104    Design options could include an escalation design of adding an 
105  experimental therapy to standard of care compared to standard of care 
106  alone for patients with ctDNA positive status (higher-risk) or a de-
107  escalation design based on ctDNA negative status (lower risk 
108  population).  The clinical trial should be randomized. 
109    Primary endpoint should be Disease-free survival (DFS) if only 
110  adjuvant therapy is given or Event-free survival (EFS) if neoadjuvant 
111  therapy is given (with or without adjuvant therapy), or OS (Overall 
112  Survival).5 
113    There should not be any early interim analyses of the primary 
114  endpoints due to limited events.  Later interim analyses may be 
115  considered however these should be pre-specified near the start of the 
116  trial, adjusted for the multiple testing and set at a reasonable point with 
117  robust data maturity.  For example, it would be expected that most 
 
5 See guidance for industry Clinical Trials Endpoints for the Approval of Cancer Drugs and Biologics (December 
2018). 
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
118  patients should have completed treatment prior to any interim analyses 
119  being conducted. 
120   
121  C.  ctDNA as a Measure of Response  
122   
123    ctDNA could be used in early phase clinical trials to aid in signal 
124  finding of drug activity and to potentially aid sponsors in their drug 
125  development plans.  
126    FDA encourages Sponsors to develop evidence regarding the 
127  usefulness of ctDNA response in addition to or supporting pathologic 
128  complete response information after neoadjuvant therapy. 
129   
130  D.  ctDNA as an Early Endpoint in Clinical Trials: 
131  Although not currently validated for use, changes in ctDNA in response to a drug 
132  may have the potential to be used as an early endpoint to support drug approval in 
133  the early-stage cancer setting.  
134   
135    Further data are required to support the use of ctDNA as an endpoint 
136  reasonably likely to predict long term outcome (DFS/EFS/OS).  
137    Trials that collect ctDNA data before and after drug treatment should 
138  also collect long term outcome data to characterize the association 
139  between ctDNA clearance and outcome.  
140    Various statistical criteria have been proposed for validating an 
141  endpoint and often meta-analytical approaches have been used. 6  An 
142  appropriate meta-analysis to validate ctDNA at a trial level association 
143  should include only randomized trials.  Sponsors should discuss and 
144  provide details of any proposed meta-analysis plan to validate use of 
145  ctDNA in a particular context of use with the FDA.  
146    The plan should include details of trial designs, inclusion and 
o
147  exclusion criteria, ctDNA assessment methods, and disease 
148  setting.  A justification for the suitability of pooling the studies 
149  should be provided.  
150    Trials should include a patient population representative of the 
o
151  population in which the endpoint ultimately will be used. 
152    An adequate number of randomized trials with sufficient 
o
153  follow-up time should be included and justified.  
154    Analysis based on individual patient-level data should allow an 
o
155  assessment of individual-level association.  
156    Prespecified criteria for concluding association based on both 
o
157  trial-level and individual -level association measures, including 
158  prespecified timing and window of ctDNA assessment should 
159  be provided.  
 
6 For additional information on meta-analyses, see the draft guidance for industry Meta-analyses of Randomized 
Controlled Clinical Trials to Evaluate the Safety of Human Drugs or Biological Products (November 2018).  When 
final, this guidance will represent FDAs current thinking on this topic.  
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
160    Long-term clinical endpoints, such as EFS/DFS and OS that 
o
161  have been clearly and consistently defined across studies 
162  should be included.  
163    Sponsors should explore the effects of missing data on trial 
o
164  results.  
165   
166   
167  IV.   ASSAY CONSIDERATIONS 
168   
169  A.  Types of Molecular Residual Disease Panels 
170  MRD panels can utilize tumor-informed methods, tumor-naive methods, or a 
171  smaller panel of candidate genes each with its own strengths and limitations as 
172  summarized below:  
173   
174    Tumor-informed panels are constructed by sequencing the tumor and 
175  then selecting a set of variants to follow.   
176    Limitations of this approach include lag time between tumor 
o
177  testing and ctDNA panel creation, and sensitivity and 
178  specificity may depend on clinical cutoffs and analytical 
179  sensitivity of the device as well as the number of tumor 
180  informed targets assayed.   
181    Tumor-naive or tumor-agnostic panels are those that are not 
182  informed by sequencing or by mutations of the primary tumor. This 
183  approach uses panel-based next generation Sequencing (NGS) to 
184  ascertain MRD.  
185    Limitations include tumor markers not covered by the ctDNA 
o
186  panel and additional characterization of panels would be 
187  needed to understand what percentage of patients are trackable 
188  with such techniques.   
189    Whole genome sequencing (WGS) could potentially be used in 
o
190  a tumor-naive fashion.  This would allow the use of other 
191  biomarkers besides mutations, epigenetic alterations (e.g. 
192  methylation) or fragmentomic analysis of ctDNA to capture 
193  tumor derived ctDNA signals.   
194   
195  Multiple markers on a candidate gene panel could help assure that the MRD assay will 
196  serve its function, even with the development of additional cytogenetic changes.  
197   
198  B.  Sampling Considerations 
199  There are several sampling considerations related to the clinical trial design and 
200  the intended use patient population that should be taken into account.  
201   
202    The shedding of ctDNA is affected by histology, grade, stage, and size 
203  of the tumor thus timing of ctDNA testing should be discussed with 
204  the FDA and should be supported by performance characteristics of 
205  the test, disease characteristics and tumor biology.   
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
206    A set time point should be chosen for enrollment into the study and 
207  pre-specified.  
208    If a sponsor wishes to use multiple ctDNA time points to determine 
209  eligibility (e.g. screening paradigm evaluating if intervention at early 
210  detection of recurrence would influence outcome) this should be 
211  supported by scientific data/rationale.  Sensitivity analyses based on 
212  different time windows could be explored (but should be 
213  predetermined and discussed in advance).  
214    The timing of ctDNA testing should be the same across study arms. 
215    A baseline pre-treatment sample should be collected to allow for 
216  consideration of the impact of variation in tumor shedding rates on 
217  assay performance.  In addition, this sample will allow for 
218  interpretation of the post-treatment sample for study enrollment.   
219    All sites in the study should follow standardized protocols for sample 
220  collection, storage, and processing and handling. 
221   
222  C.  Assay analytical validation considerations for marketing applications 
223  Analytical validation ensures that the assay measures the analyte or analytes that 
224  it is intended to measure in the intended tumor type.  Analytical validation should 
225  be conducted to establish the performance characteristics of the assay.  Validation 
226  studies should be acceptable in terms of the assays sensitivity, specificity, 
227  accuracy, precision, and other relevant performance characteristics using a 
228  specified technical protocol, which may include specimen collection, handling, 
229  and storage procedures.7  The acceptance criteria for the validation studies should 
230  be adequately justified to support clinical use. 
231   
232    MRD assay validation should encompass the entire assay system from 
233  sample collection (e.g., blood collection in the specific collection tube 
234  that will be used with the final market ready assay) to the output of the 
235  assay including the detection threshold (cut-off) that determines 
236  positive vs negative patients.  The cutoff should be established 
237  appropriately (e.g., both in terms of allelic frequencies or mutant 
238  molecules of the variants per ml of plasma and number of variants that 
239  are required to be positive in personalized panels for MRD positivity).  
240    The assay cutoff should be established to optimize assay sensitivity 
241  and specificity for the clinical use.  Analytical performance should be 
242  robust to detect MRD positivity accurately and reproducibly. 
243    The assay should have high sensitivity and negative predictive value 
244  (NPV) for supporting de-escalation of treatment and high specificity 
245  and positive predictive value (PPV) for supporting escalation of 
246  treatment.  
247    The validation approach of an MRD test will depend on the type of 
248  MRD testing modality.  As noted in section IV A., there are different 
 
7 Summary of Safety and Effectiveness Data (SSED) for the Guardant360 CDx PMA P200010: 
https://www.accessdata.fda.gov/cdrh_docs/pdf20/P200010B.pdf 
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
249  types of MRD testing approaches that are currently under 
250  development.  For tumor-naive NGS-based MRD panels, panel-based 
251  validation of fixed panel content will be needed; however, for tumor-
252  informed NGS-based personalized panels, the panel content will vary 
253  for each patient and therefore the assay validation will be based on 
254  each personalized assay. The validation strategy to support the device 
255  marketing application should be discussed with CDRH/FDA. 
256    Samples from clinical trials (clinical specimens) are recommended to 
257  be used for key assay validation studies such as confirmation of the 
258  assay limit of detection (LoD), assay precision, analytical accuracy, 
259  assay input studies.  In some analytical validation studies since a large 
260  volume of sample will be needed, clinical samples may be 
261  supplemented by contrived samples.  In general, when using contrived 
262  samples in assay validation studies, the functional equivalency 
263  between the contrived and clinical samples should be demonstrated 
264  and rationale should be provided if contrived samples are used to 
265  substitute or supplement clinical samples in certain studies.  
266    For fixed panels, cell lines carrying the specific alterations (i.e., cell 
267  line DNA spiked into an appropriate matrix) may be used as contrived 
268  samples.  For personalized assays, cell lines that represent a 
269  distribution of the number and type of variants based on early clinical 
270  study data should be developed.  
271    Assay precision should be demonstrated using samples across the 
272  detection range of the assay including samples at the assay cutoff and 
273  samples with the minimum analyte requirements. 
274    An appropriate set of reference materials should be developed to allow 
275  for comparability across multiple MRD assays. 
276   
277  V.  INVESTIGATIONAL DEVICE CONSIDERATIONS 
278   
279    The investigational ctDNA device used in the trial is subject to FDAs 
280  investigational device exemption (IDE) regulations as well as 21 CFR parts 50 
281  and 56.8 
282    Whether the sponsor needs to submit an IDE application is dependent on 
283  whether the device used in the trial is considered significant risk (SR), non-
284  significant risk (NSR), or exempt.9 
285    Sponsors can submit a Study Risk Determination pre-submission through 
286  CDRHs Q-submission program.10  
 
8 See 21 CFR 812. 
9 See guidance for industry Information Sheet Guidance for IRBs, Clinical Investigators, and Sponsors. Significant 
Risk and Nonsignificant Risk Medical Device Studies (January 2006). 
10 See guidance for industry and FDA staff Requests for Feedback and Meetings for Medical Device Submissions: 
The Q-Submission Program (January 2021).  
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
287    The sponsor may also seek a risk determination through the optional 
288  streamlined submission process for investigational devices in oncology trials 
289  for new INDs.11 
 
11 See guidance for industry Investigational In Vitro Diagnostics in Oncology Trials: Streamlined Submission 
Process for Study Risk Determination (October 2019).