Leukemia (2017) 31, 1482–1490
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 0887-6924/17
www.nature.com/leu
REVIEW
Measurable residual disease testing in acute myeloid leukaemia
CS Hourigan1, RP Gale2, NJ Gormley3, GJ Ossenkoppele4 and RB Walter5,6,7
There is considerable interest in developing techniques to detect and/or quantify remaining leukaemia cells termed measurable or,
less precisely, minimal residual disease (MRD) in persons with acute myeloid leukaemia (AML) in complete remission defined by
cytomorphological criteria. An important reason for AML MRD-testing is the possibility of estimating the likelihood (and timing) of
leukaemia relapse. A perfect MRD-test would precisely quantify leukaemia cells biologically able and likely to cause leukaemia
relapse within a defined interval. AML is genetically diverse and there is currently no uniform approach to detecting such cells.
Several technologies focused on immune phenotype or cytogenetic and/or molecular abnormalities have been developed, each
with advantages and disadvantages. Many studies report a positive MRD-test at diverse time points during AML therapy identifies
persons with a higher risk of leukaemia relapse compared with those with a negative MRD-test even after adjusting for other
prognostic and predictive variables. No MRD-test in AML has perfect sensitivity and specificity for relapse prediction at the cohortor subject levels and there are substantial rates of false-positive and -negative tests. Despite these limitations, correlations between
MRD-test results and relapse risk have generated interest in MRD-test result-directed therapy interventions. However, convincing
proof that a specific intervention will reduce relapse risk in persons with a positive MRD-test is lacking and needs testing in
randomized trials. Routine clinical use of MRD-testing requires further refinements and standardization/harmonization of assay
platforms and results reporting. Such data are needed to determine whether results of MRD-testing can be used as a surrogate end
point in AML therapy trials. This could make drug-testing more efficient and accelerate regulatory approvals. Although MRD-testing
in AML has advanced substantially, much remains to be done.
Leukemia (2017) 31, 1482–1490; doi:10.1038/leu.2017.113
INTRODUCTION
He will manage the cure best who has foreseen what is to happen
from the present state of matters.
Hippocrates. The Book of Prognostics. Around 400 BCE.
Complete remission, defined as o5 percent myeloblasts in the
bone marrow determined by cytomorphology and recovery of
blood counts has been the end point for evaluating chemotherapy efficacy in acute myeloid leukaemia (AML) for 60 years.1
Choice of this end point (and not partial remission or stable
disease as is common in lymphomas and solid neoplasms) was
based on two observations. First, persons achieving a complete
remission lived longer than those with any other response.
Second, their increase in survival corresponded directly with
duration of complete remission.2 The latter observation proved
achieving complete remission translated directly into a durable
benefit and was not merely a prognostic variable for longer
survival.
However, there are several limitations of defining remission by
cytomorphology. One is imprecision in quantifying myeloblasts in
bone marrow samples using light microscopy related to the
survey of relatively few (typically 100–400) nucleated bone
marrow cells and intra- and inter-observer variability in identifying
myeloblasts. Another issue is our imperfect ability to distinguish
normal from leukaemia (or preleukaemic) myeloblasts by cytomorphological criteria. A third issue is variability in the distribution
of myeloblasts at different sites.3 Given these limitations it is not
surprising many or most persons with AML in morphological
complete remission relapse. But others, also in complete remission
defined by these criteria, do not relapse. Why? One possibility is all
leukaemia cells able to cause relapse were eradicated by therapy.
Another is some or even many leukaemia cells able to cause
relapse remain but simply do not do so within the observation
interval. There are also other possibilities.
Limitations of defining complete remission by cytomorphology
make it desirable to try to develop more sensitive techniques to
detect residual leukaemia cells, especially those able to cause
leukaemia relapse. But why? Is it to detect some or all residual
leukaemia cell(s), only leukaemia cells biologically able to cause
relapse within a specified interval or which actually cause relapse
within this interval or some other reason? These are distinct,
sometimes overlapping, but not identical goals. As such, the goal
of a technique developed to detect residual leukaemia cells
missed by cytomorphology must be clearly defined.
There are 10–13 × 109/kg nucleated bone marrow cells in a
normal adult or 0.7–0.9 × 1012 in a 70 kg person.4 If we consider
1
Myeloid Malignancies Section, Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA; 2Haematology Research Centre,
Division of Experimental Medicine, Department of Medicine, Imperial College London, London, UK; 3Division of Hematology Products, Office of Hematology and Oncology
Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA; 4Division of Hematology, VU University Medical Center,
Amsterdam, The Netherlands; 5Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; 6Department of Medicine, Division of Hematology,
University of Washington, Seattle, WA, USA and 7Department of Epidemiology, University of Washington, Seattle, WA, USA. Correspondence: Dr CS Hourigan, Myeloid
Malignancies Section, Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Room 10CRC 5-5142, 10 Center Drive, Bethesda, MD 208141476, USA or Dr RB Walter, Clinical Research Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, D2-190, Seattle, WA 98109-1024, USA.
E-mail: hourigan@nih.gov or rwalter@fredhutch.org
Received 6 February 2017; revised 15 March 2017; accepted 21 March 2017; accepted article preview online 7 April 2017; advance online publication, 21 April 2017
MRD-Testing in AML
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5% myeloblasts as the cut-off for morphological complete
remission, ~ 1010 leukaemia cells might persist in a person
declared to be in complete remission. Increasing the sensitivity
to detect leukaemia cells below the level of 5 percent was
facilitated by studies in transplantable rat leukaemias5,6 which
reported a linear relationship between survival and numbers of
transferred leukaemia cells.5–7 These data aroused interest in
developing assays to quantify minimal residual disease (MRD) in
humans.8 Although techniques to detect MRD have improved,
current assays can still miss millions of residual leukaemia cells in a
person with AML in complete remission defined by cytomorphology. We therefore believe the abbreviation MRD, first introduced
in 1980(ref. 9) and by now well-established and likely to persist, is
best referred to as measurable residual disease,10 followed by an
expression of the limit of detection and specificity of the MRD-test
being used. Here, we summarize concepts and methods to detect
MRD in persons with AML, evaluate current data, highlight
controversies and suggest future directions (Box 1).
METHODOLOGICAL CONSIDERATIONS OF MRD-TESTING
A perfect MRD-test should accurately identify the smallest
population(s) of leukaemia cells in persons with AML in
morphological complete remission which, if left untreated, cause
relapse while being indifferent towards residual leukaemia cells
that do not cause relapse. For the clinical performance of any
MRD-test, the theoretical maximal sensitivity and specificity of an
assay to detect such residual leukaemia cells, together with other
characteristics (for example, reproducibility), are important, as are
practical considerations regarding sampling details (site, volume,
timing, frequency and so on) and result interpretation, for which
many uncertainties remain.
The clinical utility of MRD tracking in chronic myeloid leukaemia
(CML), using the BCR/ABL1 fusion transcript, is well-established.11–14
This has allowed sensitive quantification of the impact of highly
effective therapy on disease burden and the stratification of patients
based on risk of treatment-failure.15–18 However AML, much more
complex than CML, encompasses a range of myeloid neoplasms
with diverse genetic abnormalities resulting in different histologies, immune phenotypes and clinical outcomes.19–23 Consequently, there is currently no uniform approach to detecting MRD
in persons with AML.
The diverse methods to quantify MRD in AML rely on the either
phenotype or molecular abnormalities of the leukaemia cells.24–30
Multi-parameter flow cytometry (MFC)-based MRD-tests focus on
the phenotype of leukaemia cells. They operate by detecting cell
population(s) which deviate from an antigen-expression pattern
typical of normal or regenerating cells of similar lineage and
maturation stage. Such deviations include cross-lineage expression, over-expression, reduced or absent expression and asynchronous expression.27,30,31 Mutations resulting in gene products
which might be neo-antigens could, in theory, also be identified
by this technique. Advantages of MFC-based MRD-detection
include wide applicability (suitable for almost all persons with
AML if a comprehensive panel of antibodies is used),32–34 ease of
quantifying abnormal cell population(s), relative sensitivity (10 − 3,
that is, 1 in 1000 cells), rapid turn-around and the ability to
distinguish live from dead cells. MFC-based MRD-detection also
allows identification of abnormal cell population(s) with immature
‘stem/progenitor’ phenotype.35,36 However, in addition to limitations common to all MRD-testing discussed below, there are other
limitations inherent to MFC MRD-testing: (1) not all leukaemia cells
have an abnormal phenotype; (2) phenotypes may change over
time with gains/losses of specific abnormalities or patterns of
abnormalities because of disease evolution, sub-clone selection,
and/or progression through the cell cycle;37,38 (3) sensitivity of
MFC-based MRD-testing is less than an optimized PCR-based
MRD-testing (discussed below); (4) MFC-based MRD-detection is
© 2017 Macmillan Publishers Limited, part of Springer Nature.
Box 1
Suggestions for MRD-testing in AML
1. Considerable efforts were, and are, needed to standardize
BCR/ABL1 testing in CML. Given the diverse genetic aetiology
and clonal heterogeneity of AML, even greater standardization efforts are likely needed before MRD-testing is
sufficiently accurate and reproducible to be integrated into
clinical standard-of-care guidelines.
2. No current AML MRD-test has perfect sensitivity and
specificity to accurately predict leukaemia relapse. Physicians
need to carefully consider relapse probability and therapy
risks before proposing an intervention based on results of
MRD-testing.
3. Flow cytometry-based AML MRD-testing is applicable to most
cases but has limitations. AML subtypes caused by, or
associated with, a canonical genetic abnormality (for
example, APL, mutated NPM1 or core-binding factor translocations) may reasonably be monitored using qPCR.
Sequencing-based methods with error correction are likely
to become available soon.
4. We believe some MRD-testing, typically at ⩾ 2 time points,
will be a feature of most high-quality clinical trials in AML in
the future.
5. MRD-testing at one time point may have insufficient
specificity for clinical decision-making whereas trends in
MRD-tests over time are likely to be more informative.
6. Randomized trials (for example, intervention based on MRD
vs no intervention) are required to determine if MRD-guided
therapy is associated with a reduced relapse risk and longer
survival.
7. Determination of end point surrogacy requires multiple
randomized trials to prove the relationship between effect of
treatment on MRD state and the effect of treatment on
survival and/or other clinical benefit end point.
not uniform between people with AML because the ability to
identify abnormal cells depends on the degree residual leukaemia
cells differ from normal cells or from residual leukaemia cells
which do not cause relapse; (5) using MFC appropriately requires
considerable expertise and experience; analysis and data interpretation have some subjective elements and therefore potential
biases (operator-dependent) making assays challenging to harmonize (let alone standardize) across laboratories.39,40 Some, but
not all, of these problems can be reduced with standardized
laboratory procedures including sample processing and instrument settings, single tube approaches with a pre-configured and
stable assay, automated interpretation software, central review
and continuous quality assessment.36,41–43
Quantitative real-time PCR (qPCR) of the chimeric BCR/ABL1
mRNA transcript is a reproducible and highly sensitive technique
to monitor MRD in CML.11,13,44,45 No analogous single canonical
mutation exists in AML, explaining, in part, much of the
enthusiasm for MFC-based MRD-testing despite the limitations
discussed. However, mutations are seen in persons with AML and
substantial work has gone into standardizing qPCR-based assays
for the most common of those that result in chimeric mRNA
transcripts, for example, PML/RARA associated with t(15;17) in
acute promyelocytic leukaemia (APL) and RUNX1/RUNX1T1 (AML1ETO) and CBFB/MYH11 associated with t(8;21) or inv(16) in the
core-binding factor leukaemias.27,46–51 Although these tests are
highly sensitive and threshold levels and/or rates of change
associated with a high probability of relapse can be defined, falsenegative results are still possible. Mutations in nucleophosmin-1
(NPM1) may be tracked effectively using qPCR as well.52–55
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Together, these tests cover around 60% of all AML cases in those
under age 60.27 Extensive effort has also gone into developing
MRD-tests targeting detection of transcripts aberrantly expressed
in AML such as Wilms tumour-1 (WT1),56,57 among others.58–61
Increased understanding of the genomic landscape of AML has
prompted considerable interest in developing MRD-tests based on
detecting and quantifying somatic mutations.62,63 This task is
complicated by several factors: (1) there is genetic clonal
heterogeneity at diagnosis with evolution over time and possibly
emergence or selection of small sub-clone(s) at relapse;64–66
(2) error rates intrinsic to most conventional next-generation
sequencing techniques allow only for low-sensitivity detection of
mutated sequences;67 (3) understanding clonality in any sample
may be limited by depth of sequencing and algorithms used for
mutation calling;68 (4) mutated genes (for example, DNMT3A, TET2
and ASXL1 amongst others) can be detected in healthy people
without haematological abnormalities, a condition termed by
some age-related clonal haematopoiesis or clonal haematopoiesis
of indeterminate potential.69–72 This is especially so in older
persons about the same age as most persons with AML; and (5)
some genetic abnormalities persist in persons in long-term
remission, possibly because of residual pre-leukaemia cells or
expansion of normal cells with age-related somatic
mutations.37,66,73–76 Despite these limitations, technical advances
and increasingly sophisticated understanding of the clonal
somatic mutation hierarchy in AML means next-generation
sequencing-based approaches may have an increasing role in
MRD-testing in the future. However, this approach needs to
address the issue that not all mutations have equal biological
consequences in AML and how this computationally demanding
and time-consuming technology can be brought into clinical
practice.
LIMITATIONS OF MRD-TESTING
As intellectually appealing as the concepts underlying MRD tests
in AML may be, there are several practical and logistical
constraints resulting in discordance between theory and
practice.10,27,28,77,78 No current MRD-test has perfect sensitivity
or specificity to accurately predict relapse risk at the cohort level
or, at the more clinically relevant, individual level where therapy
decisions are made. Some persons with a negative MRD-test
relapse (false-negatives) whereas others with a positive MRD-test
do not (false-positives) and are cured, at least operationally. Why is
this so? Besides insufficient sensitivity of the assay, one reason for
false-negative MRD-test results is inconsistent expression of the
designated target(s) or marker(s) of the MRD-test on leukaemia
cells. AML is often an oligo-clonal disease (phenotypically,33
genetically64,79 and in terms of gene expression/epigenetics80)
with clonal selection operating naturally and under pressure from
therapy, such that the target of a previously informative MRD-test
may be less useful at later time points, even in the same person.
Other reasons include inhomogeneous distribution of leukaemia
cells in the bone marrow and/or small sample size(s).3,81
It is a common misperception that technical improvements in
MRD-assays will eventually eliminate false-negative MRD-tests by
providing a complete accounting of the remaining whole-body
disease burden. Rather, the ability to detect low levels of residual
leukaemia cells in AML is limited primarily by the character and
size of the biological sample tested and not MRD-assay
sensitivity.81 Many persons with negative MRD-tests will have
residual leukaemia which may, without additional therapy,
become detectable either by subsequent MRD-testing or by
relapse. This phenomenon is observed in trials of therapy
discontinuation in persons with CML after long-term durable
MRD-negative tests where 50–60 percent of people relapse within
6 months of stopping tyrosine kinase-inhibitor therapy.82–84
Leukemia (2017) 1482 – 1490
The frequency of MRD-testing may also impact test
performance.85 False-positive and false-negative MRD-test results
are more likely with single time point measurements than when
trends in MRD-test results are considered.86 Re-testing can
decrease the likelihood of false-positive (and negative) results.
Sometimes, however, repeat MRD-testing may result in discordance, even in the absence of intervention, which can be bidirectional: a negative-to-positive MRD-test or the converse.
Discordances have many explanations but sampling error is the
dominant issue. Requiring concordant results to declare a person
MRD-positive or -negative increases specificity but decreases
sensitivity.81 Orthogonal validation using alternative methodologies may have utility in such circumstance but may be impractical.
Sequential monitoring can be particularly helpful as a strategy to
increase sensitivity if changes in MRD-levels (for example,
increasing transcript levels) are used as the read-out, and
ordinarily a single discordant datapoint would be insufficient to
make an estimate of clinically relevant changes in residual
leukaemia burden.52,87 The optimal interval and duration of
sequential MRD-testing is unknown and may depend on variables
such as the type of AML85,87 or interval since achieving remission.
Given the potential risk of harm from unnecessary additional
treatment prompted by a false-positive MRD-test some have
advocated for a confirmatory second positive MRD-test within
2–4 weeks of a positive MRD-test before predicting relapse.28
MRD-test results can be falsely positive because of the assay (for
example, technical errors or laboratory contamination) or can fail
to reflect relapse-free clinical outcome because of eradication of
biologically important leukaemia cells with subsequent therapy
(ies) (or, possibly occasionally by immune-mediated anti-leukaemia effects, such as that reported after allotransplants), short
observation interval or early death from causes other than
relapse.81 Important biological reasons to consider for falsepositive MRD-tests are the expression of the MRD marker(s) on
normal cells, pre-leukaemia cells or leukaemia cells unable to
cause relapse.81 Just as the detection of cytogenetic or genetic
abnormalities in an otherwise healthy person is not intrinsically
disease-defining,72,88 detection of some abnormalities targeted by
MRD-testing in a person who has completed therapy does not
necessarily indicate residual AML or relapse risk,73–76,89–91 a
principle more generally seen in oncology.92 There is also the
problem of leukaemia cells resident in unsampled tissues such as
the central nervous system and skin, sites of relapse which could
become more important if MRD-test result-directed therapies are
successful at eliminating bone marrow-based relapses. Lastly,
although results of MRD-testing when done by a laboratory on
stored samples predict clinical outcomes, clearly there is
considerable variation between operators and centres in MRDtesting. This is particularly so for MFC-based MRD-assays,93 which
may be mitigated in part by standardization,94,95 formalized
proficiency testing40 and automated analyses.96 Cross study
comparisons of MRD-testing are limited by the lack of an
independent reference standard for interlaboratory proficiency
testing. Many studies of MRD-testing in AML use the terms MRDnegative and/or -positive, which vary with the sensitivity of the
MRD-test and are impossible to evaluate critically in the absence
of quantitation. Standard laboratory operating procedures including pre-analytical workflow, threshold definitions and reporting
guidelines are used successfully in CML but have not yet been
agreed upon for AML.16,45,97
MRD-TESTING AS TOOL TO PREDICT RELAPSE
Many studies in persons with AML in complete remission,
regardless of the technique used to assess MRD state, report a
robust correlation between a positive MRD-test, a higher risk of
relapse and shorter survivals compared with persons with a
negative MRD-test. These differences are noted as early as 2 weeks
© 2017 Macmillan Publishers Limited, part of Springer Nature.
MRD-Testing in AML
CS Hourigan et al
after beginning induction chemotherapy but are also seen
thereafter, for example, after completing the first or second cycles
of induction chemotherapy, during and after post-remission
chemotherapy and before and after a haematopoietic cell
transplant.49,52,59,61,62,98–114 Comparisons between studies are
complicated by differences in sensitivity, specificity and timing
of MRD-testing and other variables.8,24,26–28,30,115,116 Unsurprisingly, the likelihood of having a positive MRD-test in
cytomorphologically defined complete remission is associated
with the cytogenetic/molecular prognostic-risk category and
with other adverse prognostic factors such as older age,
an antecedent haematologic disorder, prior chemotherapy
and/or radiation therapy or a multiple drug resistance (MDR)
phenotype.112,117–120 Nevertheless, multivariable regression
modelling consistently indicate a positive result of MRD-testing
is an independent prognostic variable for relapse and
survival.52,99–102,104,105,120 An association of MRD-test results and
survival is not directly biologically related but may reflect the
importance of relapse-related deaths on survival rates in many,
but not all, therapy settings. Often the MRD-test result is the most
important factor for relapse and survival in univariate analyses and
is the only prognostic factor in multivariate analyses. Hence,
therapy-response measured by MRD-testing is a stronger predictor of leukaemia relapse than pre-treatment variables and, at
the cohort level, refines risk-stratification beyond that provided by
response assessment by cytomorphology.
Although the prognostic value of MRD-test results in those in
complete remission for predicting subsequent leukaemia relapse
is convincing, the relevance of the rate of change in MRD levels
during therapy is less clear. Studies in children and adults with
AML report persons achieving a complete remission with a
negative MRD-test after the first cycle of induction chemotherapy
have lower cumulative incidences of relapse than those achieving
this state after additional therapy.101,102,104,105 In contrast, in one
study99,100 about 10 percent of subjects with a positive MRD-test
by MFC after induction chemotherapy became MRD-test negative
following post-remission therapy and multivariate analyses
showed an independent association with relapse-free survival
and survival only for post-consolidation but not post-induction
MRD-test results. Similarly, a recent study of the prognostic value
of MRD-testing in children on day 15 after starting induction
chemotherapy and repeated immediately before starting postremission therapy reported a positive MRD-test at the later but not
former time point was an independent prognostic variable for
event-free survival and survival.114 In those followed by RUNX1RUNX1T1 qPCR the most informative landmark for prediction of
relapse and survival outcomes appears to be MRD-test result in
bone marrow samples after completing consolidation
therapy47,50,51 whereas for subjects followed by testing for
mutated NPM1 by qPCR it appears assessing blood samples after
the second cycle of chemotherapy is most accurate.52 This
discordance regarding optimal timing and sample source highlights the need for additional studies—or analyses of data from
from completed studies—to clarify how data from kinetics of
MRD-test result changes during therapy are best used in specific
subtype and therapy contexts to predict clinical outcomes and for
perhaps therapy decision-making in AML. In contrast, for those in
cytomorphological remission after therapy, the rates of increase in
qPCR MRD-test levels associated with relapse are well-defined
with characteristic kinetics in different AML subtypes.49,52,85,86,121
Although current MRD-tests are an important tool for estimating relapse risk in persons with AML in complete remission, a
recent analysis in adults with newly diagnosed de novo AML
treated with intensive chemotherapy on the SWOG S0106 trial
reported MRD-test results on achieving complete remission
improves survival prediction on an subject-level only
minimally.112 Better prediction accuracy is achieved studying
more immediate end points such as 6- and 12-month relapse-free
© 2017 Macmillan Publishers Limited, part of Springer Nature.
survival.112 These data caution against placing too much emphasis
on results of MRD-tests to predict subsequent risk and timing of
relapse at the individual level especially when MRD-testing is done
only at one time point. Features of MRD-testing, which might
affect reproducible correlation with survival in AML, include the
sample tested (blood vs bone marrow, volume), hematopoietic
recovery at time of testing (complete remission vs remission with
incomplete haematopoietic recovery), when during therapy (for
example, after induction vs after post-remission therapy) and how
often (for example, once vs repeated at intervals) the test was
done and other technical parameters.
With many studies claiming results of MRD-testing assessment
of complete remission in AML are better than cytomorphology for
predicting relapse, the question arises as to what extent
conventional assessments of complete remission will continue to
be used in the future. Two studies in children report a poor
correlation between MRD-test results and cytomorphology with a
substantial proportion of subjects with ⩾ 5% myeloblasts but a
negative MRD-test by MFC and/or molecular assays. Others with
o5% myeloblasts had a positive MRD-test.102,103 As in other
studies, a positive MRD-test was strongly associated with relapse
risk whereas myeloblast levels (o 5 vs ⩾ 5%) added no additional
prognostic information. Put otherwise, outcomes of subjects with
a positive MRD-test and o5% myeloblasts and those ⩾ 5%
myeloblasts were similar.103 In addition, in two series of adults
with AML receiving myeloablative allotransplants in complete
remission but with a positive MRD-test had outcomes similar to
persons not in morphological complete remission.59,110,122 These
data suggest results of MRD-testing could supplement or replace
our current cytomorphological based definition of remission.23
Such a change would have significant implications for therapy
algorithms of persons with AML. However, we believe that before
this happens there should be more convincing data that a MRDbased definition of complete remission correlates with increased
survival (or other clinical benefit) in diverse AML populations and
disease state and therapy settings.
MRD-TESTING AS TOOL FOR THERAPY DECISION-MAKING
The close association between MRD-test results and relapse risk
has generated substantial interest in using results of MRD-testing
to direct therapy decisions in persons with AML, for example,
intensify therapy in persons with a positive MRD-test or deintensify therapy in those with a negative MRD-test. Inherent in a
strategy of giving more therapy to someone with a positive MRDtest is the hope the intervention(s) will decrease relapse risk AND
improve survival. Several studies report most persons with AML
and a positive MRD-test relapse within 3–6 months of the
determination. The implication is one would detect relapse by
cytomorphological criteria if one simply waited 3–6 months and
repeated a conventional blood or bone marrow study. Thus,
inherent in the strategy of giving more therapy to someone with a
positive MRD-test is the unproved belief a lead-time of 3–6 months
is important, that is, long-term treatment outcomes will be better
if therapy is given before there is cytomorphological relapse.
The concept of MRD-test-directed therapy has precedent in
persons with acute lymphoblastic leukaemia, where the data from
several non-randomized prospective trials indicate better outcomes with this tactic.123–126 Results of randomized studies are
contradictory. The data from the UKALL 2003 trial in children and
young adults support using results of MRD-testing to direct
therapy-intensity.127,128 In contrast, data from a recent AIEOP-BFM
ALL 2000 trial reported increased relapses when therapy-intensity
was reduced in children with a negative MRD-test.129 In APL,
results of MRD-testing are only available from non-randomized
studies. These data suggest therapy based on results of MRDtesting can prevent clinical relapse.130–132 However, outcomes of
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modern therapy of APL are so good that results of MRD-testing
play only a minor role in current clinical practice.
There are no data from randomized studies on the efficacy of
MRD-test directed therapy in non-APL AML. Results from three
prospective, non-randomized multi-centre studies suggest better
outcomes when anti-leukaemia therapy was selected based on
the results of MRD-tests.101,133,134 However, these studies have
important limitations including selection biases, subject and
disease heterogeneity and others which preclude accepting these
data as proof of the value of MRD-testing in directing AML
therapy. There are two additional single centre studies in which
therapy was directed by results of MRD-testing.135,136 Although
both suggest the possibility MRD-directed therapy could decrease
or delay relapse these data should be interpreted cautiously
because the studies were uncontrolled. Even if relapses can be
delayed or avoided in some persons, MRD-directed therapy may
not result in better survival because some persons who relapse
can be rescued with chemotherapy and/or an allotransplant.137
Moreover, pre-emptive therapies based on results of MRD-testing
have toxicities and their impact not only on survival but also
quality-of-life and on the possibility to receive a future transplant
needs careful consideration.
There are many potential biases complicating interpretation of
results of non-randomized clinical trials and rather than concluding these studies prove therapy decisions based on MRD-test data
results in better outcomes in AML, these studies are an impetus to
conduct well-designed, appropriately-controlled trials with sufficient statistical power and observation intervals to evaluate the
potential value of MRD-testing in guiding therapy decisions. There
are many opportunities for the testing of the value, if any, of MRDtesting based therapy-interventions in AML. Value should not be
assumed a priori. Although switching from MRD-test-positivity to
-negativity is a reasonable immediate therapy goal, only
randomized trials can definitively determine whether converting
to MRD-negativity with additional therapy is associated with a
reduced relapse risk and, perhaps, longer survival.
MRD AS SURROGATE END POINT FOR DRUG DEVELOPMENT
AND REGULATORY APPROVAL
From a regulatory perspective, drug approval requires the
demonstration of clinical benefit. Currently, survival is the end
point used as evidence for the clinical benefit of new drugs or new
drug combinations for the therapy of AML. Reduction in AMLassociated symptoms is another potential basis for approval, but
use of this end point is challenging because of the lack of
validated instruments and methodological limitations.138 Obvious
disadvantages of using a survival end point for the purpose of
drug approval include the relatively long duration needed to
complete follow-up, particularly when investigating younger
individuals with AML, and confounding by post-remission or
rescue therapies, for example, transplants.138–140 Use of an early
post-therapy surrogate end point for survival has been suggested
as a way to accelerate AML drug development.138,139
In the regulatory context, a surrogate end point is a marker
thought to predict a clinical outcome such as survival but is not
itself a measure of clinical benefit.141 MRD-test results are an
indirect measure of numbers of residual leukaemia cells and
becoming MRD-test negative may be a plausible indicator of
reduced (or delayed) relapse risk. The question arises whether
becoming MRD-test negative is a biologically plausible surrogate
for improved survival. Perhaps. However, besides biological
plausibility, reliability of a surrogate end point also depends on:
(1) the robustness of the prognostic value of the surrogate for the
clinical outcome; and (2) proof the surrogate quantitatively
captures the effect of therapy on survival or other clinical benefit
end point.142
Leukemia (2017) 1482 – 1490
There has been a long reliance on the use of surrogates to
support drug approvals in oncology (for example, overall response
rates or time-to-progression). In the US, surrogate end points can
be used to support either regular or accelerated approval,
depending on the evidence in support of the surrogate.143
Although MRD-testing could theoretically be used as a surrogate
to support drug approval in AML, surrogacy would first have to be
established.
For regulatory purposes and specifically, the qualification or
acceptance of a surrogate, MRD would need to be proved with
statistical rigour to be a valid surrogate for survival.144,145 The
randomized trials needed to support end point surrogacy differ in
design from those needed to evaluate the value of MRD-test
result-directed therapy. The clinical data needed to support
surrogacy must allow for the determination of the relationship
between the treatment effect on MRD-testing and treatment
effect on the accepted clinical benefit outcome (that is, overall
survival).141,142 The challenge is accumulating a data set large
enough to establish and confirm the optimal parameters of MRDtesting as a correlate of survival, independent of disease state (first
complete remission vs later complete remissions), molecular
subgroup and intensity of induction -or post-remission therapy.
In contrast to cytomorphology-defined complete remission, there
are no convincing analyses showing MRD-defined complete
remission correlates with survival for different AML populations,
for different subtypes of AML, for different disease states or after
different therapies.
Most evaluations of MRD-test results as a surrogate end point in
AML were done in persons receiving induction chemotherapy.
Using MRD-test results to direct therapy, for example, treating
persons in morphological complete remission who switch from a
negative to a positive MRD-test as is done in APL is another area of
interest.146 There are no convincing data that converting from a
positive to negative MRD-test correlates with survival. Until
conversion of MRD-test results is validated as a surrogate
specifically in this setting, randomized trials evaluating the efficacy
of MRD-test result based therapy-interventions need to include a
survival end point or co-end point.
Although some investigators consider event-free survival as
conventionally-defined (cytomorphological relapse or death) a
surrogate for survival in AML, attempts to validate this end point
have been inconsistent.147,148 Whether a positive MRD-test or a
previously negative MRD-test becoming positive should be
considered an event in event-free survival analyses is an
interesting question requiring testing in clinical trials. Although
follow-up of trials using an event-free survival end point might be
briefer compared with a survival end point, a disadvantage is the
need for frequent sampling and testing.143
CONCLUSIONS
Technological advances using diverse techniques have resulted in
MRD-assays with reasonably high sensitivity and specificity. Except
perhaps those AML subtypes caused by or associated with a
canonical genetic abnormality (for example, PML/RARA, mutated
NPM1 or core-binding factor translocations) no single approach to
detect or quantify MRD has been proven superior. Each assay has
advantages and disadvantages needing consideration. Although
MRD-testing is becoming more available, standardization and
harmonization is needed to facilitate comparisons between
studies and for the determination of the value of MRD-testing to
predict relapse, direct therapy and as a potential surrogate end
point for drug approvals. Work toward this goal by the European
LeukemiaNet is ongoing and tools to further this process are
being developed.36,149
Despite the limitations we discuss, many studies report that
results of MRD-testing can inform on relapse risk during and after
AML therapy for cohorts of persons in morphological complete
© 2017 Macmillan Publishers Limited, part of Springer Nature.
MRD-Testing in AML
CS Hourigan et al
1487
remission. For individuals, however, results of testing at a single
landmark time point may only slightly increase the accuracy of
relapse prediction compared to that achieved using current riskstratification approaches.112 The possibility of false-positive and
-negative MRD-tests and of sampling error must also be
considered. Repeat MRD-testing has been shown to improve the
accuracy of relapse prediction and such confirmatory testing may
be an important feature of any clinical use of MRD-testing in
AML.48 Whether and how one should respond to results of the
data from MRD-testing remains to be determined by appropriately
designed clinical trials as it is currently unproven that intervention
will improve survival. An important variable is physician and
patient tolerance for an incorrect MRD-test result and consequences of acting thereon. For example, if therapy prompted by
the MRD-test result has little or no risk of adverse events one
might be willing to accept a false-positive -MRD-test. However, if
the consequences could be dire, tolerance for a false-positive
MRD-test should be less. The treating physician must evaluate
data from cohorts of similar persons, together with an understanding of the limitations of current MRD-testing including
characteristics of the particular test being used, and other personspecific objective and subjective variables, to generate an
integrated relapse risk assessment for the individual they are
evaluating.150
Conventional cytomorphology-based assessment of complete
remission in AML has been repeatedly shown to encompass
widely diverse levels of residual leukaemia cells and to be
associated with a range of clinical outcomes. Consequently, we
believe some estimation of leukaemia state by MRD-testing
(typically at least two informative time points) will likely become
a feature of future AML trials. The small incremental cost increase
of integrating MRD-testing into clinical trials and the lack of a
universally-accepted assay for all AML subtypes need not be
prohibitive. Whether such determinations should be done
routinely outside the context of clinical trials is controversial as
many questions about the optimal use of this information remain.
Although randomized clinical trials evaluating the value of
MRD-testing using different techniques in heterogeneous populations of persons with AML at diverse times during therapy and
across different therapies are clearly needed, data from all clinical
trials could potentially prove useful if carefully annotated with
details of the performance characteristics of the MRD-test used.
The importance of randomized assessments of the value of MRDtesting in AML to predict relapse, direct -therapy and determine if
MRD-test results are a valid surrogate for survival cannot be
overemphasized. If established as a surrogate end point, use of
MRD-testing in AML trials could make drug-testing more time and
cost effective and expedite regulatory approvals. Although there
may be many perceived hurdles for the conduct of such studies,
data from such randomized trials will be critical to determine the
clinical utility of MRD-testing in AML.
CONFLICT OF INTEREST
CSH receives research funding from Merck Sharpe and Dohme and SELLAS Life
Sciences Group AG. RPG is a part-time employee of Celgene Corp. The remaining
authors declare no conflict of interest.
ACKNOWLEDGEMENTS
Frederick R Appelbaum (Fred Hutchinson Cancer Research Center), Anton Hagenbeek
(University of Utrecht), Jacob M Rowe (Sharee Zedek Medical Centre), Charles A
Schiffer (Wayne State University), Jerald P Radich (Fred Hutchinson Cancer Research
Center), Paresh Vyas (University of Oxford) and Donna Przepiorka (US Food and Drug
Administration) kindly reviewed the typescript. This work was supported in part by
the Intramural Research Programs of the National Heart, Lung, and Blood Institute of
the National Institutes of Health. RPG acknowledges support from the National
Institute of Health Research (NIHR) Biomedical Research Centre funding scheme. RBW
is a Leukemia & Lymphoma Society Scholar in Clinical Research. The opinions
© 2017 Macmillan Publishers Limited, part of Springer Nature.
expressed here are ours and do not represent the official position of the National
Institutes of Health, US Food and Drug Administration, or the United States
Government.
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