Abstract

Mounting evidence indicates that the presence of measurable (“minimal”) residual disease (MRD), defined as posttherapy persistence of leukemic cells at levels below morphologic detection, is a strong, independent prognostic marker of increased risk of relapse and shorter survival in patients with acute myeloid leukemia (AML) and can be used to refine risk-stratification and treatment response assessment. Because of the association between MRD and relapse risk, it has been postulated that testing for MRD posttreatment may help guide postremission treatment strategies by identifying high-risk patients who might benefit from preemptive treatment. This strategy, which remains to be formally tested, may be particularly attractive with availability of agents that could be used to specifically eradicate MRD. This review examines current methods of MRD detection, challenges to adopting MRD testing in routine clinical practice, and recent recommendations for MRD testing in AML issued by the European LeukemiaNet MRD Working Party. Inclusion of MRD as an end point in future randomized clinical trials will provide the data needed to move toward standardizing MRD assays and may provide a more accurate assessment of therapeutic efficacy than current morphologic measures.

Introduction

More than 50% of adult patients with acute myeloid leukemia (AML) relapse after attaining morphologically defined complete remission (CR) with induction chemotherapy.1-3  Assessment of posttreatment remission is traditionally based primarily on cytomorphology, with AML relapse conventionally defined as ≥5% blasts in the bone marrow not attributable to other causes.4-6  Microscopic assessment of bone marrow or peripheral blood morphology relies on examination of a relatively small number of cells (200-500) and its reliability is dependent, in part, on sample quality and the pathologist’s expertise.7  As primarily derived from data in younger adults, the risk of relapse following allogeneic hematopoietic stem cell transplantation (allo-HSCT) for consolidation after first CR is 15% to 20% for patients with favorable-risk disease, 20% to 25% for intermediate-risk disease, 30% to 40% for poor-risk disease, and 40% to 50% for very poor-risk disease.8  Among older patients (age ≥60 years) with AML, the respective pooled 2- and 5-year survival estimates following allo-HSCT are 44% and 35% for relapse-free survival (RFS) and 45% and 38% for overall survival (OS).9 

Currently, pretreatment factors such as age, cytogenetics, and the presence of certain gene mutations are used to estimate posttreatment risk of relapse based on data from large patient cohorts.5,6,10-12 Table 1 lists prognostic implications of specific mutations in AML as described by the European LeukemiaNet (ELN)10  and the National Comprehensive Cancer Network guidelines.6  The risk of relapse has been linked to the postchemotherapy persistence of “minimal residual disease” (MRD), which has been defined as leukemic cells at levels below morphologic detection.12  Flow cytometric and molecular techniques for assessment of residual leukemia are more sensitive than morphologic assessment, and consensus is growing that MRD might more aptly be called “measurable residual disease,” because the presence of any disease detected by these methodologies after treatment is associated with a worse prognosis13,14  and detectable leukemia even in morphologic remission may not be “minimal.” MRD monitoring has become part of routine clinical practice in the management of patients with acute lymphoblastic leukemia, acute promyelocytic leukemia (APL), and chronic myeloid leukemia.15-18  Mounting evidence indicates that the presence of MRD is a strong, independent prognostic marker of increased risk of relapse and shorter survival in patients with AML compared with patients with a negative MRD test.4,19-23  This recurrent observation has raised interest in routine MRD testing in AML. To guide the development of a standardized or harmonized approach to MRD testing, the ELN MRD Working Party recently issued consensus recommendations for the measurement and application of MRD in AML (Table 2).24 

Table 1.

Risk status stratification by genetic abnormality per ELN 2017 and NCCN 2017 guidelines

Risk category*ELN criteria10 NCCN criteria6 
Favorable t(8;21)(q22;q22.1); RUNX1-RUNX1T1 Core binding factor: inv(16), or t(16;16), or t(8;21), or t(15;17) 
inv(16)(p13.1;q22) or t(16;16)(p13.1;q22); CBFB-MYH11 Normal cytogenetics: NPM1 mutation in absence of FLT3-ITD or isolated biallelic (double) CEBPA mutation 
Mutated NPM1 without FLT3-ITD or with FLT3-ITDlow§  
Biallelic mutated CEBPA  
Intermediate Mutated NPM1 and FLT3-ITDhigh§ Normal cytogenetics 
Wild-type NPM1 without FLT3-ITD or with FLT3-ITDlow§ (without adverse-risk genetic lesions) +8 alone 
t(9;11)(p21.3;q23.3); MLLT3-KMT2A|| t(9;11) 
Cytogenetic abnormalities not classified as favorable or adverse Other nondefined 
 Core binding factor with KIT mutation 
Poor/adverse t(6;9)(p23;q34.1); DEK-NUP214 Complex (≥3 clonal chromosomal abnormalities) 
t(v;11q23.3); KMT2A rearranged Monosomal karyotype 
t(9;22)(q34.1;q11.2); BCR-ABL1 −5, 5q–, –7, 7q– 
inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2); GATA2,MECOM(EVI1) 11q23 – non t(9;11) 
−5 or del(5q); –7; –17/abn(17p) inv(3), t(3;3) 
Complex karyotype, monosomal karyotype# t(6;9) 
Wild-type NPM1 and FLT3-ITDhigh t(9;22) 
Mutated RUNX1** Normal cytogenetics: with FLT3-ITD mutation†† 
Mutated ASXL1** TP53 mutation 
Mutated TP53‡‡  
Risk category*ELN criteria10 NCCN criteria6 
Favorable t(8;21)(q22;q22.1); RUNX1-RUNX1T1 Core binding factor: inv(16), or t(16;16), or t(8;21), or t(15;17) 
inv(16)(p13.1;q22) or t(16;16)(p13.1;q22); CBFB-MYH11 Normal cytogenetics: NPM1 mutation in absence of FLT3-ITD or isolated biallelic (double) CEBPA mutation 
Mutated NPM1 without FLT3-ITD or with FLT3-ITDlow§  
Biallelic mutated CEBPA  
Intermediate Mutated NPM1 and FLT3-ITDhigh§ Normal cytogenetics 
Wild-type NPM1 without FLT3-ITD or with FLT3-ITDlow§ (without adverse-risk genetic lesions) +8 alone 
t(9;11)(p21.3;q23.3); MLLT3-KMT2A|| t(9;11) 
Cytogenetic abnormalities not classified as favorable or adverse Other nondefined 
 Core binding factor with KIT mutation 
Poor/adverse t(6;9)(p23;q34.1); DEK-NUP214 Complex (≥3 clonal chromosomal abnormalities) 
t(v;11q23.3); KMT2A rearranged Monosomal karyotype 
t(9;22)(q34.1;q11.2); BCR-ABL1 −5, 5q–, –7, 7q– 
inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2); GATA2,MECOM(EVI1) 11q23 – non t(9;11) 
−5 or del(5q); –7; –17/abn(17p) inv(3), t(3;3) 
Complex karyotype, monosomal karyotype# t(6;9) 
Wild-type NPM1 and FLT3-ITDhigh t(9;22) 
Mutated RUNX1** Normal cytogenetics: with FLT3-ITD mutation†† 
Mutated ASXL1** TP53 mutation 
Mutated TP53‡‡  
*

The prognostic value of a marker is treatment-dependent and may change with new therapies.

Presence of KIT mutations in patients with t(8:21) and, to a lesser extent, inv(16), confers a high risk of relapse; these patients should be considered intermediate risk and considered for HSCT if available.

Other cytogenetic findings in addition to these do not alter risk status.

§

Low, low allelic ratio (<0.5); high, high allelic ratio (≥0.5); recent studies indicate that AML with NPM1 mutation and FLT3-ITD low allelic ratio may also have a more favorable prognosis and patients should not routinely be assigned to allogeneic HCT.75,76 

||

Presence of t(9;11)(p21.3;q23.3) takes precedence over rare, concurrent adverse-risk gene mutations.

3 or more chromosomal abnormalities in the absence of 1 of the World Health Organization–designated recurring translocations or inversions: t(8;21), inv(16) or t(16;16), t(9;11), t(v;11)(v;q23.3), t(6;9), inv(3) or t(3;3); AML with BCR-ABL1.

#

Defined by the presence of 1 single monosomy (excluding loss of X or Y) with at least 1 additional monosomy or structural chromosome abnormality (excluding core-binding factor AML).

**

These should not be used as adverse prognostic markers if they occur with favorable-risk AML subtypes.

††

FLT3-ITD mutations are considered to confer a significantly poorer outcome in patients with normal karyotype; there is controversy about whether FLT3-TKD mutations carry equally poor prognosis.

‡‡

TP53 mutations are significantly associated with AML with complex and monosomal karyotype.

NCCN, National Comprehensive Cancer Network.

Table 2.

ELN recommendations for MRD testing

Recommendations
Flow cytometry  
 1 Use the following markers in a MRD panel: 
CD7, CD11b, CD13, CD15, CD19, CD33, CD34, CD45, CD56, CD117, HLA-DR (backbone: CD45, CD34, CD117, CD13, CD33, FSC/SSC). 
If necessary, add a “monocytic tube” containing: 
CD64/CD11b/CD14/CD4/CD34/HLA-DR/CD33/CD45. 
 2 Integrate the classic LAIP approach with the DfN approach. To trace all aberrancies (at and beyond diagnosis, including newly formed postdiagnosis aberrancies), apply a full panel both at diagnosis and follow-up. 
 3 Aspirate 5-10 mL BM and use the first pull for MRD assessment. At present PB, with its lower MRD content, should not be used for MRD assessment. 
Pull as low as desirable BM volume because contamination with PB increases with BM volume. 
 4 Estimate the contamination with PB, especially when a first pool of BM was impossible. 
 5 Use 500 000 to 1 000 000 white blood cells, use the best aberrancy available and relate it to CD45+ white blood cells. 
 6 To define “MRD-negative” and “MRD-positive” patient groups, a cutoff of 0.1% is recommended. 
 7 If true MRD <0.1% is found, report this as “MRD-positive <0.1%, may be consistent with residual leukemia.” If applicable, the comment “this level has not been clinically validated” should be added. 
 8 In a multicenter setting, transport and storage of full BM at room temperature for a period of 3 d is acceptable. 
 9 Single-center studies with no extensive experience on MFC MRD are strongly discouraged. 
Molecular biology  
 1 Molecular MRD analysis is indifferent to the anticoagulant used during cell sampling; thus, heparin or EDTA can be used as anticoagulant. 
 2 Aspirate 5-10 mL BM and use the first pull for molecular MRD assessment. 
 3 WT1 expression should not be used as an MRD marker unless no other MRD marker is available in the patient. 
 4 Do not use mutations in FLT3-ITD, FLT3-TKD, NRAS, KRAS, DNMT3A, ASXL1, IDH1, IDH2, or MLL-PTD and expression levels of EVI1 as single MRD markers. However, these markers may be useful when used in combination with a second MRD marker. 
 5 We define molecular progression in patients with molecular persistence as an increase of MRD copy numbers ≥1 log10 between any 2 positive samples. Absolute copy numbers should be reported in addition to the fold increase to enable the clinician to make his or her own judgments. 
 6 We define molecular relapse as an increase of the MRD level ≥1 log10 between 2 positive samples in a patient who previously tested negative. The conversion of negative to positive MRD in PB or BM should be confirmed 4 wk after the initial sample collection in a second sample from both BM and PB. If MRD increases in the follow-up samples ≥1 log10, molecular relapse should be diagnosed. 
Clinical  
 1 Refine morphology-based CR by assessment of MRD, because CRMRD is a new response criterion according to the AML ELN recommendation 2017. 
Use MRD to refine risk assessment before consolidation treatment, the postinduction time point closest to consolidation treatment is recommended. 
 2 MRD monitoring should be considered part of the standard of care for AML patients. 
Monitoring beyond 2 years of follow-up should be based on the relapse risk of the patient and decided individually. 
Patients with mutant NPM1, RUNX1-RUNX1T1, CBFB-MYH11, or PML-RARA should have molecular assessment of residual disease at informative clinical time points. 
 3 Not to assess molecular MRD in subtypes other than APL, CBF AML, and NPM1-mutated AML. 
 4 For AML patients not included in the molecularly defined subgroups here, MRD should be assessed using MFC. 
During the treatment phase, we recommend molecular MRD assessment at minimum at diagnosis, after 2 cycles of standard induction/consolidation chemotherapy, and after the end of treatment in PB and BM. 
During follow-up of patients with PML-RARA, RUNX1-RUNX1T1, CBFB- MYH11, mutated NPM1, and other molecular markers we recommend molecular MRD assessment every 3 mo for 24 mo after the end of treatment in BM and in PB. Alternatively, PB may be assessed every 4-6 wk. 
 5 Failure to achieve an MRD-negative CR or rising MRD levels during or after therapy are associated with disease relapse and inferior outcomes and should prompt consideration of changes in therapy. 
 6 In APL, the most important MRD end point is achievement of PCR-negativity for PML-RARA at the end of consolidation treatment. 
For patients with PML-RARA fusion and low-/intermediate-risk Sanz score who are treated with ATO and ATRA, MRD analysis should be continued until the patient is in CRMRD in BM and then should be terminated. 
 7 Detectable levels of PML-RARA by PCR during active treatment of APL should not change the treatment plan for an individual patient. 
 8 A change in status of PML-RARA by PCR from undetectable to detectable, and confirmed by a repeat sample, should be regarded as an imminent disease relapse in APL. 
 9 Patients with CBF AML should have an initial assessment of MRD after 2 cycles of chemotherapy, followed by serial measurements every 3 mo for at least the first 2 y after the end of treatment. 
 10 MRD should be assessed pretransplant. 
 11 MRD should be performed posttransplant. 
 12 All clinical trials should require molecular and/or MFC assessment of MRD at all times of evaluation of response. 
Recommendations
Flow cytometry  
 1 Use the following markers in a MRD panel: 
CD7, CD11b, CD13, CD15, CD19, CD33, CD34, CD45, CD56, CD117, HLA-DR (backbone: CD45, CD34, CD117, CD13, CD33, FSC/SSC). 
If necessary, add a “monocytic tube” containing: 
CD64/CD11b/CD14/CD4/CD34/HLA-DR/CD33/CD45. 
 2 Integrate the classic LAIP approach with the DfN approach. To trace all aberrancies (at and beyond diagnosis, including newly formed postdiagnosis aberrancies), apply a full panel both at diagnosis and follow-up. 
 3 Aspirate 5-10 mL BM and use the first pull for MRD assessment. At present PB, with its lower MRD content, should not be used for MRD assessment. 
Pull as low as desirable BM volume because contamination with PB increases with BM volume. 
 4 Estimate the contamination with PB, especially when a first pool of BM was impossible. 
 5 Use 500 000 to 1 000 000 white blood cells, use the best aberrancy available and relate it to CD45+ white blood cells. 
 6 To define “MRD-negative” and “MRD-positive” patient groups, a cutoff of 0.1% is recommended. 
 7 If true MRD <0.1% is found, report this as “MRD-positive <0.1%, may be consistent with residual leukemia.” If applicable, the comment “this level has not been clinically validated” should be added. 
 8 In a multicenter setting, transport and storage of full BM at room temperature for a period of 3 d is acceptable. 
 9 Single-center studies with no extensive experience on MFC MRD are strongly discouraged. 
Molecular biology  
 1 Molecular MRD analysis is indifferent to the anticoagulant used during cell sampling; thus, heparin or EDTA can be used as anticoagulant. 
 2 Aspirate 5-10 mL BM and use the first pull for molecular MRD assessment. 
 3 WT1 expression should not be used as an MRD marker unless no other MRD marker is available in the patient. 
 4 Do not use mutations in FLT3-ITD, FLT3-TKD, NRAS, KRAS, DNMT3A, ASXL1, IDH1, IDH2, or MLL-PTD and expression levels of EVI1 as single MRD markers. However, these markers may be useful when used in combination with a second MRD marker. 
 5 We define molecular progression in patients with molecular persistence as an increase of MRD copy numbers ≥1 log10 between any 2 positive samples. Absolute copy numbers should be reported in addition to the fold increase to enable the clinician to make his or her own judgments. 
 6 We define molecular relapse as an increase of the MRD level ≥1 log10 between 2 positive samples in a patient who previously tested negative. The conversion of negative to positive MRD in PB or BM should be confirmed 4 wk after the initial sample collection in a second sample from both BM and PB. If MRD increases in the follow-up samples ≥1 log10, molecular relapse should be diagnosed. 
Clinical  
 1 Refine morphology-based CR by assessment of MRD, because CRMRD is a new response criterion according to the AML ELN recommendation 2017. 
Use MRD to refine risk assessment before consolidation treatment, the postinduction time point closest to consolidation treatment is recommended. 
 2 MRD monitoring should be considered part of the standard of care for AML patients. 
Monitoring beyond 2 years of follow-up should be based on the relapse risk of the patient and decided individually. 
Patients with mutant NPM1, RUNX1-RUNX1T1, CBFB-MYH11, or PML-RARA should have molecular assessment of residual disease at informative clinical time points. 
 3 Not to assess molecular MRD in subtypes other than APL, CBF AML, and NPM1-mutated AML. 
 4 For AML patients not included in the molecularly defined subgroups here, MRD should be assessed using MFC. 
During the treatment phase, we recommend molecular MRD assessment at minimum at diagnosis, after 2 cycles of standard induction/consolidation chemotherapy, and after the end of treatment in PB and BM. 
During follow-up of patients with PML-RARA, RUNX1-RUNX1T1, CBFB- MYH11, mutated NPM1, and other molecular markers we recommend molecular MRD assessment every 3 mo for 24 mo after the end of treatment in BM and in PB. Alternatively, PB may be assessed every 4-6 wk. 
 5 Failure to achieve an MRD-negative CR or rising MRD levels during or after therapy are associated with disease relapse and inferior outcomes and should prompt consideration of changes in therapy. 
 6 In APL, the most important MRD end point is achievement of PCR-negativity for PML-RARA at the end of consolidation treatment. 
For patients with PML-RARA fusion and low-/intermediate-risk Sanz score who are treated with ATO and ATRA, MRD analysis should be continued until the patient is in CRMRD in BM and then should be terminated. 
 7 Detectable levels of PML-RARA by PCR during active treatment of APL should not change the treatment plan for an individual patient. 
 8 A change in status of PML-RARA by PCR from undetectable to detectable, and confirmed by a repeat sample, should be regarded as an imminent disease relapse in APL. 
 9 Patients with CBF AML should have an initial assessment of MRD after 2 cycles of chemotherapy, followed by serial measurements every 3 mo for at least the first 2 y after the end of treatment. 
 10 MRD should be assessed pretransplant. 
 11 MRD should be performed posttransplant. 
 12 All clinical trials should require molecular and/or MFC assessment of MRD at all times of evaluation of response. 

Reprinted from Schuurhuis et al.24 

ATO, arsenic trioxide; ATRA, all-trans retinoic acid; BM, bone marrow; HLA-DR, HLA–antigen D related; PB, peripheral blood.

A prominent strategy to detect MRD is immunophenotypic evaluation by multiparameter flow cytometry (MFC). It is now well established that MRD detected by MFC is an independent prognostic factor for relapse, RFS, and OS.4,19-22  In studies of patients <65 years of age with AML who were fit to receive cytosine arabinoside plus anthracycline-based induction and consolidation chemotherapy, MRD-negative status as detected by MFC was identified as the most important independent predictor of RFS and OS.25,26  Similarly, a retrospective exploratory analysis of data from the Southwest Oncology Group S0106 study showed that MFC-detected MRD after completion of induction chemotherapy could be used to stratify younger patients by risk of AML recurrence, and that MRD status was the single most important predictor of OS and RFS in individual patients.27  Data in older patients with AML have also demonstrated the prognostic relevance of MRD monitoring by MFC in patients undergoing traditional cytotoxic chemotherapy induction.28  Detection of MRD by MFC during morphological CR before allo-HSCT has also been linked to a substantially higher likelihood of relapse and worse survival.29,30  Molecular approaches to detect MRD are equally informative for prognosis. For example, among patients with nucleophosmin 1 (NPM1)–mutated AML who had undergone intensive chemotherapy, the persistence of NPM1-mutated transcripts detected using real-time polymerase chain reaction (RT-PCR) was an independent predictor of relapse or death22  and of outcomes of allo-HSCT.31,32  Another study using whole-genome or exome sequencing of samples from a cohort of adults with AML found that detection of persistent leukemia-associated mutations in bone marrow cells during remission ∼30 days after start of chemotherapy was linked to a significantly increased risk of relapse, shorter event-free survival (EFS), and poorer OS.33 

Thus, adding MRD evaluation to other posttreatment assessments (eg, morphologic evaluations) could help guide postremission treatment strategies by identifying patients at high risk of relapse who might benefit from preemptive therapy,34  an appealing treatment concept that will require formal validation. Although not proven to date, the concept of MRD eradication aiding decision-making and improving outcomes of patients with AML is plausible. This is supported by experience with bispecific antibodies such as blinatumomab in ALL.35 

This review examines current methods of MRD detection, some challenges in adopting MRD testing in routine clinical practice, and describes some of the recent recommendations from the ELN MRD Working Party consensus statement for the detection of MRD in AML.24 

MRD detection methods

Technologies to measure MRD based on immunophenotype, cytogenetic abnormalities, and molecular mutations each have advantages and disadvantages. The clinical usefulness of MRD detection depends on the choice of MRD marker (eg, specific gene mutation, surface antigen), which in some cases might be a therapeutic target.19,36  An ideal MRD test should discriminate between cells that would not cause relapse from the smallest clinically significant populations of leukemic cells that hold the potential to cause relapse.37  Current MRD testing methods have not achieved this ideal state, except for PCR testing in APL.38 Table 3 summarizes the advantages and limitations of available methods of MRD detection. MFC and RT quantitative PCR (RT-qPCR) are the most commonly used technologies; more recently, next-generation sequencing (NGS) is being used for molecular assessments.6,7,23,37,39,40  Fluorescence in situ hybridization or chromosome banding analysis can detect leukemic cells with cytogenetic abnormalities41  but, because a smaller number of cells are interrogated, are generally less sensitive than MFC or molecular methods. Table 4 lists potential markers for monitoring MRD in AML and the testing technologies used to detect them.15,24,42 

Table 3.

Pros and cons of methods used to detect MRD in AML

TechnologySensitivityProsCons
MFC ∼10−4 to 10−5 Wide applicability (>90%) Challenging and somewhat subjective interpretation requires experienced pathologist 
Relatively quick (results ≤1 d) Sensitivity dependent on antibody panel used 
Single result interpretable Limited harmonization and standardization across laboratories 
High specificity when using defined LAIP Leukemic phenotype not necessarily stable over time (eg, initial LAIP may not identify subclones leading to relapse) 
Can detect cells with leukemia-stem cell phenotype  
Can distinguish between live and dead cells  
Ease of data storage  
Provides information about whole sample cellularity  
NGS ∼10−3 to 10−5 Relatively easy to perform Limited standardization 
Sensitive Error rate leads to low sensitivity of mutated sequences 
Applicable to specific subgroups Mutated genes can be detected in healthy people without hematologic abnormalities 
 Persistence of some genetic abnormalities in patients in long-term remission 
 Risk of contamination 
RT-qPCR ∼10−3 to 10−5 Wide applicability Results may take multiple days 
May be run by any certified laboratory with RT-qPCR capacity Expensive (computationally demanding and time-consuming) 
High sensitivity (≥MFC) Requires high-level expertise 
Well standardized Requires setting of threshold limits 
Quality assurance routinely incorporated Interpretation often requires trend of results 
 Different mutations have different biological consequences in AML 
 Molecular targets applicable to only ∼50% of all AML cases and <35% in older patents 
FISH ∼1 to 10−2 Superior to PCR-based assays for detection of numeric cytogenetic abnormalities (gains and losses of whole chromosomes or deletions/duplications) Considerably less sensitive than PCR or MFC 
Not useful for patients with normal karyotype 
Quality-assured probes are expensive; technique is labor intensive 
Chromosome banding analysis NA More common in routine clinical practice Labor-intensive, comparatively costly, low-throughput technique reliant on highly trained technical staff 
Evaluates the dividing proportion of cells ∼10× less sensitive than FISH 
 Not useful for patients with normal karyotype 
TechnologySensitivityProsCons
MFC ∼10−4 to 10−5 Wide applicability (>90%) Challenging and somewhat subjective interpretation requires experienced pathologist 
Relatively quick (results ≤1 d) Sensitivity dependent on antibody panel used 
Single result interpretable Limited harmonization and standardization across laboratories 
High specificity when using defined LAIP Leukemic phenotype not necessarily stable over time (eg, initial LAIP may not identify subclones leading to relapse) 
Can detect cells with leukemia-stem cell phenotype  
Can distinguish between live and dead cells  
Ease of data storage  
Provides information about whole sample cellularity  
NGS ∼10−3 to 10−5 Relatively easy to perform Limited standardization 
Sensitive Error rate leads to low sensitivity of mutated sequences 
Applicable to specific subgroups Mutated genes can be detected in healthy people without hematologic abnormalities 
 Persistence of some genetic abnormalities in patients in long-term remission 
 Risk of contamination 
RT-qPCR ∼10−3 to 10−5 Wide applicability Results may take multiple days 
May be run by any certified laboratory with RT-qPCR capacity Expensive (computationally demanding and time-consuming) 
High sensitivity (≥MFC) Requires high-level expertise 
Well standardized Requires setting of threshold limits 
Quality assurance routinely incorporated Interpretation often requires trend of results 
 Different mutations have different biological consequences in AML 
 Molecular targets applicable to only ∼50% of all AML cases and <35% in older patents 
FISH ∼1 to 10−2 Superior to PCR-based assays for detection of numeric cytogenetic abnormalities (gains and losses of whole chromosomes or deletions/duplications) Considerably less sensitive than PCR or MFC 
Not useful for patients with normal karyotype 
Quality-assured probes are expensive; technique is labor intensive 
Chromosome banding analysis NA More common in routine clinical practice Labor-intensive, comparatively costly, low-throughput technique reliant on highly trained technical staff 
Evaluates the dividing proportion of cells ∼10× less sensitive than FISH 
 Not useful for patients with normal karyotype 

FISH, fluorescence in situ hybridization; NA, not available. QA, quality assurance.

Table 4.

Testing technologies and potential markers for monitoring MRD in AML

Testing technologiesPotential markers
MFC CD2 CD34 
CD4 CD45 
CD7 CD56 
CD13 CD123 
CD15 CD117 
CD19 HLA-DR 
CD33  
NGS NPM1 
RUNX1 
FLT3-ITD 
IDH1/IDH2 
RT-qPCR CBFB/MYH11 
FLT3-ITD 
IDH1/IDH2 
NPM1 
RUNX1/RUNX1T1 
t(10;11) – KMT2A-MLLT10 
t(11;19) – KMT2A-ELL or KMT2A-MLLT1 
t(6;11) – KMT2A-MLLT4 
t(9;11) – KMT2A-MLLT3 
WT1 
Testing technologiesPotential markers
MFC CD2 CD34 
CD4 CD45 
CD7 CD56 
CD13 CD123 
CD15 CD117 
CD19 HLA-DR 
CD33  
NGS NPM1 
RUNX1 
FLT3-ITD 
IDH1/IDH2 
RT-qPCR CBFB/MYH11 
FLT3-ITD 
IDH1/IDH2 
NPM1 
RUNX1/RUNX1T1 
t(10;11) – KMT2A-MLLT10 
t(11;19) – KMT2A-ELL or KMT2A-MLLT1 
t(6;11) – KMT2A-MLLT4 
t(9;11) – KMT2A-MLLT3 
WT1 

MLL/MLLT3, mixed lineage leukemia.

MFC

MFC uses panels of fluorochrome-labeled monoclonal antibodies to identify aberrantly expressed antigens located on (or within) leukemic cells.15  Instruments that have multiple lasers to detect different fluorochromes, with combinations of multiple monoclonal antibodies, have increased the sensitivity of MFC to detect 10−3 to 10−5 leukemic cells within the white blood cell compartment.43  There are 2 main approaches used to detect MRD by MFC; 1 involves identification of leukemia-associated immunophenotypes (LAIPs) that differ from the majority of normal hematopoietic cells and the other approach entails identification of different-from-normal (DfN) patterns.44  LAIP are cells with abnormal patterns of antigens; examples include cross-lineage antigen expression (eg, expression of lymphoid markers in myeloid blasts), asynchronous antigen expression (eg, coexpression of antigens that are not usually found together during normal cellular differentiation), and over- or underexpression of antigens compared with normal levels.45-47  An extensive panel of monoclonal antibodies is required to detect all potentially abnormal LAIP antigen expression patterns in AML, which can number up to 100.26  A standard fixed monoclonal antibody panel is used to identify DfN patterns at all stages of disease/treatment with MFC.44  An advantage of this method is that it does not restrict MRD determination to specific LAIP present at diagnosis and takes immunophenotypic shifts over time into account.44  LAIP detection by MFC is more commonly used than the fixed-antibody method,42  but differences between the LAIP and DfN approaches may be minimized if sufficiently large antibody panels (≥8 colors) are used for detection.24  Because phenotypes may change over time by gaining or losing specific abnormalities or patterns of abnormalities during disease evolution, a prior MRD target (as defined by a specific LAIP) may be less useful at later time points for the same patient.15,37,48  Therefore, the ELN MRD Working Party suggests using a “LAIP-based DfN approach” to monitoring MRD (ie, using the same antibody-fluorochrome combinations with a minimum set of markers during follow-up assessments as those used at diagnosis to track emerging aberrancies) (Table 2).24  Researchers continue to work on improving MFC methodology and technology. For example, 1 group has developed a 1-tube assay with a single fluorescence channel that appears to work as well as standard 7-tube antibody panel to accurately quantify CD34+CD38 leukemic stem cells.49 

RT-qPCR

RT-qPCR is used to amplify leukemia-associated genetic abnormalities. Optimized RT-qPCR assays are more sensitive than MFC, with a detection range of 10−4 to 10−6.24,43  Additionally, quantitative assays that measure number of leukemic transcripts can be informative of whether transcript levels are rising or falling and can potentially inform further therapy, although benefits of MRD-directed therapy in AML are not yet firmly established.44  Viable targets for molecular MRD monitoring include leukemic fusion genes such as promyelocytic leukemia gene retinoic acid receptor-α (PML-RARA), core-binding factor subunit β myosin heavy chain 11 (CBFB-MYH11), and runt-related transcription factor 1 (RUNX1)/RUNX1 translocated to 1 (RUNX1T1), and mutant NPM1.24  Wilms’ tumor gene (WT1) should not be used as an MRD marker because of poor sensitivity and specificity unless no other MRD markers are available.24  The ELN MRD Working Party recommends against use of Fms-like tyrosine kinase internal tandem duplication (FLT3-ITD), FLT3-TKD, NRAS, KRAS, IDH1, IDH2, MLL-PTD, and expression levels of EVI1 as single markers of MRD because they are prone to frequent losses or gains; however, these mutations may have prognostic significance if accompanied by other MRD markers.24  Given available molecular targets, RT-qPCR assessment of MRD is thought to be applicable to only ∼50% of all AML cases and less than ∼35% in older patients (Figure 1), whereas MFC can detect MRD in ∼90% of patients when a comprehensive antibody panel is used.19,28,44,46,47,50  Limitations of RT-qPCR–based MRD assays are their dependence on specific mutations, requiring individual reference standard curves based on target serial dilutions.51  Digital PCR, a high-throughput technology that generates absolute quantification, can clonally amplify target nucleic acids and does not require a reference standard curve, has greater assay sensitivity than RT-qPCR.52  For example, digital PCR can detect a variety of NPM1 mutation subtypes without the need for multiple plasmid standards.53,54 

Figure 1.

Proportions of leukemia-specific MRD targets detectable by RT-qPCR for patients with AML by age group. Reprinted from Grimwade and Freeman.19 

Figure 1.

Proportions of leukemia-specific MRD targets detectable by RT-qPCR for patients with AML by age group. Reprinted from Grimwade and Freeman.19 

NGS

Next-generation DNA sequencing technologies, which allow parallel and repeated sequencing of millions of small DNA fragments, can be used to evaluate a few genes or an entire genome.55  The ability of NGS to assay large numbers of mutated genes could help trace the evolution of malignant clones, which cannot be done with RT-qPCR.15  Studies have demonstrated the feasibility of NGS to monitor mutations for which targeted therapies are available, such as FLT3-ITD56  and IDH1/2,57  and mutations with prognostic relevance, such as CEBPA and NPM1 in patients with AML.10  A recent study compared a targeted 28-gene NGS panel for detection of common AML mutations (with variant allele frequency [VAF] ≥5%) and a 10-color MFC assay of different-from-normal MRD in patients with AML before allo-HSCT.58  Results of the 2 assays were concordant in 71% of patients. For patients in CR or CR with incomplete hematologic recovery (CRi), MRD measured by NGS was much greater than the estimated percentage of aberrant blasts detected by MFC, suggesting that residual mutations persisted in non-blast compartments during remission. Patients found to be MRD-positive with both assays had the highest risk of relapse compared with patients who were negative by both assays and with patients who had discordant assay results.58  Similarly, in 340 patients with AML in CR or CR with CRi, there was a 69.1% concordance of MRD detection in the bone marrow using a 54-gene NGS evaluation and an MFC assay; however, persistent mutations were detected by NGS only in 64 patients.23  Four-year relapse rate was highest among patients with MRD detected by both methods (73.3%), followed by those with MRD only on NGS (52.3%), those with MRD only on MFC (49.8%), and those who were MRD-negative on both assays (26.7%).23 

Factors that complicate the use of NGS to monitor MRD in patients with AML include the genetic clonal heterogeneity at AML diagnosis and during the course of the disease. The predominant leukemic clone at presentation might not be the clone that causes clinical relapse and mortality.59  Moreover, determination of clonality in a given sample can be influenced by the depth of sequencing and the algorithm used to identify mutations.37  NGS currently has an intrinsic error rate that limits its sensitivity for most single-nucleotide variants to ∼1% to 2% of all reads.15  NGS typically generates shorter sequence lengths; for example, 1 of the most commonly used technologies, Illumina’s sequencing by synthesis, routinely produces read lengths of 75 to 100 base pairs from libraries with insert sizes of 200 to 500 base pairs. Thus, assembly of longer repeats and duplications may suffer from the short read length.60  Further, NGS technology is computationally demanding, time-consuming, and still expensive (although it is expected that costs may drop in the future), which might make it difficult to apply in clinical practice. Reflecting the current state of development, the ELN MRD Working Party suggests NGS techniques for MRD measurement are best reserved for clinical trials at this time.24 

Challenges to clinical application of MRD testing

AML is genetically diverse and, currently, there is no uniform approach to detecting the leukemic cells that are biologically capable of and likely to cause relapse.37  The genetic heterogeneity of AML and lack of universal antigenic surface markers on leukemic stem cells increase the challenge of standardizing MRD detection protocols. Additional challenges to adopting MRD testing in routine clinical practice for patients with AML have included the absence of interlaboratory standardization or consensus regarding optimal key parameters, including type of specimen, MRD target, timing of MRD assessment, technology (eg, MFC vs RT-qPCR), testing protocols, and lack of established cutoff values,42,61  although the ELN MRD Working Party recommendations address several of these issues.24  Variables that affect the ability to detect MRD are assay sensitivity, the skills and expertise of personnel, biologic properties of the leukemic cells, and the quality and number of viable cells used for analyses.15,36,37  The ELN 2017 Recommendations for Diagnosis and Treatment of AML highlight that MRD testing should be performed in experienced, centralized diagnostic laboratories. Because sensitivities vary by type of MRD marker and testing method, reported results should specify the test applied, assay sensitivity, and cutoff values.10  Currently, the National Comprehensive Cancer Network AML 2018 Clinical Practice Guidelines for AML do not recommend MRD monitoring, but note that ongoing research is moving MRD monitoring to the forefront for all patients with AML.6 

Another subject of debate is whether routine clinical sampling for MRD testing should be performed on peripheral blood or bone marrow. The ELN MRD consensus report recommends testing both bone marrow and blood for molecular MRD during treatment.24  Bone marrow sampling generally offers greater sensitivity because MRD levels in peripheral blood are lower than in bone marrow.19  Nevertheless, peripheral blood sampling is less expensive and less painful for patients who may be unwilling to undergo the more frequent bone marrow sampling required to monitor MRD during various courses of treatment.14  MRD analysis of peripheral blood requires a minimum of 20 mL of blood; in patients with white blood cell counts <1 × 109/L, more blood may be necessary to improve sensitivity.24  The ELN MRD Working Party suggests aspirating 5 to 10 mL of bone marrow using the first pull, noting that contamination from peripheral blood increases as bone marrow sample volume increases.24  Additionally, peripheral blood may be preferable for PCR-based gene expression MRD monitoring because of high background “noise” in bone marrow.14  A study of younger adults (ages 18-60 years) with AML found that as detected by RT-qPCR, the presence of mutant NPM1 MRD in peripheral blood in first remission was a strong predictor of relapse, independent of cytogenetics and FLT3-ITD status, and might have application in selecting patients who would benefit from allo-HSCT.32 

The choice of an MRD target can be confounded by the persistence of genetic abnormalities in patients in long-term remission.62  For example, mutated DNMT3A with up to 50% VAF can persist in patients who have been in remission for several years.62  Another concern is that some commonly mutated genes in AML, such as TET2, DNMT3A, and ASXL1, can also be mutated in healthy people with no hematologic abnormalities, especially as people age. This phenomenon has been called age-related clonal hematopoiesis of indeterminate potential. Whole exome sequencing of samples from 12 380 people with no hematologic malignancies indicated 10% of persons age >65 years showed evidence of clonal hematopiesis.63  These mutations in older patients with AML may not be the drivers of leukemogenesis.63-65  However, the presence of AML-associated mutations may be indicative of early events in the development of hematologic malignancies in some cases because they significantly increase the risk of eventually developing one.65,66 

In a recent study, samples from 430 patients with AML in CR or CRi who had at least 1 mutation at diagnosis were obtained between 21 days and 4 months from the start of a second treatment cycle for analysis by targeted NGS and by MFC.23  Mutations in TET2, DNMT3A, and ASXL1 (DTA) were present during remission in >50% of patients. Detection of DTA mutations was not associated with a higher 4-year relapse rate than that of patients without these mutations unless they were accompanied by other non-DTA mutations.23  The researchers speculated that DTA mutations may have persisted in nonleukemic clones that repopulated the bone marrow after induction chemotherapy.23 

A study of patients with de novo AML who had received up to 2 rounds of induction chemotherapy evaluated MRD status at 30 days after treatment using digital sequencing of leukemia-specific mutations in 50 patients in morphologic remission.33  Although all patients showed normal morphology at day 30, some patients’ samples showed clearance of all mutations; others showed clearance of only a few of the mutations, which returned at relapse; a third group of patients showed clearance in a subset of the mutations at day 30, but the founding clone mutations persisted in almost every cell.33  Patients with EFS durations >12 months were significantly less likely to have persistent disease as indicated by VAF at day 30 than those with EFS ≤12 months (P = .01); for patients who relapsed, day 30 VAF had increased because cells containing those mutations reexpanded.33  These data suggest MRD testing at 30 days posttreatment can provide more important prognostic information than morphologic status, but repeated testing and trends in MRD status over time may be more informative.37  The optimum interval duration for sequential MRD testing is unknown and may depend on disease characteristics. To avoid false-positive results, some have suggested that a confirmatory MRD test should be performed at 2 to 4 weeks after a positive MRD test before making predictions about impending relapse.37 

The optimal time for MRD testing may depend on the type of MRD. Ommen and colleagues showed the kinetics of molecular relapse can differ markedly among leukemias characterized by NPM1, PML-RARA, RUNX1-RUNX1T1, and CBFB-MYH11 AML.67  The investigators developed a model to predict the time between molecular relapse and hematologic relapse. They found that CBFB-MYH11 AML displayed a slower clone regrowth than AML with the other molecular signatures and recommended MRD testing for CBFB-MYH11 be performed every 6 months, whereas testing for PML-RARA MRD was recommended for every 2 months.67  There is a continued need to establish the optimal intervals for MRD assessment to predict impending relapse. Currently, the ELN MRD Working Party recommends MRD testing at diagnosis, after 2 cycles of chemotherapy at the closest time point before consolidation treatment, and during follow-up of patients with PML-RARA, RUNX1-RUNX1T1, CBFB-MYH11, mutated NPM1, and other molecular markers. Molecular MRD assessment should be conducted in bone marrow and peripheral blood every 3 months for 24 months after the end of treatment, or in peripheral blood every 4 to 6 weeks.24  MRD testing should be performed before and after bone marrow transplant.24 

More studies are also needed to determine relevant MRD thresholds, which will of necessity vary according to the technology used for assessment and the type of tissue under study. In the study described previously, day 30 samples were assessed for MRD using digital sequencing that included probes covering all exons of 264 recurrently mutated genes in AML.33  The VAF threshold was set at 2.5% in bone marrow; investigators noted that because the vast majority of AML-associated somatic mutations are heterozygous, a 2.5% VAF threshold suggests that at least 5% of bone marrow cells under examination would contain the mutation(s).33  Indeed, detection of persistent AML-associated mutations in ≥5% of cells in day 30 remission samples was significantly associated with reduced OS and increased risk of relapse compared with patients who attained mutational clearance.33  Studies measuring LAIP by MFC use lower detection thresholds, for example, from 0.01% to 1.0%,26  and thresholds may depend on the types of LAIP under study.28  The ELN MRD Working Party suggests a threshold of 0.1% to distinguish between MRD positivity and negativity; however, they noted that MRD LAIP levels <0.1% may still signal residual leukemia.24 

Remission maintenance therapies

The correlation between MRD status and relapse risk has generated substantial interest in using results of MRD testing to direct therapy decisions for AML patients; for example, therapy might be initiated or intensified when MRD is present, reduced, or discontinued for those who are MRD-negative. Initiating or intensifying treatment of patients with MRD may lessen risk of relapse and improve OS, although this remains to be proven.14,37  The risks associated with any therapy must be considered when making decisions based on MRD status. At present, there are few published studies that have evaluated MRD stratification and/or therapeutic strategies to eradicate MRD in patients with AML.

Azacitidine has been shown to increase expression of epigenetically silenced leukemia antigens and induce a CD8+ T-cell response to tumor antigens posttransplant, potentially augmenting a graft-versus-leukemia effect.68-71  At least 2 studies have evaluated preemptive use of azacitidine after SCT based on detection of MRD. The RELAZA phase 2 study evaluated azacitidine after allo-HSCT in 20 patients with CD34+ AML or myelodysplastic syndromes and signs of MRD, defined as decreases of peripheral blood CD34+ donor chimerism to <80%, without concomitant signs of hematologic relapse.72  After 4 cycles of azacitidine, 10 patients (80%) were MRD-negative; of these, 4 remained MRD-negative at a median follow-up of 347 days. The investigators noted that tracking MRD after allo-HSCT via peripheral blood CD34+ donor chimerism monitoring allowed preemptive use of azacitidine only when MRD was detected, avoiding unnecessary toxicity in patients in CR at low risk of relapse.72  In another study, 10 patients with mutant NPM1 AML and normal karyotype in first or second CR after intensive chemotherapy or autologous or allo-HSCT who showed evidence of molecular relapse (defined as a 1% increase in mutant NPM1 transcripts in bone marrow) or persistent MRD in sequential RT-PCR analyses of bone marrow or peripheral blood received azacitidine treatment.73  Molecular response was defined as a 1-log reduction in MRD from the pretreatment value. At a median follow-up of 10 months (range, 2-12), patients had received a median of 5 azacitidine treatment cycles. Of the 10 patients, 7 showed a molecular response and remained in CR.73 

On a promising note, because of growing acceptance of the prognostic value of MRD, several ongoing AML studies are prospectively evaluating the effect of interventions on MRD. Some of these studies require detectable MRD as an eligibility criterion.

Conclusions

The ELN MRD Working Party recommends MRD testing as part of the standard of care for AML patients.24  Its recently published guidelines promote widespread adoption of MRD testing for monitoring of therapeutic efficacy and/or the prognoses of patients with AML; however, some questions remain. The MRD Working Party notes that the predictive power of several mutations is low or needs to be clarified. They recommend further study of the clinical implications of detectable flow cytometric MRD levels <0.1%. Moreover, different MRD thresholds after induction chemotherapy may have variable meaning in differing patient risk groups and the clinical significance of MRD for patients treated with nonintensive therapies such as hypomethylating agents requires more study.24,74  Ultimately, the molecular heterogeneity of AML and clonal architecture may prevent a “1-size-fits-all” approach to MRD detection.19  Incorporation of MRD as an end point of ongoing and future randomized clinical trials should provide the data needed to move toward improved understanding of the influence of MRD markers in patients not included in molecularly defined AML subgroups (eg, APL, CBF-AML, AML with BCR-ABL1, AML with NPM1 mutations). These studies will also help determine whether MRD assessment will prove to be a more accurate measure of therapeutic efficacy than current morphologic measures.

Acknowledgments

The authors received editorial support during manuscript development from Sheila Truten and Kelly Dittmore of Medical Communication Company, Inc. (Wynnewood, PA), who were funded by Celgene Corporation. The authors directed manuscript development and are fully responsible for all content and editorial decisions.

Authorship

Contribution: F.R. prepared the first draft of the manuscript; and all authors contributed equally to revising the manuscript and all approved manuscript content and submission to the journal.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Farhad Ravandi, Section of Developmental Therapeutics, Department of Leukemia, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; e-mail: fravandi@mdanderson.org.

References

References
1.
Breems
DA
,
Van Putten
WL
,
Huijgens
PC
, et al
.
Prognostic index for adult patients with acute myeloid leukemia in first relapse
.
J Clin Oncol
.
2005
;
23
(
9
):
1969
-
1978
.
2.
Walter
RB
,
Kantarjian
HM
,
Huang
X
, et al
.
Effect of complete remission and responses less than complete remission on survival in acute myeloid leukemia: a combined Eastern Cooperative Oncology Group, Southwest Oncology Group, and M. D. Anderson Cancer Center Study
.
J Clin Oncol
.
2010
;
28
(
10
):
1766
-
1771
.
3.
Burnett
AK
,
Milligan
D
,
Goldstone
A
, et al
;
United Kingdom National Cancer Research Institute Haematological Oncology Study Group
.
The impact of dose escalation and resistance modulation in older patients with acute myeloid leukaemia and high risk myelodysplastic syndrome: the results of the LRF AML14 trial
.
Br J Haematol
.
2009
;
145
(
3
):
318
-
332
.
4.
Chen
X
,
Xie
H
,
Wood
BL
, et al
.
Relation of clinical response and minimal residual disease and their prognostic impact on outcome in acute myeloid leukemia
.
J Clin Oncol
.
2015
;
33
(
11
):
1258
-
1264
.
5.
Fey
MF
,
Buske
C
,
Group
EGW
;
ESMO Guidelines Working Group
.
Acute myeloblastic leukaemias in adult patients: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up
.
Ann Oncol
.
2013
;
24
(
suppl 6
):
vi138
-
vi143
.
6.
National Comprehensive Cancer Network
.
NCCN Guidelines: acute myeloid leukemia, version 1.2018
.
7.
DeAngelo
DJ
,
Stein
EM
,
Ravandi
F
.
Evolving therapies in acute myeloid leukemia: progress at last?
Am Soc Clin Oncol Educ Book
.
2016
;
35
:
e302
-
e312
.
8.
Cornelissen
JJ
,
Blaise
D
.
Hematopoietic stem cell transplantation for patients with AML in first complete remission
.
Blood
.
2016
;
127
(
1
):
62
-
70
.
9.
Rashidi
A
,
Ebadi
M
,
Colditz
GA
,
DiPersio
JF
.
Outcomes of allogeneic stem cell transplantation in elderly patients with acute myeloid leukemia: a systematic review and meta-analysis
.
Biol Blood Marrow Transplant
.
2016
;
22
(
4
):
651
-
657
.
10.
Döhner
H
,
Estey
E
,
Grimwade
D
, et al
.
Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel
.
Blood
.
2017
;
129
(
4
):
424
-
447
.
11.
Arber
DA
,
Orazi
A
,
Hasserjian
R
, et al
.
The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia
.
Blood
.
2016
;
127
(
20
):
2391
-
2405
.
12.
Ossenkoppele
G
,
Schuurhuis
GJ
.
MRD in AML: time for redefinition of CR?
Blood
.
2013
;
121
(
12
):
2166
-
2168
.
13.
Goldman
JM
,
Gale
RP
.
What does MRD in leukemia really mean?
Leukemia
.
2014
;
28
(
5
):
1131
.
14.
Percival
ME
,
Lai
C
,
Estey
E
,
Hourigan
CS
.
Bone marrow evaluation for diagnosis and monitoring of acute myeloid leukemia
.
Blood Rev
.
2017
;
31
(
4
):
185
-
192
.
15.
Tomlinson
B
,
Lazarus
HM
.
Enhancing acute myeloid leukemia therapy - monitoring response using residual disease testing as a guide to therapeutic decision-making
.
Expert Rev Hematol
.
2017
;
10
(
6
):
563
-
574
.
16.
van Dongen
JJ
,
van der Velden
VH
,
Brüggemann
M
,
Orfao
A
.
Minimal residual disease diagnostics in acute lymphoblastic leukemia: need for sensitive, fast, and standardized technologies
.
Blood
.
2015
;
125
(
26
):
3996
-
4009
.
17.
De Angelis
F
,
Breccia
M
.
Molecular monitoring as a path to cure acute promyelocytic leukemia
.
Rare Cancers Ther
.
2015
;
3
(
1-2
):
119
-
132
.
18.
Paschka
P
,
Müller
MC
,
Merx
K
, et al
.
Molecular monitoring of response to imatinib (Glivec) in CML patients pretreated with interferon alpha. Low levels of residual disease are associated with continuous remission
.
Leukemia
.
2003
;
17
(
9
):
1687
-
1694
.
19.
Grimwade
D
,
Freeman
SD
.
Defining minimal residual disease in acute myeloid leukemia: which platforms are ready for “prime time”?
Blood
.
2014
;
124
(
23
):
3345
-
3355
.
20.
San Miguel
JF
,
Vidriales
MB
,
López-Berges
C
, et al
.
Early immunophenotypical evaluation of minimal residual disease in acute myeloid leukemia identifies different patient risk groups and may contribute to postinduction treatment stratification
.
Blood
.
2001
;
98
(
6
):
1746
-
1751
.
21.
Buccisano
F
,
Maurillo
L
,
Gattei
V
, et al
.
The kinetics of reduction of minimal residual disease impacts on duration of response and survival of patients with acute myeloid leukemia
.
Leukemia
.
2006
;
20
(
10
):
1783
-
1789
.
22.
Ivey
A
,
Hills
RK
,
Simpson
MA
, et al
;
UK National Cancer Research Institute AML Working Group
.
Assessment of minimal residual disease in standard-risk AML
.
N Engl J Med
.
2016
;
374
(
5
):
422
-
433
.
23.
Jongen-Lavrencic
M
,
Grob
T
,
Hanekamp
D
, et al
.
Molecular minimal residual disease in acute myeloid leukemia
.
N Engl J Med
.
2018
;
378
(
13
):
1189
-
1199
.
24.
Schuurhuis
GJ
,
Heuser
M
,
Freeman
S
, et al
.
Minimal/measurable residual disease in AML: a consensus document from the European LeukemiaNet MRD Working Party
.
Blood
.
2018
;
131
(
12
):
1275
-
1291
.
25.
Ravandi
F
,
Jorgensen
J
,
Borthakur
G
, et al
.
Persistence of minimal residual disease assessed by multiparameter flow cytometry is highly prognostic in younger patients with acute myeloid leukemia
.
Cancer
.
2017
;
123
(
3
):
426
-
435
.
26.
Terwijn
M
,
van Putten
WL
,
Kelder
A
, et al
.
High prognostic impact of flow cytometric minimal residual disease detection in acute myeloid leukemia: data from the HOVON/SAKK AML 42A study
.
J Clin Oncol
.
2013
;
31
(
31
):
3889
-
3897
.
27.
Othus
M
,
Wood
BL
,
Stirewalt
DL
, et al
.
Effect of measurable (‘minimal’) residual disease (MRD) information on prediction of relapse and survival in adult acute myeloid leukemia
.
Leukemia
.
2016
;
30
(
10
):
2080
-
2083
.
28.
Freeman
SD
,
Virgo
P
,
Couzens
S
, et al
.
Prognostic relevance of treatment response measured by flow cytometric residual disease detection in older patients with acute myeloid leukemia
.
J Clin Oncol
.
2013
;
31
(
32
):
4123
-
4131
.
29.
Walter
RB
,
Buckley
SA
,
Pagel
JM
, et al
.
Significance of minimal residual disease before myeloablative allogeneic hematopoietic cell transplantation for AML in first and second complete remission
.
Blood
.
2013
;
122
(
10
):
1813
-
1821
.
30.
Walter
RB
,
Gooley
TA
,
Wood
BL
, et al
.
Impact of pretransplantation minimal residual disease, as detected by multiparametric flow cytometry, on outcome of myeloablative hematopoietic cell transplantation for acute myeloid leukemia
.
J Clin Oncol
.
2011
;
29
(
9
):
1190
-
1197
.
31.
Karas
M
,
Steinerova
K
,
Lysak
D
, et al
.
Pre-transplant quantitative determination of NPM1 mutation significantly predicts outcome of allogeneic hematopoietic stem cell transplantation in patients with normal karyotype AML in complete remission
.
Anticancer Res
.
2016
;
36
(
10
):
5487
-
5498
.
32.
Balsat
M
,
Renneville
A
,
Thomas
X
, et al
.
Postinduction minimal residual disease predicts outcome and benefit from allogeneic stem cell transplantation in acute myeloid leukemia with NPM1 mutation: a study by the Acute Leukemia French Association Group
.
J Clin Oncol
.
2017
;
35
(
2
):
185
-
193
.
33.
Klco
JM
,
Miller
CA
,
Griffith
M
, et al
.
Association between mutation clearance after induction therapy and outcomes in acute myeloid leukemia
.
JAMA
.
2015
;
314
(
8
):
811
-
822
.
34.
Hourigan
CS
,
Karp
JE
.
Minimal residual disease in acute myeloid leukaemia
.
Nat Rev Clin Oncol
.
2013
;
10
(
8
):
460
-
471
.
35.
Topp
MS
,
Gökbuget
N
,
Zugmaier
G
, et al
.
Long-term follow-up of hematologic relapse-free survival in a phase 2 study of blinatumomab in patients with MRD in B-lineage ALL
.
Blood
.
2012
;
120
(
26
):
5185
-
5187
.
36.
Paietta
E
.
Should minimal residual disease guide therapy in AML?
Best Pract Res Clin Haematol
.
2015
;
28
(
2-3
):
98
-
105
.
37.
Hourigan
CS
,
Gale
RP
,
Gormley
NJ
,
Ossenkoppele
GJ
,
Walter
RB
.
Measurable residual disease testing in acute myeloid leukaemia
.
Leukemia
.
2017
;
31
(
7
):
1482
-
1490
.
38.
Grimwade
D
,
Jovanovic
JV
,
Hills
RK
, et al
.
Prospective minimal residual disease monitoring to predict relapse of acute promyelocytic leukemia and to direct pre-emptive arsenic trioxide therapy
.
J Clin Oncol
.
2009
;
27
(
22
):
3650
-
3658
.
39.
Ravandi
F
,
Jorgensen
JL
.
Monitoring minimal residual disease in acute myeloid leukemia: ready for prime time?
J Natl Compr Canc Netw
.
2012
;
10
(
8
):
1029
-
1036
.
40.
Chatterjee
T
,
Mallhi
RS
,
Venkatesan
S
.
Minimal residual disease detection using flow cytometry: applications in acute leukemia
.
Med J Armed Forces India
.
2016
;
72
(
2
):
152
-
156
.
41.
Wolff
DJ
,
Bagg
A
,
Cooley
LD
, et al
;
American College of Medical Genetics Laboratory Quality Assurance Committee
.
Guidance for fluorescence in situ hybridization testing in hematologic disorders
.
J Mol Diagn
.
2007
;
9
(
2
):
134
-
143
.
42.
Mosna
F
,
Capelli
D
,
Gottardi
M
.
Minimal residual disease in acute myeloid leukemia: still a work in progress?
J Clin Med
.
2017
;
6
(
6
):
E57
.
43.
Del Principe
MI
,
Buccisano
F
,
Maurillo
L
, et al
.
Minimal residual disease in acute myeloid leukemia of adults: determination, prognostic impact and clinical applications
.
Mediterr J Hematol Infect Dis
.
2016
;
8
(
1
):
e2016052
.
44.
Ossenkoppele
G
,
Schuurhuis
GJ
.
MRD in AML: does it already guide therapy decision-making?
Hematology Am Soc Hematol Educ Program
.
2016
;
2016
:
356
-
365
.
45.
Kern
W
,
Bacher
U
,
Haferlach
C
,
Schnittger
S
,
Haferlach
T
.
The role of multiparameter flow cytometry for disease monitoring in AML
.
Best Pract Res Clin Haematol
.
2010
;
23
(
3
):
379
-
390
.
46.
San Miguel
JF
,
Martínez
A
,
Macedo
A
, et al
.
Immunophenotyping investigation of minimal residual disease is a useful approach for predicting relapse in acute myeloid leukemia patients
.
Blood
.
1997
;
90
(
6
):
2465
-
2470
.
47.
Zeijlemaker
W
,
Schuurhuis
GJ
.
Minimal residual disease and leukemic stem cells in acute myeloid leukemi. InTech. 2013. Available at: http://cdn.intechopen.com/pdfs/39017/InTech-Minimal_residual_disease_and_leukemic_stem_cells_in_acute_myeloid_leukemia.pdf. Accessed 3 May 2018
.
48.
Zeijlemaker
W
,
Gratama
JW
,
Schuurhuis
GJ
.
Tumor heterogeneity makes AML a “moving target” for detection of residual disease
.
Cytometry B Clin Cytom
.
2014
;
86
(
1
):
3
-
14
.
49.
Zeijlemaker
W
,
Kelder
A
,
Oussoren-Brockhoff
YJ
, et al
.
A simple one-tube assay for immunophenotypical quantification of leukemic stem cells in acute myeloid leukemia
.
Leukemia
.
2016
;
30
(
2
):
439
-
446
.
50.
Bahia
DM
,
Yamamoto
M
,
Chauffaille
ML
, et al
.
Aberrant phenotypes in acute myeloid leukemia: a high frequency and its clinical significance
.
Haematologica
.
2001
;
86
(
8
):
801
-
806
.
51.
Nolan
T
,
Hands
RE
,
Bustin
SA
.
Quantification of mRNA using real-time RT-PCR
.
Nat Protoc
.
2006
;
1
(
3
):
1559
-
1582
.
52.
Ommen
HB
.
Monitoring minimal residual disease in acute myeloid leukaemia: a review of the current evolving strategies
.
Ther Adv Hematol
.
2016
;
7
(
1
):
3
-
16
.
53.
Drandi
D
,
Kubiczkova-Besse
L
,
Ferrero
S
, et al
.
Minimal residual disease detection by droplet digital PCR in multiple myeloma, mantle cell lymphoma, and follicular lymphoma: a comparison with real-time PCR
.
J Mol Diagn
.
2015
;
17
(
6
):
652
-
660
.
54.
Mencia-Trinchant
N
,
Hu
Y
,
Alas
MA
, et al
.
Minimal residual disease monitoring of acute myeloid leukemia by massively multiplex digital PCR in patients with NPM1 mutations
.
J Mol Diagn
.
2017
;
19
(
4
):
537
-
548
.
55.
Behjati
S
,
Tarpey
PS
.
What is next generation sequencing?
Arch Dis Child Educ Pract Ed
.
2013
;
98
(
6
):
236
-
238
.
56.
Bibault
JE
,
Figeac
M
,
Hélevaut
N
, et al
.
Next-generation sequencing of FLT3 internal tandem duplications for minimal residual disease monitoring in acute myeloid leukemia
.
Oncotarget
.
2015
;
6
(
26
):
22812
-
22821
.
57.
Debarri
H
,
Lebon
D
,
Roumier
C
, et al
.
IDH1/2 but not DNMT3A mutations are suitable targets for minimal residual disease monitoring in acute myeloid leukemia patients: a study by the Acute Leukemia French Association
.
Oncotarget
.
2015
;
6
(
39
):
42345
-
42353
.
58.
Getta
BM
,
Devlin
SM
,
Levine
RL
, et al
.
Multicolor flow cytometry and multigene next-generation sequencing are complementary and highly predictive for relapse in acute myeloid leukemia after allogeneic transplantation
.
Biol Blood Marrow Transplant
.
2017
;
23
(
7
):
1064
-
1071
.
59.
Ramos
NR
,
Mo
CC
,
Karp
JE
,
Hourigan
CS
.
Current approaches in the treatment of relapsed and refractory acute myeloid leukemia
.
J Clin Med
.
2015
;
4
(
4
):
665
-
695
.
60.
Alkan
C
,
Sajjadian
S
,
Eichler
EE
.
Limitations of next-generation genome sequence assembly
.
Nat Methods
.
2011
;
8
(
1
):
61
-
65
.
61.
NCCN
.
NCCN guidelines, version 1.2017. Acute myeloid leukemia; 2017
.
62.
Pløen
GG
,
Nederby
L
,
Guldberg
P
, et al
.
Persistence of DNMT3A mutations at long-term remission in adult patients with AML
.
Br J Haematol
.
2014
;
167
(
4
):
478
-
486
.
63.
Malcovati
L
,
Cazzola
M
.
The shadowlands of MDS: idiopathic cytopenias of undetermined significance (ICUS) and clonal hematopoiesis of indeterminate potential (CHIP)
.
Hematology Am Soc Hematol Educ Program
.
2015
;
2015
:
299
-
307
.
64.
Jaiswal
S
,
Natarajan
P
,
Silver
AJ
, et al
.
Clonal hematopoiesis and risk of atherosclerotic cardiovascular disease
.
N Engl J Med
.
2017
;
377
(
2
):
111
-
121
.
65.
Steensma
DP
,
Bejar
R
,
Jaiswal
S
, et al
.
Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes
.
Blood
.
2015
;
126
(
1
):
9
-
16
.
66.
Genovese
G
,
Kähler
AK
,
Handsaker
RE
, et al
.
Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence
.
N Engl J Med
.
2014
;
371
(
26
):
2477
-
2487
.
67.
Ommen
HB
,
Schnittger
S
,
Jovanovic
JV
, et al
.
Strikingly different molecular relapse kinetics in NPM1c, PML-RARA, RUNX1-RUNX1T1, and CBFB-MYH11 acute myeloid leukemias
.
Blood
.
2010
;
115
(
2
):
198
-
205
.
68.
Goodyear
OC
,
Dennis
M
,
Jilani
NY
, et al
.
Azacitidine augments expansion of regulatory T cells after allogeneic stem cell transplantation in patients with acute myeloid leukemia (AML)
.
Blood
.
2012
;
119
(
14
):
3361
-
3369
.
69.
Goodyear
O
,
Agathanggelou
A
,
Novitzky-Basso
I
, et al
.
Induction of a CD8+ T-cell response to the MAGE cancer testis antigen by combined treatment with azacitidine and sodium valproate in patients with acute myeloid leukemia and myelodysplasia
.
Blood
.
2010
;
116
(
11
):
1908
-
1918
.
70.
Jabbour
E
,
Giralt
S
,
Kantarjian
H
, et al
.
Low-dose azacitidine after allogeneic stem cell transplantation for acute leukemia
.
Cancer
.
2009
;
115
(
9
):
1899
-
1905
.
71.
Craddock
C
,
Labopin
M
,
Robin
M
, et al
.
Clinical activity of azacitidine in patients who relapse after allogeneic stem cell transplantation for acute myeloid leukemia
.
Haematologica
.
2016
;
101
(
7
):
879
-
883
.
72.
Platzbecker
U
,
Wermke
M
,
Radke
J
, et al
.
Azacitidine for treatment of imminent relapse in MDS or AML patients after allogeneic HSCT: results of the RELAZA trial
.
Leukemia
.
2012
;
26
(
3
):
381
-
389
.
73.
Sockel
K
,
Wermke
M
,
Radke
J
, et al
.
Minimal residual disease-directed preemptive treatment with azacitidine in patients with NPM1-mutant acute myeloid leukemia and molecular relapse
.
Haematologica
.
2011
;
96
(
10
):
1568
-
1570
.
74.
Boddu
P
,
Jorgensen
J
,
Kantarjian
H
, et al
.
Achievement of a negative minimal residual disease state after hypomethylating agent therapy in older patients with AML reduces the risk of relapse
.
Leukemia
.
2018
;
32
(
1
):
241
-
244
.
75.
Gale
RE
,
Green
C
,
Allen
C
, et al
;
Medical Research Council Adult Leukaemia Working Party
.
The impact of FLT3 internal tandem duplication mutant level, number, size, and interaction with NPM1 mutations in a large cohort of young adult patients with acute myeloid leukemia
.
Blood
.
2008
;
111
(
5
):
2776
-
2784
.
76.
Schlenk
RF
,
Kayser
S
,
Bullinger
L
, et al
;
German-Austrian AML Study Group
.
Differential impact of allelic ratio and insertion site in FLT3-ITD-positive AML with respect to allogeneic transplantation
.
Blood
.
2014
;
124
(
23
):
3441
-
3449
.
77.
Kayser
S
,
Schlenk
RF
,
Grimwade
D
,
Yosuico
VE
,
Walter
RB
.
Minimal residual disease-directed therapy in acute myeloid leukemia
.
Blood
.
2015
;
125
(
15
):
2331
-
2335
.
78.
Paietta
E
.
Assessing minimal residual disease (MRD) in leukemia: a changing definition and concept?
Bone Marrow Transplant
.
2002
;
29
(
6
):
459
-
465
.
79.
Salipante
SJ
,
Fromm
JR
,
Shendure
J
,
Wood
BL
,
Wu
D
.
Detection of minimal residual disease in NPM1-mutated acute myeloid leukemia by next-generation sequencing
.
Mod Pathol
.
2014
;
27
(
11
):
1438
-
1446
.