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 CS Hourigan et al 1483 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 Leukemia (2017) 1482 – 1490 MRD-Testing in AML CS Hourigan et al 1484 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 Leukemia (2017) 1482 – 1490 1485 MRD-Testing in AML CS Hourigan et al 1486 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. 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