Hematologic Malignancies: 
Regulatory Considerations for 
Use of Minimal Residual 
Disease in Development of Drug 
and Biological Products for 
Treatment 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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) 
 
January 2020 
Clinical/Medical
  Hematologic Malignancies: 
Regulatory Considerations for 
Use of Minimal Residual 
Disease in Development of Drug 
and Biological Products for 
Treatment 
Guidance for Industry 
 
Additional copies are available from: 
 
Office of Communications, Division of Drug Information 
Center for Drug Evaluation and Research 
Food and Drug Administration 
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor 
 Silver Spring, MD 20993-0002 
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353; Email: druginfo@fda.hhs.gov 
https://www.fda.gov/drugs/guidance-compliance-regulatory-information/guidances-drugs 
and/or 
Office of Communication, Outreach, and Development 
Center for Biologics Evaluation and Research 
Food and Drug Administration 
10903 New Hampshire Ave., Bldg. 71, Room 3128 
Silver Spring, MD  20993-0002 
Phone: 800-835-4709 or 240-402-8010; Email: ocod@fda.hhs.gov 
https://www.fda.gov/vaccines-blood-biologics/guidance-compliance-regulatory-information-biologics/biologics-
guidances 
 
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) 
 
January 2020 
Clinical/Medical 
 
  TABLE OF CONTENTS 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  BACKGROUND ............................................................................................................... 2 
III.  DEVELOPMENT OF MRD AS A BIOMARKER FOR REGULATORY USE ........ 3 
A.  Regulatory Uses of Biomarkers .................................................................................................... 3 
B.  Mechanisms for Novel Surrogate Endpoint Acceptance or Qualification ................................ 4 
C.  Meta-Analyses for Validation of MRD as a Surrogate Endpoint .............................................. 5 
D.  MRD as an Endpoint in Clinical Trials ....................................................................................... 7 
E.  MRD for Patient Selection or Enrichment .................................................................................. 8 
IV.  TECHNOLOGY CONSIDERATIONS .......................................................................... 8 
A.  Assay Considerations ..................................................................................................................... 8 
1.  Cellular Technology Platforms ....................................................................................................................9 
2.  Molecular Technology Platforms .................................................................................................................9 
3.  All Technology Platforms ........................................................................................................................... 10 
B.  Sampling Considerations............................................................................................................. 10 
V.  DISEASE-SPECIFIC CONSIDERATIONS ................................................................ 11 
A.  Acute Lymphoblastic Leukemia ................................................................................................. 11 
B.  Acute Myeloid Leukemia ............................................................................................................ 11 
C.  Acute Promyelocytic Leukemia .................................................................................................. 12 
D.  Chronic Lymphocytic Leukemia ................................................................................................ 13 
E.  Chronic Myeloid Leukemia ........................................................................................................ 13 
F.  Multiple Myeloma ........................................................................................................................ 14 
VI.  REGULATORY SUBMISSIONS THAT USE MRD .................................................. 15 
GLOSSARY................................................................................................................................. 18 
  Contains Nonbinding Recommendations 
1  Hematologic Malignancies: Regulatory Considerations for Use of 
2  Minimal Residual Disease in Development of Drug and Biological 
3  Products for Treatment 
4  Guidance for Industry1 
5   
6   
7   
8  This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on 
9  this topic.  It does not establish any rights for any person and is not binding on FDA or the public.  You 
10  can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations.  
11  To discuss an alternative approach, contact the FDA office responsible for this guidance as listed on the 
12  title page. 
13   
14   
15   
16  I.  INTRODUCTION 
17   
18  This guidance is intended to help sponsors planning to use minimal residual disease (MRD) as a 
19  biomarker in clinical trials conducted under an investigational new drug application (IND) or to 
20  support marketing approval of drugs and biological products2 for treating specific hematologic 
21  malignancies. 
22   
23  The use of MRD as a biomarker in drug development is distinct from FDAs requirement for 
24  investigating, clearing, or approving an in vitro diagnostic device for clinical use in measuring 
25  MRD.  Manufacturers interested in developing a specific MRD assay for clinical use should 
26  consult the Office of In Vitro Diagnostics and Radiological Health in the Center for Devices and 
27  Radiological Health (CDRH). 
28   
29  In general, FDAs guidance documents do not establish legally enforceable responsibilities.  
30  Instead, guidances describe the Agencys current thinking on a topic and should be viewed only 
31  as recommendations, unless specific regulatory or statutory requirements are cited.  The use of 
32  the word should in Agency guidances means that something is suggested or recommended, but 
33  not required. 
34   
                                                 
1 This guidance has been prepared by the Oncology Center of Excellence in cooperation with the Center for Drug 
Evaluation and Research and the Center for Biologics Evaluation and Research at the Food and Drug 
Administration. 
 
2 For the purposes of this guidance, all references to drug products include both human drugs and biological 
products. 
1  Contains Nonbinding Recommendations 
35   
36  II.  BACKGROUND 
37   
38  Despite development of treatments that eliminate morphologically detectable malignant cells, 
39  some patients with hematologic malignancies who have achieved complete remission or 
40  complete response (CR), even of considerable duration, will experience relapse.  Conventional 
41  morphologic detection for hematologic malignancies has a threshold limit of one tumor cell in 
42  100 cells.  Technology exists that can detect the persistence of malignancy at orders of 
43  magnitude below the limit of conventional morphologic detection, a level of disease burden 
44  known as MRD.  These technologies measure cell characteristics such as genetic mutations, cell 
45  surface markers, or specific DNA gene rearrangements. 
46   
47  MRD as a general measure of tumor burden has multiple potential regulatory and clinical uses as 
48  a biomarker.  Depending upon the clinical setting, MRD may be used to reflect a patients 
49  response to treatment or as a prognostic tool to assess a patients risk of future relapse.  As such, 
50  MRD can be used to enrich clinical trial populations or guide allocation into specific treatment 
51  arms in clinical trials.  There are challenges within each context of use that need to be addressed, 
52  such as the underlying disease, patient heterogeneity, therapeutic context, target of therapy, or a 
53  combination of disease parameters, to allow effective use of MRD in regulatory decision-
54  making. 
55   
56  MRD assessments can vary among laboratories and technologies, which can result in discrepant 
57  results. Many clinical laboratories develop their own protocols that can affect MRD 
58  measurements.  Technologies can have different performance characteristics.  Sample collection 
59  procedures can also differ.  However, standardized methodologies can ensure that results 
60  obtained between technologies and laboratories are consistent.  This includes standardized 
61  posttreatment timing for when a bone marrow (BM) or blood sample is collected, standardized 
62  sample processing, predetermined MRD thresholds, and accurate reporting of the performance 
63  characteristics of the test (e.g., accuracy, precision, specificity, sensitivity).  For example, 
64  reporting MRD negative results without information regarding limit of detection is not 
65  meaningful. 
66   
67  The evidence to support the clinical validity of MRD as a biomarker varies across hematologic 
68  cancer types and patient populations.  To gain a better understanding of the state of the science of 
69  MRD, FDA cosponsored public workshops on MRD in acute lymphoblastic leukemia (ALL), 
70  chronic lymphocytic leukemia (CLL), and acute myeloid leukemia (AML) as well as a 
71  symposium on MRD in multiple myeloma (MM) from 2012 through 2014.  In addition, a public 
72  workshop, Minimal Residual Disease as a Surrogate Endpoint in Hematologic Cancer Trials,3 
73  was held on September 7, 2016, under a cooperative agreement with FDA to discuss the clinical, 
74  statistical, and technical barriers to implementing use of MRD in clinical trials.  As a result of 
75  these workshops and an analysis4 of marketing applications showing inconsistent quality of 
                                                 
3 See https://healthpolicy.duke.edu/events/minimal-residual-disease-surrogate-endpoint-hematologic-cancer-trials. 
 
4 Gormley N, V Bhatnagar, LA Ehrlich, B Kanapuru, H-Z Lee, AE McKee, A Farrell, and R Pazdur., 2017, FDA 
Analysis of MRD Data in Hematologic Malignancy Applications, J Clin Oncol, 35:2541. 
2  Contains Nonbinding Recommendations 
76  MRD data, FDA identified a need to provide sponsors with guidance on use of MRD as a 
77  biomarker in regulatory submissions. 
78   
79   
80  III.  DEVELOPMENT OF MRD AS A BIOMARKER FOR REGULATORY USE 
81   
82  A.  Regulatory Uses of Biomarkers 
83   
84  The term biomarker is commonly understood as referring to a characteristic that is measured as 
85  an indicator of normal biologic processes, pathogenic processes, or responses to an exposure or 
86  intervention, including therapeutic interventions.5  MRD can be used as a biomarker.  The 
87  terminology listed below is derived from the BEST Resource6 definitions and the draft guidance 
88  for industry and FDA staff Qualification Process for Drug Development Tools (December 
89  2019)7 but has been slightly modified to reflect applicability to MRD.  Sponsors can potentially 
90  use MRD status as any of the following types of biomarkers: 
91   
92    Diagnostic biomarker: A biomarker used to detect or confirm presence of a disease or 
93  condition of interest or to identify individuals with a subtype of the disease. 
94   
95    Prognostic biomarker: A biomarker used to identify likelihood of a clinical event, 
96  disease recurrence or progression in patients who have the disease or medical condition 
97  of interest.  A prognostic biomarker informs about the natural history of the disease in 
98  that particular patient in the absence of a therapeutic intervention. 
99   
100    Predictive biomarker: A biomarker used to identify individuals who are more likely 
101  than similar individuals without the biomarker to experience a favorable or unfavorable 
102  effect from exposure to a drug product. 
103   
104    Efficacy-response biomarker: A biomarker that is used to show that a response has 
105  occurred in an individual who has been exposed to a drug product. 
106   
107    Monitoring biomarker: A biomarker measured serially and used to detect a change in 
108  degree or extent of the disease. 
109   
                                                 
5 See FDA-NIH Biomarker Working Group, 2018, BEST (Biomarkers, EndpointS, and other Tools) Resource, 
accessed September 9, 2019, https://www.ncbi.nlm.nih.gov/books/NBK338448/.  See also Section 507 of the 
Federal Food, Drug, and Cosmetic Act, which defines biomarker for purposes of that section, in relevant part, as a 
characteristic (such as a physiologic, pathologic, or anatomic characteristic or measurement) that is objectively 
measured and evaluated as an indicator of normal biologic processes, pathologic processes, or biological responses 
to a therapeutic intervention. 
 
6 FDA-NIH Biomarker Working Group, 2018, BEST (Biomarkers, Endpoints, and other Tools) Resource. 
 
7 When final, this guidance will represent the FDAs current thinking on this topic.  For the most recent version of a 
guidance, check the FDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-
documents. 
3  Contains Nonbinding Recommendations 
110  An efficacy-response biomarker could be a surrogate endpoint.  A surrogate endpoint does not 
111  measure the clinical benefit of primary interest; instead, it predicts the clinical benefit based on 
112  epidemiologic, therapeutic, pathophysiologic, or other scientific evidence.  Specifically, a 
113  surrogate endpoint predicts a specific clinical outcome of the patient at some later time and can 
114  be used as the basis of marketing application approval decisions. 
115   
116  Understanding which of these biomarker attributes applies to the proposed use of MRD is 
117  important to consider when validating MRD for that proposed use and for the trial design.  There 
118  are challenges within each MRD context of use that should be adequately justified, such as 
119  underlying disease, patient heterogeneity, therapeutic context, target of therapy, or a combination 
120  of disease parameters. 
121   
122  B.  Mechanisms for Novel Surrogate Endpoint Acceptance or Qualification 
123   
124  Two mechanisms exist to obtain the Agencys feedback on the use of a novel surrogate endpoint 
125  to support approval.  One mechanism is through the formal drug development tool (DDT) 
126  qualification process, specifically the biomarker qualification process.  The DDT qualification 
127  process is an initiative undertaken in response to FDAs Critical Path Initiative and updated 
128  under the 21st Century Cures Act, adding section 507 to the Federal Food, Drug, and Cosmetic 
129  Act.  The purpose of the DDT qualification process is to qualify a DDT for a specific context of 
130  use, such that a sponsor and FDA can rely on the DDT to have a specific interpretation and 
131  application in drug development and regulatory review.  FDA will make information about a 
132  DDT that has been formally qualified for a specific context of use publicly available to expedite 
133  drug development and review of regulatory applications.  A qualified DDT can be included in 
134  submissions of INDs, new drug applications (NDAs), or biologics license applications (BLAs) 
135  without the need for FDA to reconsider and reconfirm the suitability of the DDT.  Qualifying a 
136  biomarker requires robust scientific evidence, and there is a higher evidentiary standard if the 
137  biomarker is to be used as a surrogate endpoint.8 
138   
139  A second mechanism to obtain the Agencys feedback on the use of a novel surrogate endpoint 
140  to support approval is through discussions with the specific Center for Drug Evaluation and 
141  Research (CDER) or Center for Biologics Evaluation and Research (CBER) review division.  In 
142  this setting, the pharmaceutical sponsor or interested group meets with the FDA review division 
143  to present scientific data in support of the proposed surrogate endpoint.  These data may be from 
144  previous clinical trials conducted by the sponsor, a meta-analysis of several trials conducted in 
145  the disease area, or other data, including product-nonspecific data, that support the use of the 
146  proposed surrogate endpoint.  An example of this mechanism for a surrogate endpoint reasonably 
147  likely to predict clinical benefit is pathologic complete response in neoadjuvant treatment of 
148  breast cancer.9  An example of a validated surrogate endpoint that used this mechanism is the 
                                                 
8 For additional information on the DDT qualification process, see the DDT Qualification Programs web page at 
www.fda.gov/Drugs/DevelopmentApprovalProcess/DrugDevelopmentToolsQualificationProgram/default.htm and 
the draft guidance for industry and FDA staff Qualification Process for Drug Development Tools.  When final, this 
guidance will represent the FDAs current thinking on this topic. 
 
9 See the guidance for industry Pathological Complete Response in Neoadjuvant Treatment of High-Risk Early- 
Stage Breast Cancer: Use as an Endpoint to Support Accelerated Approval (October 2014).  We update guidances 
 
4  Contains Nonbinding Recommendations 
149  surrogate of complete response at 30 months in follicular lymphoma.  A surrogate endpoint that 
150  is reasonably likely to predict clinical benefit can be used to support accelerated approval, and a 
151  validated surrogate endpoint can support traditional approval.10  To explore this approach 
152  further, sponsors should request a meeting with the relevant review division. 
153   
154  With either approach, the strength of evidence to support surrogacy depends on (1) biological 
155  plausibility of the relationship, (2) demonstration in epidemiological studies of the prognostic 
156  value of the surrogate endpoint for the clinical outcome, and (3) evidence from clinical trials that 
157  treatment effects on the surrogate endpoint correspond to effects on the clinical outcome.11 
158   
159  C.  Meta-Analyses for Validation of MRD as a Surrogate Endpoint 
160   
161  Various statistical criteria have been proposed for validating a surrogate endpoint; often, meta-
162  analytical approaches have been used.  The terminology and definitions below provide further 
163  detail about statistical principles relevant to the validation of a surrogate endpoint. 
164   
165    Individual-level association is the strength of the association between the surrogate and 
166  the true clinical endpoint. 
167   
168    Trial-level association is the strength of the association between the effects of treatment 
169  on the surrogate and the true endpoint. 
170   
171  Although single-arm trial data may be used to demonstrate individual-level association and 
172  assess efficacy outcome of interest in subgroups by MRD level for the purposes of hypothesis 
173  generation, the meta-analysis to validate MRD at the trial level should include only randomized 
174  trials.  The issues pertinent to meta-analyses in general have been discussed in another 
175  guidance.12 
176   
177  Sponsors should discuss with the Agency and provide details of the meta-analysis plan, which 
178  should include but not be limited to considering the following aspects: 
179   
180    Details of trial designs, inclusion and exclusion criteria, MRD assessment, and disease 
181  setting.  Sponsors should justify poolability of the data. 
182   
                                                 
periodically.  To make sure you have the most recent version of a guidance, check the FDA guidance web page at 
https://www.fda.gov/regulatory-information/search-fda-guidance-documents. 
 
10 For additional information on expedited programs and surrogate endpoints used to support accelerated or 
traditional approval, see the guidance for industry Expedited Programs for Serious ConditionsDrugs and 
Biologics (May 2014). 
 
11 See the ICH guidance for industry E9 Statistical Principles for Clinical Trials (September 1998). 
 
12 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. 
5  Contains Nonbinding Recommendations 
183    Inclusion of trials that include a patient population representative of the population in 
184  which the surrogate endpoint ultimately will be used. 
185   
186    Inclusion of an adequate number of randomized trials with sufficient follow-up time.  
187  Sponsors should justify the number of trials to be included in the meta-analysis. 
188   
189    Inclusion of trials that demonstrate both positive and negative results.  For example, 
190  sponsors should present randomized trials that failed to meet their primary endpoint, and 
191  trials that had divergent MRD and event-free survival/progression-free survival/overall 
192  survival (EFS/PFS/OS) results, if available, should also be presented.  Sponsors should 
193  explain the divergent results if possible. 
194   
195    Analysis based on individual patient-level data to allow an assessment of individual-level 
196  surrogacy. 
197   
198    Prespecified criteria for concluding surrogacy based on both trial-level and patient-level 
199  association measures, including prespecified timing and window of the MRD assessment.  
200  If a fixed time point is not feasible, the MRD assessments in a window of the trial should 
201  be prespecified.  Sponsors should explore sensitivity analyses based on different time 
202  windows.  Sponsors should discuss with the Agency the time window chosen.  For 
203  example, sponsors can prespecify for patients with newly diagnosed ALL the MRD 
204  assessment at the time of the first complete response (CR1), 28 days plus or minus a 
205  window of a specific number of days. 
206   
207    Inclusion of long-term clinical endpoints, such as EFS/PFS and OS that have been clearly 
208  and consistently defined across studies.  Sponsors should explore alternative event 
209  definitions for EFS/PFS or alternative censoring schemes for EFS/PFS/OS as sensitivity 
210  analyses. 
211   
212    Discussion of missing MRD assessments and reasons for missing data (e.g., caused by 
213  sample collection issues, loss to follow-up).  Sponsors should explore the effects of 
214  missing data on the results. 
215   
216    Consideration of the statistical handling of unevaluable samples. 
217   
218    Potential confounding factors, which sponsors should incorporate into the planned 
219  validation analyses. 
220   
221    Sensitivity analyses to demonstrate robustness of the surrogate (e.g., alternative statistical 
222  methods for evaluation of association,13 cross validation) and subgroup analyses. 
                                                 
13 Shi Q, CR Flowers, W Hiddemann, R Marcus, M Herold, A Hagenbeek, E Kimby, H Hochster, U Vitolo, BA 
Peterson, E Gyan, M Ghielmini, T Nielsen, S De Bedout, T Fu, N Valente, NH Fowler, E Hoster, M Ladetto, F 
Morschhauser, E Zucca, G Salles, and DJ Sargent., 2017, Thirty-Month Complete Response as a Surrogate End 
Point in First-Line Follicular Lymphoma Therapy: An Individual Patient-Level Analysis of Multiple Randomized 
Trials, J Clin Oncol, 35(5):552560. 
 
6  Contains Nonbinding Recommendations 
223   
224    Discussion and analyses using different MRD cutoffs (e.g., 10-4, 10-5).  For assisting in 
225  the interpretation of the results, sponsors can present analyses such as surrogate threshold 
226  effect.14 
227   
228  Even if a meta-analysis supports validation of MRD as a surrogate endpoint, applying these 
229  results to a new trial requires a certain amount of extrapolation.  Some caveats regarding the use 
230  of MRD as a surrogate endpoint include the following: 
231   
232    Even if MRD can be validated as a surrogate endpoint, the use of MRD may not be 
233  applicable to subgroups of the patient population or future trial populations if there are 
234  important differences (e.g., prior therapy, disease status, line of treatment) between the 
235  population evaluated in the meta-analysis and the to-be-enrolled population.  This may 
236  represent a different context of use, and as such, any differences should be justified.  
237  Sponsors should perform sensitivity and subgroup analyses to evaluate the strength of the 
238  surrogate endpoint in different disease settings or patient characteristics. 
239   
240    When a new drug product is under investigation, it may not be reasonable to assume that 
241  the quantitative relationship between the drug products effects on the surrogate endpoint 
242  and the clinical benefit endpoint will be the same as previously studied drug products 
243  effects.  This is especially true for drug products that have a markedly different 
244  mechanism of action (e.g., cytotoxic therapy versus immunotherapy).  Although the 
245  credibility of this extrapolation will be primarily based on biological considerations, the 
246  meta-analyses mentioned above can provide supportive evidence.  To obtain best 
247  estimates of the relationship between the surrogate and clinical benefit endpoints, the 
248  meta-analysis should include drug products with varying mechanisms of action and 
249  evaluate the relationship in mechanistic subtypes. 
250   
251  D.  MRD as an Endpoint in Clinical Trials 
252   
253  MRD analyses should be based on the intent-to-treat (ITT) population.  A patient may not have 
254  an MRD assessment because of a missed assessment, test failure, or not meeting clinical criteria 
255  for assessment (i.e., lack of CR).  For ITT-based analyses, sponsors should consider any patient 
256  without an MRD assessment as not responsive to treatment.  Analyses based on the MRD 
257  evaluable population are appropriate for sensitivity analyses.  
258   
259  Missing and unevaluable assessments of MRD should be kept to a minimum.  Sponsors should 
260  collect and summarize reasons for missing MRD assessments and consult with the Agency 
261  before finalizing the statistical analysis plan.  Sponsors also should perform further exploratory 
262  or sensitivity analyses to evaluate comparability of the results using different evaluation 
263  populations. 
264   
                                                 
 
14 Burzykowski T and Buyse M, 2006, Surrogate Threshold Effect: An Alternative Measure for Meta-Analytic 
Surrogate Endpoint Validation, Pharm Stat, 5(3):173186. 
 
7  Contains Nonbinding Recommendations 
265  Sponsors can also include MRD in the clinical trial as a secondary or an exploratory endpoint.  If 
266  MRD-negative response (e.g., MRD-negative CR) is used as a secondary endpoint and is 
267  planned for inclusion in the prescribing information, it should be included as a key secondary 
268  endpoint with appropriate control for multiplicity.15 
269   
270  E.  MRD for Patient Selection or Enrichment 
271   
272  Many clinical risk classifications may not be able to accurately predict relapse in patients with 
273  hematologic malignancies, which may result in inappropriate use or timing of treatments.  MRD 
274  has been regarded as an important prognostic factor for predicting disease recurrence, which may 
275  improve risk classification.  Sponsors can use MRD level to serve as a stratification factor, select 
276  patients at high risk, or enrich the trial population.16 
277   
278  FDA recommends that sponsors consult the Agency about incorporating any MRD assay into a 
279  trial before submitting the protocol for trials that include MRD for patient selection or as an 
280  endpoint (primary or key secondary). 
281   
282   
283  IV.  TECHNOLOGY CONSIDERATIONS 
284   
285  A.  Assay Considerations 
286   
287  Currently, four general technologies are used for MRD assessment in hematologic malignancies: 
288  multiparametric flow cytometry (MPFC), next-generation sequencing (NGS), quantitative 
289  reverse transcription polymerase chain reaction (RT-qPCR) of specific gene fusions, and allele-
290  specific oligonucleotide polymerase chain reaction (ASO-PCR).  These cellular (MPFC) and 
291  molecular (NGS, RT-qPCR, and ASO-PCR) technology platforms have different advantages and 
292  limitations in terms of sample input, cost, robustness, and reproducibility. 
293   
294  FDA is agnostic as to which technology platform is used in clinical trials assessing MRD.  
295  However, sponsors should fully prespecify the selected platform (in terms of assay procedure, 
296  reagents, and analysis) and analytically validate the platform for its context of use.  Also, in the 
297  context of a clinical trial, ideally sponsors should use a single technology to assess MRD to 
298  compare results directly.  Although use of multiple technologies is discouraged, if the sponsor 
299  includes multiple technologies in the trial and plans for the primary analysis to be based on data 
300  from multiple technologies, the sponsor should prespecify the methodology for combining these 
301  technologies into a single MRD determination and discuss this with the Agency. 
302   
303  Analytical validation ensures that the assay measures the analyte or analytes that it is intended to 
304  measure in the intended tissue type.  The process of analytical validation is defined as 
305  establishing that the performance characteristics of the assay are acceptable in terms of the 
                                                 
15 See the draft guidance for industry Multiple Endpoints in Clinical Trials (January 2017).  When final, this 
guidance will represent the FDAs current thinking on this topic. 
 
16 See the guidance for industry Enrichment Strategies for Clinical Trials to Support Determination of Effectiveness 
of Human Drugs and Biological Products (March 2019). 
8  Contains Nonbinding Recommendations 
306  assays sensitivity, specificity, accuracy, precision, and other relevant performance 
307  characteristics using a specified technical protocol, which may include specimen collection, 
308  handling, and storage procedures.  Analytical validation is concerned with the assays technical 
309  performance and does not address clinical utility. 
310   
311  MRD assay validation should encompass the entire assay system from sample collection (e.g., 
312  BM aspirate versus blood) to system output (e.g., decision-making threshold for MRD positive 
313  versus negative) and should use relevant clinical samples.  Where technically feasible, the 
314  detection threshold of the MRD assay should be at least 10-fold below the clinical decision-
315  making threshold (the definition of MRD).  For example, if MRD positive or negative is defined 
316  as detection of greater or less than 1 x 10-5 cells, respectively, then the assay should be optimized 
317  and validated to have an analytical sensitivity of at least 1 x 10-6.  If this level of detection is not 
318  feasible with the proposed assay, sponsors should provide appropriate justification that the assay 
319  is adequate to fulfill its intent in the trial.  Additionally, to ensure that the assay performance 
320  achieved in validation testing is replicated in the clinical trial, sponsors should strictly adhere to 
321  the assay protocol in all clinical trial laboratory sites.  The following sections detail specific 
322  considerations for the different technology platforms. 
323   
324  1.  Cellular Technology Platforms 
325   
326  When using cellular technology platforms for MRD assessments in clinical trials, sponsors 
327  should do the following: 
328   
329    Prespecify the total number of events to be collected to support the quantitative 
330  assessment of MRD 
331   
332    Use a consistent panel of antibodies and fluorochromes, as no single antigen is specific 
333  for any neoplasm 
334   
335    Consider sample stability, which may limit the utility of flow cytometry 
336   
337    Use a consistent analysis template (e.g., gating strategy) 
338   
339    Determine whether the therapy affects the expression and therefore detectability of the 
340  specific antigens targeted by the antibody panels of the flow cytometry assay 
341   
342    Evaluate the potential for the flow assay to detect normal BM cells that are regenerating 
343  after chemotherapy to reduce the likelihood that those cells are misinterpreted as 
344  abnormal cells 
345   
346  2.  Molecular Technology Platforms 
347   
348  When using molecular technology platforms for MRD assessments in clinical trials, sponsors 
349  should do the following: 
350   
351    Prespecify nucleic acid quantity (e.g., micrograms) and quality metrics 
9  Contains Nonbinding Recommendations 
352   
353    Consider the need for an internal control when a cell number is derived from DNA 
354  content calculations because poor DNA quality may cause artificially low MRD levels 
355   
356    Store diagnostic samples to be used for clone identification in case of assay changes 
357   
358    Consider how to account for shifts in clonality as assessed by molecular markers (i.e., the 
359  specific molecular marker may be lost as a result of treatment while the disease remains 
360  present) 
361   
362    Track assay failures (i.e., failures of the assay to identify the relevant clone for a patient) 
363  and consider this failure rate for clinical endpoint calculations 
364   
365  3.  All Technology Platforms 
366   
367  When using any technology platform for MRD assessments in clinical trials, sponsors should do 
368  the following: 
369   
370    Prespecify preanalytical procedures and ensure that the sample collection and storage 
371  procedures used are appropriate to obtain the desired cell population 
372   
373    Take hemodilution into account (specifically, the amount of blood needed for the 
374  procedure to obtain the required number of events or amount of nucleic acid) and request 
375  that investigators use the first BM pull for MRD assessments  
376   
377    For all testing, especially if centralized testing is not used, assay protocols and result 
378  interpretation should be standardized to ensure MRD measurements are comparable 
379  between laboratories 
380   
381  B.  Sampling Considerations 
382   
383  Target levels of MRD for use in a regulatory setting are disease-specific and dependent upon the 
384  proposed use of the biomarker.  In a clinical trial, the protocol should prespecify the 
385  measurement of MRD, which sponsors should conduct at prespecified times using a consistent 
386  and validated assay.  The MRD assessment at a prespecified postinduction therapy time point is 
387  anticipated to be a sensitive measure of CR to induction therapy in either a frontline or 
388  relapsed/refractory setting.  Consistent time-point specification would provide an opportunity to 
389  assess the kinetics of an MRD response and its duration, which may provide supportive evidence 
390  of drug activity.  The timing of MRD assessment also is important when considering using MRD 
391  before allogeneic hematopoietic cell transplantation to predict transplant outcomes. 
392   
393   
10  Contains Nonbinding Recommendations 
394  V.  DISEASE-SPECIFIC CONSIDERATIONS 
395   
396  A.  Acute Lymphoblastic Leukemia 
397   
398  MRD has emerged as one of the most significant prognostic factors in ALL, independent of 
399  patient age, B- or T-cell origin, or genetic subtype.  Additional considerations for using MRD in 
400  ALL treatment trials include the following: 
401   
402    BM is the preferred substrate for measuring MRD.  If blood samples are used for 
403  assessing MRD in the clinical trial, sponsors should include justification for using blood 
404  rather than BM. 
405   
406    CR with recovery of blood counts is the preferred time point to assess MRD.  For 
407  regimens for which the efficacy-response evaluation is based on a calendar-driven time 
408  point rather than waiting for blood count recovery, at least an M1 marrow (marrow with 
409  leukemic blasts less than 5%) should be documented for patients being assessed for 
410  MRD. 
411   
412    When MRD is used as an efficacy endpoint for ALL, the absence of extramedullary 
413  disease should be documented concurrently with assessment of BM and blood counts.  
414  However, FDA does not expect the conduct of invasive procedures to test for 
415  extramedullary disease if the procedures are not within the clinical standard of care at the 
416  time of the efficacy evaluation. 
417   
418    FDA has accepted an MRD level of 0.1% or more to define patients with ALL in first or 
419  second CR with high risk of relapse.  For trials that use MRD levels of less than 0.1% 
420  with CR for patient selection, the submission should include information to justify use of 
421  the lower MRD level. 
422   
423    For new drugs that have a demonstrated durable CR in patients with relapsed or 
424  refractory ALL, FDA has accepted MRD of less than 0.01% as supporting evidence of 
425  efficacy.  As technologies improve and new clinical findings emerge, the level of MRD 
426  needed to support an efficacy claim may change. 
427   
428  B.  Acute Myeloid Leukemia 
429   
430  The molecular heterogeneity of AML poses substantial challenges to the use of MRD as a 
431  biomarker.  Additional considerations for use of MRD in AML treatment trials include the 
432  following: 
433   
434    BM is the preferred substrate for measuring MRD.  If blood samples are used for 
435  response assessment of MRD in the clinical trial, sponsors should include justification for 
436  using blood rather than BM. 
437   
438    CR with recovery of blood counts is the preferred time point to assess MRD.  If 
439  assessments are made at CR without count recovery or at lesser responses (e.g., complete 
11  Contains Nonbinding Recommendations 
440  remission with incomplete hematologic recovery), sponsors should include data to justify 
441  the plan. 
442   
443    For the marker (e.g., cell surface or genetic mutation) selected to assess MRD, sponsors 
444  should provide data showing that the marker reflects the leukemia and not underlying 
445  clonal hematopoiesis (false-positive result).  Sponsors should also describe the false-
446  negative rate that might result from relapse from a marker-negative clone.  If multiple 
447  markers and/or multiple platforms are used, sponsors should provide an analysis of the 
448  risk of false-positive and false-negative results for each marker individually and for the 
449  panel as a whole. 
450   
451    For studies of targeted therapies (e.g., IDH1, IDH2, or FLT3 inhibitors) for which the 
452  MRD marker is the target of the therapy, sponsors can use nonclinical data to identify the 
453  mutations in the marker that are known to be sensitive to the therapy and those that are 
454  known to be resistant to the therapy.  If using only the target of therapy as the MRD 
455  marker, sponsors should provide justification for not using other MRD markers to avoid 
456  false-negative results when clonality changes. 
457   
458  C.  Acute Promyelocytic Leukemia 
459   
460  The standard-of-care use of MRD testing and monitoring is established for the initial treatment 
461  of patients with acute promyelocytic leukemia (APL) using tretinoin with arsenic and/or 
462  anthracycline.  Whether the same guidelines for use of MRD apply to other drug classes needs to 
463  be confirmed as new drugs are evaluated for initial or salvage therapy.  Additional specific 
464  considerations include the following: 
465   
466    BM is the preferred substrate for measuring MRD.  If blood samples are used for 
467  response assessment of MRD in the clinical trial, sponsors should include justification for 
468  using blood rather than BM. 
469   
470    CR following recovery of blood counts is the preferred time point to assess MRD.  If 
471  assessments are made at CR without count recovery or at lesser responses, sponsors 
472  should include data to justify the plan. 
473   
474    MRD should be assessed at the end of consolidation rather than at the end of induction, 
475  when differentiating agents are used, to avoid false-positive results.  For new drug 
476  products for treatment of APL, sponsors should use data from early-phase trials to 
477  establish the optimal timing for MRD assessment in the pivotal trials. 
478   
479    Patients with low-risk APL who achieve confirmed MRD negativity after 
480  arsenic/tretinoin-based therapy are generally considered cured and require no further 
481  monitoring.  For new drug products for treatment of APL, long-term monitoring may be 
482  required in the pivotal trial if data from early-phase trials are not sufficient to confirm 
483  that MRD negativity is also durable with the new drug product. 
484   
12  Contains Nonbinding Recommendations 
485    An MRD level less than 0.01% is generally considered negative after first-line 
486  arsenic/tretinoin- or idarubicin/tretinoin-based induction.  For new drug products for 
487  treatment of APL, sponsors should use data from early-phase trials to confirm this 
488  threshold for defining MRD negativity for the new drug product. 
489   
490    Although an MRD level less than 0.01% is generally considered negative after first-line 
491  treatment, marketing applications for treatment of molecular relapse may need clinical 
492  outcomes (i.e., EFS) if data are not available to support a proposed MRD threshold as the 
493  sole criterion for response to salvage therapy. 
494   
495  D.  Chronic Lymphocytic Leukemia 
496   
497  Current literature suggests that there is an association between MRD negativity and OS in 
498  patients with CLL treated with chemoimmunotherapy.  The therapeutic paradigm with small 
499  molecule inhibitors of the B-cell receptor signaling pathway and other novel products continue to 
500  rapidly evolve in this area.  Additional specific considerations include the following: 
501   
502    MRD status should be measured by a standardized method with a quantitative lower limit 
503  of detection sufficient to evaluate the prospective cutoff in the trial and at least less than 
504  10-4 (0.01%).  Currently, MRD is most commonly assessed using RT-qPCR and flow 
505  cytometric methods, but NGS can also reliably assess MRD in CLL. 
506   
507    A challenge in MRD testing is that CLL is a multicompartmental disease involving the 
508  BM, blood, lymph nodes, liver, and spleen; after treatment, one or more of these sites 
509  may serve as a reservoir for residual disease.  Sponsors should carefully consider for 
510  assessment the sample source, which should be the same throughout the trial.  This is 
511  especially important as therapeutic intervention differentially affects MRD measurement 
512  in peripheral blood and BM, as has been demonstrated with certain therapeutics (e.g., 
513  anti-CD20 monoclonal antibodies, alemtuzumab). 
514   
515    The timing of when to test for MRD has yet to be standardized and the time to response 
516  and response durations may vary by type of therapeutic regimen.  Sponsors should 
517  prespecify the timing and method of MRD testing and provide adequate justification in 
518  the protocol.  MRD should also be measured at the end-of-treatment response assessment 
519  to fully capture the treatment effect. 
520   
521    MRD should be assessed in patients who are in CR.  If MRD assessments are to be made 
522  in patients in other response categories (e.g., partial response), sponsors should include 
523  data to justify the plan. 
524   
525  E.  Chronic Myeloid Leukemia 
526   
527  There have been dramatic improvements in clinical outcomes in patients with chronic myeloid 
528  leukemia (CML) from targeting the BCR-ABL1 oncoprotein.  The detection and monitoring of 
529  MRD has become standard of care in patients with CML.  Specific considerations include the 
530  following: 
13  Contains Nonbinding Recommendations 
531   
532    Monitoring MRD in CML should use assays with results based on the International Scale 
533  (IS) with the standardized baseline set to 100%.  Molecular response is expressed as log 
534  reduction from 100%. 
535   
536    Currently, RT-qPCR(IS) is the preferred assay to monitor response to therapy.  In 
537  general, RT-qPCR assays with a sensitivity of more than 4.5-log reduction from the 
538  standardized baseline are recommended for measuring BCR-ABL1 transcripts. 
539   
540    Major molecular response (MMR) is defined as BCR-ABL(IS) of less than 0.1% or more 
541  than 3-log reduction in BCR-ABL1 mRNA from the standardized baseline if RT-
542  qPCR(IS) is not available. 
543   
544    There is evidence that achieving an MMR predicts superior long-term clinical outcomes 
545  (PFS/EFS). 
546   
547    Achieving MMR has become a consensus goal of CML therapy, and durable MMR can 
548  be a measure of clinical benefit. 
549   
550    In addition, MRD can be used to select and monitor patients who are eligible for 
551  treatment discontinuation of tyrosine kinase inhibitor therapy. 
552   
553  F.  Multiple Myeloma 
554   
555  Significant improvements in clinical outcomes of MM have spurred interest in the use of MRD 
556  as a potential surrogate endpoint to expedite drug development.  Multiple trials have evaluated 
557  the relationship between MRD status and PFS/OS. 
558   
559  Additional specific considerations for use of MRD in trials of new drug products for treatment of 
560  MM include the following: 
561   
562    Most published literature to date has evaluated MRD in the newly diagnosed 
563  posttransplant setting.  Fewer studies have evaluated MRD in the setting of 
564  relapsed/refractory disease or newly diagnosed patients with myeloma who are not 
565  eligible for transplant.  The relationship between MRD and clinical benefit and the test 
566  performance characteristics should be demonstrated in each disease setting (e.g., relapsed 
567  refractory, newly diagnosed, nontransplant eligible, smoldering MM) to validate MRD as 
568  a surrogate endpoint in MM.  This is especially important in disease settings, such as 
569  smoldering myeloma, in which there is a lower disease burden and the potential for 
570  toxicity or other nondisease-related factors influence long-term outcomes. 
571   
572    MRD should be assessed only in patients who are in CR.  If MRD assessments are to be 
573  made in patients in other response categories (e.g., partial response, very good partial 
574  response), sponsors should include data to justify the plan. 
575   
14  Contains Nonbinding Recommendations 
576    MRD is currently assessed using MPFC and NGS methods in the BM.  These 
577  methodologies are not able to detect extramedullary disease.  There has been interest in 
578  using imaging techniques (e.g., positron emission tomography/computed tomography, 
579  magnetic resonance imaging) in combination with MRD to assess response.  When 
580  considering using MRD in MM clinical trials, sponsors should discuss with FDA how 
581  extramedullary disease will be assessed and whether imaging should be incorporated into 
582  the assessment of response. 
583   
584    At this time, the relationship between MRD and clinical benefit in patients with different 
585  cytogenetic abnormalities and their associated risks is unclear.  When considering using 
586  MRD in clinical trials, sponsors should consider the patients cytogenetic risk.  For 
587  example, given the prognostic effect of cytogenetics, the trial may benefit from 
588  stratification to ensure that there is no imbalance between the arms that would affect the 
589  MRD assessment.  Alternatively, trials may be designed to intervene in patients who are 
590  MRD positive and have poor risk cytogenetics because this may represent a group at risk 
591  for particularly poor outcomes. 
592   
593   
594  VI.  REGULATORY SUBMISSIONS THAT USE MRD 
595   
596  As indicated above, FDA views MRD as a biomarker that is a reliable quantitation of tumor 
597  burden, independent of assay.  As such, FDA does not foresee the need to codevelop an MRD 
598  assay with a drug product.17  However, for FDA to adequately assess the safety of a proposed 
599  clinical trial that uses MRD (e.g., for patient selection) or to determine the credibility of a 
600  clinical trial outcome based in part on MRD, submissions that use MRD for regulatory purposes 
601  or for critical treatment purposes should include sufficient information to address the following 
602  two main issues: 
603   
604    Is MRD, as assessed (sample, timing, threshold, etc.), a clinically valid biomarker for the 
605  context of use (disease, disease status, type of therapy, etc.)? 
606   
607    Is the MRD assay used (or to be used) in the clinical trial analytically valid for the range 
608  of results that are important to the trial? 
609   
610  When the MRD assay used is FDA-cleared or -approved for the specific malignancy and 
611  specimen type, identifying the assay with the required number of cells to be evaluated or the 
612  DNA input requirements will be sufficient to address the analytical validity in most cases.  When 
613  the MRD assay is not FDA-cleared or -approved, FDA would expect additional information, 
614  such as those listed in Table 1, to be submitted for review. 
615   
                                                 
17 A potential exception might be when the MRD marker is the direct target of the drug product under study, such as 
for selecting patients for treatment in a clinical trial of an Fms-related tyrosine kinase 3 (FLT3) inhibitor when the 
MRD assay is for an FLT3 mutation.  In such a circumstance, sponsors should consult with FDA about the need for 
a companion diagnostic early in clinical development. 
15  Contains Nonbinding Recommendations 
616  Table 1. Information to Help Review of Regulatory Submissions That Use MRD 
IND Clinical Trial Submission  NDA or BLA Submission 
1. Justification that MRD as used is clinically valid   1. Justification that MRD as used is clinically valid 
for the proposed context and   for the context of the proposed claim and 
   
2. Letter of authorization to cross-reference the  2. Letter of authorization to cross-reference the 
investigational device exemption or other device- investigational device exemption or other device-
related regulatory submission for information  related regulatory submission for information 
about the assay or  about the assay or 
 A statement of intended use;   A statement of intended use; 
 The specific test method (including instruments,   The specific test method (including instruments, 
reagents, and specimen handling);  reagents, and specimen handling); 
 Confirmation that the lab has a process in place   Confirmation that the lab has a process in place 
for reagent control;  for reagent control; 
 A brief discussion of how the test method was   A brief discussion of how the test method was 
validated analytically for each specimen type;  validated analytically for each specimen type; 
and  and 
 A summary of the performance obtained for   A summary of the performance obtained for 
accuracy, precision, specificity, and sensitivity;  accuracy, precision, specificity, and sensitivity; 
and  and 
   
3. Indicate in the clinical trial informed consent  3. A SAS XPORT file (xpt file extension) with results 
document that the MRD assay is investigational.  of MRD testing.  For each result, specify the 
sample type, date of sample, assay used, input 
quantity, assay sensitivity, and assay result. 
617   
618   
619  For an IND clinical trial submission, when using an MRD assay that is not FDA-cleared or -
620  approved for the intended use and the trial is considered a significant risk device trial (e.g., 
621  eligibility criterion, allocation to a specific treatment, departures from standard of care, etc.), 
622  FDA may require an investigational device exemption to use the assay in the clinical trial.18  
623  Sponsors can submit a letter of authorization to cross-reference the investigational device 
624  exemption, which will then provide the necessary information regarding the assay.  When the 
625  trial is considered a nonsignificant risk device study, the sponsor should submit abbreviated 
626  information about the assay (see Table 1) to the IND for review to allow FDA to confirm that 
627  results from the device will be interpretable.  An NDA or BLA submission should include 
628  similar information about the assay (see Table 1) in addition to a data file with the results of 
629  MRD testing. 
630   
631  Although general principles outlined in this guidance should help sponsors with crucial questions 
632  about potential MRD use for marketing applications, FDA recommends that sponsors meet with 
                                                 
18 As an alternative, sponsors can consider the streamlined approach to codeveloping the MRD assay with the drug 
product under the IND as described in the draft guidance Investigational In Vitro Diagnostics in Oncology Trials: 
Streamlined Submission Process for Study Risk Determination (April 2018).  When final, this guidance will 
represent the FDAs current thinking on this topic.  See 21 CFR 812; for information on risk determination for 
investigational use of devices, see the guidance for industry and FDA staff Requests for Feedback and Meetings for 
Medical Device Submissions: The Q-Submission Program (May 2019). 
16  Contains Nonbinding Recommendations 
633  FDA before starting a drug development pathway incorporating MRD assessment intended to 
634  support an NDA or a BLA.  FDA will ensure that these meetings include a multidisciplinary 
635  team of review staff from CBER, CDER, and CDRH, as needed.  Sponsors can submit protocols 
636  using MRD after these meetings and request a special protocol assessment for eligible protocols, 
637  if they choose, that provides confirmation of the acceptability of assessments, endpoints, and 
638  protocol design to support drug marketing applications.  Ultimately, marketing approval depends 
639  not only on the design of clinical trials but also on FDA review of the results and data from all 
640  studies in the drug marketing application. 
641   
642   
643     
17  Contains Nonbinding Recommendations 
644  GLOSSARY 
645   
646  ALL    acute lymphoblastic leukemia 
647  AML    acute myeloid leukemia 
648  APL    acute promyelocytic leukemia 
649  ASO-PCR  allele-specific oligonucleotide polymerase chain reaction 
650  BLA    biologics license application 
651  BM    bone marrow 
652  CBER   Center for Biologics Evaluation and Research 
653  CDER   Center for Drug Evaluation and Research 
654  CLL    chronic lymphocytic leukemia 
655  CML    chronic myeloid leukemia 
656  CR    complete response or complete remission 
657  DDT    drug development tool 
658  EFS    event-free survival 
659  IDE    investigational device exemption 
660  IND    investigational new drug application 
661  IS    International Scale 
662  ITT    intent-to-treat 
663  MM    multiple myeloma 
664  MMR    major molecular response 
665  MPFC   multiparametric flow cytometry 
666  MRD    minimal residual disease 
667  NDA    new drug application 
668  NGS    next-generation sequencing 
669  OS    overall survival 
670  PFS    progression-free survival 
671  RT-qPCR  quantitative reverse transcription polymerase chain reaction 
18