Monitoring Minimal Residual Disease in Acute Myeloid Leukemia: Ready for Prime Time?

Authors: Farhad Ravandi MDa and Jeffrey L. Jorgensen MD, PhDa
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  • a From the Departments of Leukemia and Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, Texas.

Predicting the outcome of therapy in patients with acute myeloid leukemia (AML) is currently necessary for making treatment decisions. Pretreatment covariates, such as clinical and molecular predictors, have helped identify which patients are more or less likely to survive their disease using the currently available regimens. Progress in establishing optimized flow cytometry and quantitative polymerase chain reaction assays for detecting minimal residual leukemia has provided new potential tools for predicting outcome. However, the most important next step in using these techniques toward personalized treatment of AML would be developing effective and safe strategies for eradicating the residual leukemic cells that are likely chemoresistant. With further refinement and standardization of the assays, and the development of novel, effective, and molecularly targeted agents, monitoring of minimal residual disease is likely to be incorporated into AML guidelines.

NCCN: Continuing Education

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This activity has been designated to meet the educational needs of physicians and nurses involved in the management of patients with cancer. There is no fee for this article. No commercial support was received for this article. The National Comprehensive Cancer Network (NCCN) is accredited by the ACCME to provide continuing medical education for physicians.

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All clinicians completing this activity will be issued a certificate of participation. To participate in this journal CE activity: 1) review the learning objectives and author disclosures; 2) study the education content; 3) take the post-test with a 70% minimum passing score and complete the evaluation at http://education.nccn.org/node/1174; and 4) view/print certificate.

Release date: August 7, 2012; Expiration date: August 7, 2013.

Learning Objectives

Upon completion of this activity, participants will be able to:

  • Describe the benefits and limitations of MRD monitoring in AML.
  • Outline how pretreatment covariates, such as clinical and molecular predictors, can determine prognosis/survival.

Predicting Relapse in AML: Potential for Modification of Therapy

Current treatment strategies in acute myeloid leukemia (AML) involve the administration of induction chemotherapy with the goal of achieving a morphologic complete remission (CR), wherein myeloblasts constitute less than 5% of marrow cellularity and evidence exists of adequate recovery of marrow function with normalization of peripheral blood counts.1,2 Although achieving CR remains the first requisite for long-term leukemia-free survival, it is clear that most, if not all, patients in morphologic CR after initial therapy harbor leukemic cells that, if not dealt with effectively, will lead to relapse. Disease recurrence provides a greater therapeutic challenge because it likely involves selection of clones resistant to initial therapy.3 Therefore, a major part of treatment for AML is focused on postremission strategies, including consolidation chemotherapy and allogeneic stem cell transplantation, to diminish the probability of relapse. Clearly, if a universally effective and nontoxic strategy for eliminating residual leukemia was available, identifying patients more likely to relapse would not be necessary. However, despite its curative potential, allogeneic stem cell transplantation is not uniformly successful and can be associated with significant morbidity and mortality, and therefore requires appropriate selection of patients most likely to relapse.

In the past several decades, several clinical predictors of relapse, such as age, WBC count at presentation, cytogenetics, and, more recently, molecular aberrations, have been established that have assisted in the postremission decision-making process, particularly regarding allogeneic stem cell transplantation.4 Although these predictors are continuously refined, such as through the identification of new molecular abnormalities both in cytogenetically normal and abnormal AML, they are by no means absolute in predicting the outcome.5 Therefore, more than 35% of patients with “favorable risk” cytogenetics experience relapse, and despite better identification of patients with cytogenetically normal AML who have a higher likelihood of early relapse (such as those with FLT3 internal tandem duplication [ITD]), which patients are not likely to relapse still cannot be predicted with certainty. Therefore, clearly, more precise tools are needed to predict disease recurrence in AML, allowing better patient allocation to postremission strategies.

Quantification of minimal residual leukemia can provide an estimate of the reduction of disease burden, reflecting the inherent leukemia biology and drug-resistance mechanisms, the adequacy of the treatment, and other host-drug interactions influencing the response. Techniques include multiparameter flow cytometry (MFC) with a sensitivity of 1 in 103 to 104 cells, and real-time quantitative polymerase chain reaction (RQ-PCR) with a sensitivity as low as 1 in 106 molecular targets.6 The RQ-PCR assay can be applied to detect recurring fusion transcripts, such as PML-RARa, Cb-MYH11, and RUNX1-RUNX1T1; overexpressed genes, such as WT1 and FRAME; or mutated genes, such as NPM16,7 (Table 1).

Assessment of minimal residual disease (MRD) is now well established in the management of patients with acute lymphoblastic leukemia (ALL), with several studies clearly showing the utility of different assays in identifying MRD and allowing tailored therapy for individuals.8,9 Unfortunately, except in cases of acute promyelocytic leukemia (APL) and perhaps core binding factor leukemias, relatively few groups have begun to routinely monitor MRD as a component of AML therapy. This may reflect the lack of standardization of the available techniques among different laboratories, the diagnostically challenging nature of some assays such as MFC, and/or the lack of clearly effective alternative intensification or eradication strategies for most patients identified to be MRD-positive. With further refinement and standardization of the assays and with the development of novel, effective, and molecularly targeted agents, MRD monitoring is likely to be incorporated into AML guidelines in the future.

Multiparameter Flow Cytometry

The assays used to detect MRD in leukemia include PCR to identify and/or quantitate recurrent fusion transcripts or mutated or overexpressed genes, and MFC to detect leukemia-associated immunophenotypes (LAIPs).7,1012 LAIPs consisting of aberrant patterns of antigen expression can be identified in the leukemic blasts of most patients with AML at diagnosis.13,14 They include asynchronous expression of antigens (ie, aberrant expression of antigens across maturation stages, such as coexpression of early and late antigens), cross-lineage

Table 1

Currently Available Techniques for Minimal Residual Disease Monitoring

Table 1
expression of lymphoid markers, and overexpression or lack of expression of lineage-appropriate markers.10 Additionally, abnormal forward and side scatter patterns are commonly displayed by leukemia cells, further assisting in their detection. However, in contrast to most cases of ALL, LAIPs in AML may be heterogeneous, with several blast populations with different LAIPs often identified in a single case.10 Therefore, using comprehensive panels of antibodies, one or more LAIPs can be identified in virtually all patients with AML and used for MRD monitoring. The sensitivity of assays based on MFC is in the region of 1 in 103 to 105 nucleated bone marrow cells, depending on the antibody panels and the number of LAIPs identifiable in a particular case.10,11,15

Some authors have suggested considering this analysis only in patients whose blasts express high-frequency LAIPs, using an arbitrary percentage cutoff of antigen expression levels on blasts, whereas others have suggested that all observed LAIPs should be considered in the analysis regardless of the percentage of blasts on which they are expressed.15 The former approach increases specificity and sensitivity, and the latter increases the proportion of patients suitable for MRD monitoring.10,15 Other authors have commented on the potential for phenotypic shifts between the diagnosis and relapse samples.1618 Using 9 panels of 3 antibodies, Baer et al.17 examined samples at diagnosis and relapse from 136 patients with AML and showed phenotypic changes in the leukemia cells in 91% of patients. Similarly, Voskova et al.18 found a complete change in LAIPs in approximately 20% of AMLs, with 80% having at least one LAIP similar to the ones present at diagnosis. Furthermore, Buccisano at al.15 reported on 12 patients experiencing relapse among 47 MRDnegative patients, all with the same phenotype at diagnosis and relapse. Although the data on the stability of the immunophenotype of the leukemia cells at relapse are inconsistent, experts recommend using comprehensive panels of antibodies at diagnosis and in MRD assays to identify baseline LAIPs, and then to detect as many conserved and newly presenting LAIPs as possible during posttherapy monitoring.10,15

Another major obstacle to using MFC in MRD monitoring is the difficulty in standardization, because different laboratories select different antibody combinations, which will only evolve further as new reagents become available. Interpretation is often challenging because of partial overlap of the blast phenotype with subsets of normal myeloid and monocytic cells, particularly during marrow recovery after chemotherapy.

Several studies have evaluated the role of MRD detection with MFC in predicting the outcome in patients with AML, and have shown correlations between clinical outcomes and the leukemia load as detected by MFC after induction and/or consolidation.13,1924 San Miguel et al.20 evaluated MRD with MFC in the first bone marrow obtained at CR from 126 patients with AML who expressed an aberrant phenotype at diagnosis. The investigators were able to identify 4 risk groups with cumulative incidence of relapse (CIR) rates of 0, 14%, 50%, and 84% at 3 years depending on the MRD level (P = .0001), and show that MRD was also predictive of overall survival (P = .003).20 In a study of 100 patients with AML, Buccisano et al.21 were able to identify a threshold of 1 in 3.5 × 104 leukemia cells as predictive of outcome after both induction and consolidation therapy. On multivariate analysis, postconsolidation MRD status independently affected relapse rate, overall survival, and relapse-free survival (P < .001, for all comparisons).21 Vinditti et al.,22,23 recently confirmed their previous observations that the level of MRD by MFC after consolidation is predictive of relapse-free and overall survival. Similar results have been reported in pediatric patients with AML, with a recent, large, multicenter study of 232 patients showing that an MRD level of 1% or higher post induction was a significant independent adverse risk factor for both event-free (P = .003) and overall survivals (P = .028).25 Importantly, modification of therapy based on MRD status was associated with an improvement in the outcome of patients compared with historical controls.

Real-Time PCR for Leukemia-Associated Fusion Transcripts

Recurring molecular markers are also targets for monitoring MRD and have been the subject of many studies over the past decade. They include gene fusion transcripts, gene mutations quantifiable by mutation-specific primers, and overexpressed genes.6,7,11 Several recurring balanced translocations, including t(15;17)(q22;q12) [PML-RARA], t(8;21)(q22;q22) [RUNX1-RUNX1T1 or AML1-ETO], and inv(16) (p13.1q22) [CBFB-MYH11], occur in approximately 20% of patients with AML and can be detected with well-established quantitative PCR assays. They are very stable between diagnosis and relapse, with only rare cases reported with a change at relapse, if that can be truly considered as a relapse.

PML-RARA is very well studied, with early reports suggesting the importance of achieving a negative PCR status using a qualitative assay, leading to the recommendation of initiation of therapy before overt relapse.2628 Recent reports have evaluated the use of quantitative assays to identify thresholds for high risk of relapse.2931 In a study of 406 patients with APL treated with a combination of all-trans retinoic acid and cytarabine and anthracycline-based chemotherapy, Grimwade et al.31 applied an internationally standardized RQ-PCR to monitor treatment response. They reported that persistent positive MRD after consolidation and recurrence of MRD after being negative were the most powerful predictors of relapse in a multivariate analysis, and recommended rigorous sequential RQ-PCR monitoring in addition to a preemptive treatment strategy to further reduce clinical relapse in patients with APL.31 Using this strategy, the 3-year cumulative incidence of morphologic relapse was only 5%.31 Importantly, bone marrow samples had approximately 1.5 log greater sensitivity than peripheral blood, and molecular relapse in the bone marrow preceded that in peripheral blood in most patients, suggesting that the former is more reliable for early prediction of relapse to allow preemptive therapy.31

Several other studies have examined the role of MRD monitoring using RQ-PCR in core binding factor leukemias and have shown that the rate of decrease of the fusion transcripts and specific log reduction levels at appropriate time points are of prognostic significance.30 Marcucci et al.32 reported that patients with inv(16) AML who had a CBFB-MYH11 fusion transcript copy number greater than 10 had a significantly shorter CR duration (P = .002) and higher risk for disease relapse (P = .05) than those with a CBFB-MYH11 fusion transcript copy number less than 10. Krauter et al.33 evaluated MRD levels using RQ-PCR for RUNX1-RUNX1T1 and CBFB-MYH11 at various time points during therapy in 37 patients with core binding factor leukemias. They reported that patients with an MRD level less than 1% of the diagnosis value had a significantly lower risk of relapse than those with an MRD value of 1% or greater at one time point after induction therapy (P < .001). Leroy et al.34 reported that a less than 3-log reduction of RUNX1-RUN1XT1 after induction therapy was associated with a higher likelihood of relapse in patients with t(8;21) AML. Others have suggested that the transcription levels after induction therapy were not significant, but that those after consolidation could be predictive of relapse.30 Several other reports have also indicated posttherapeutic threshold levels for fusion transcripts below which relapse was unlikely to occur.3537 Most recently, Corbacioglu et al.37 reported several relevant landmarks for predicting the outcome among 53 younger patients with inv(16) AML. Using RQ-PCR, achievement of negativity in at least 1 bone marrow examination during consolidation, or achievement of negativity in at least 2 bone marrow or peripheral blood samples during and after consolidation, were associated with significantly improved relapse-free and overall survival rates. Furthermore, conversion from PCR negativity to positivity with copy ratios more than 10 predicted relapse.37

Real-Time PCR for Leukemia-Associated Molecular Aberrations

Recent studies have further characterized patients with cytogenetically normal AML into molecularly distinct subgroups based on the presence of recurrent gene mutations, including FLT3-ITD or FLT3 length mutations (FLT3-LM),38 NPM1,39 MLL-PTD,40 CEBPA,41 IDH1 and -2,4244 and DNMT3A45 mutations. Several of these mutations have been evaluated for their potential as markers for MRD monitoring. Some of these mutations, such as CEBPA, are unsuitable as markers for MRD monitoring because currently available techniques lack sensitivity for their detection (ie, sequencing).

Schnittger et al.46 identified FLT3-LM in 234 patients with AML (165 with cytogenetically normal AML) and, through evaluating samples at diagnosis and follow-up, showed this marker to be reliable for MRD monitoring. Others have suggested that this mutation can be gained or lost at relapse and therefore may not be the ideal MRD target.4749

NPM1 mutations are the most common molecular aberrations in cytogenetically normal AML, occurring in more than half of these cases.39 In a recent study from the German-Austrian AML Study Group, RQ-PCR for mutated NPM1 was applied to samples from 245 patients aged 16 to 60 years at various time during therapy.50 Achievement of MRD negativity after double-induction therapy identified patients with a low 4-year CIR of 6.5% compared with those who were MRD-positive (53.0%; P < .001), which translated into significant differences in overall survival (90% vs. 51%, respectively; P = .001). Similarly, CIR was 15.7% in patients who were MRD-negative compared with 66.5% in those who were MRD-positive (P < .001) after completion of all therapy.50 Furthermore, serial posttreatment assessment of MRD allowed early detection of relapse.50 Other studies have confirmed the prognostic significance of monitoring NPM1 mutations.51 Similar concerns about the stability of NPM1 mutations between diagnosis and relapse samples have also been raised.52 However, overall, and with limited studies published to date, mutated NPM1 is considered a stable target for MRD monitoring in most patients with the mutation.7

Approximately half of patients with AML lack a leukemia-specific target suitable for MRD monitoring and this has led to considerable interest in identifying more universal targets, allowing extension of molecular monitoring to a much wider population. Apart from MFC, another approach has been to use RQ-PCR to detect transcripts that are overexpressed in leukemia blasts compared with normal cells.53 One such gene, WT1, is overexpressed in many cases of AML and has been the subject of several studies examining its utility as a target for MRD monitoring.5456 However, its suitability has been questioned, possibly because of differences in performing the RQ-PCR assay and false-negatives related to the existence of WT1 mutations in 10% of cases.7 To study this further, the investigators from the European LeukemiaNet evaluated 10 assays and applied the best-performing assay to diagnostic and followup samples from patients with AML treated with intensive chemotherapy.57 They were able to show that WT1 was sufficiently overexpressed to discriminate 2-log or higher reductions in 46% and 13% of patients' peripheral blood or bone marrow samples, respectively. In patients with informative samples (approximately half being peripheral blood), greater transcript reduction after induction and consolidation was associated with a reduced risk for relapse.57 Therefore, WT1 monitoring is potentially useful in select patients using peripheral blood samples and a standardized WT1 assay.7,57 Other overexpressed genes of potential interest include FRAME, although data on this MRD target are limited.58

The Need for Standardization

The aforementioned study on WT1 is a clear example of the need to standardize assays for MRD monitoring to be able to compare data and apply the strategy in large cooperative group studies.57 Clearly, MRD assessment using both MFC and PCR can to identify patients who are more likely to experience relapse and hence may benefit from alternate strategies, such as allogeneic stem cell transplantation, or investigational approaches, such as immunotherapy. However, before these strategies can be more widely applicable, consensus is needed among investigators regarding elements such as the technical details of sampling, cell separation, staining procedures and antibody panels for MFC, primers, and normalization of results from PCR. Therefore, quality assurance programs need to be developed to minimize interlaboratory variations and improve the precision of the analyzed data. Choice of initial time points and intervals for posttherapy monitoring also requires consensus agreement.

The Importance of Availability of Effective Therapy

Clearly, the ability to use MRD assessment to effectively improve leukemia-free survival and increase the cure rates is highly dependent on the availability of effective treatments or strategies to eradicate MRD. To date, the benefit from early salvage after molecular conversion has been shown in APL using preemptive arsenic trioxide, but not in patients with other non-APL AML.31 In a recent report, single-agent dasatinib did not prevent hematologic relapse in patients with core binding factor AML in first CR but with persistent or reappearing molecular MRD, calling into question the potential usefulness of this agent in KIT-mutated core binding factor leukemias.59 Therefore, although use of targeted therapy to eradicate MRD is attractive and promising, further data are needed before this becomes an established practice for most patients.

Using MRD to determine whether to perform an allogeneic stem cell transplantation also depends on the proposition that patients who are MRD-negative have little likelihood of experiencing relapse and that patients who are MRD-positive are likely to be cured with the procedure. Unfortunately, in previously reported studies, positive MRD before allogeneic stem cell transplantation is associated with a high likelihood of failure, and therefore this strategy (although currently the best available) cannot be considered definitive.60

Conclusions

Over the past 2 decades, improvements in laboratory diagnostics and the development of assays capable of detecting differences between normal and leukemic cells, such as the molecular aberrations specific to the leukemia cells and their respective immunophenotypes, have increased the ability to detect residual leukemia after therapy and use it to predict outcome. Recent refinements to these assays are likely to increase their relevance and importance. This is likely further enhanced through the identification of effective and target-specific drugs and agents capable of eradicating leukemia stem cells, which are the main constituents of the minimal residual leukemia population.61 The available techniques are sensitive and specific enough to detect cells with leukemia immunophenotype and the well-described molecular aberrations. A limited number of studies of MRD in AML have been published, with even fewer studies incorporating the results in the treatment decision-making process. Standardization of techniques is also needed to compare the results of studies. Despite these limitations, MRD monitoring is an important aspect of treatment for patients with AML and will be incorporated in future trials examining the role of novel and established strategies such as vaccines, molecularly targeted drugs, and early allogeneic transplantation in the treatment algorithms. Several NCCN Member Institutions have incorporated routine monitoring for MRD using both MFC and realtime PCR into their institutional treatment strategies.

The authors (Farhad Ravandi, MD, and Jeffrey L. Jorgensen, MD, PhD) have disclosed that they have no financial interests, arrangements, commercial interest conflicts, or affiliations with the manufacturers of any products discussed in this article or their competitors.

EDITOR

Kerrin M. Green, MA, Assistant Managing Editor, Journal of the National Comprehensive Cancer Network.

Ms. Green has disclosed that she has no relevant financial relationships.

CE AUTHOR

Nicole B. Fair, BS, Manager, Continuing Education and Grants

Ms. Fair has disclosed that she has no relevant financial relationships.

Kristina M. Gregory, RN, MSN, OCN, Vice President, Clinical Information Operations

Ms. Gregory has disclosed that she has no relevant financial relationships.

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Instructions for Completion

To participate in this journal CE activity: 1) review the learning objectives and author disclosures; 2) study the education content; 3) take the post-test with a 70% minimum passing score and complete the evaluation at http://education.nccn.org/node/1174; 4) view/print certificate. After reading the article, you should be able to answer the following multiple-choice questions. Credit cannot be obtained for tests completed on paper. You must be a registered user on NCCN.org. If you are not registered on NCCN.org, click on “New Member? Sign up here” link on the left hand side of the Web site to register. Only one answer is correct for each question. Once you successfully answer all post-test questions you will be able to view and/or print your certificate. Software requirements: Internet

Post-Test Questions

  1. True or False: NPM1 mutations are the most common molecular aberrations in cytogenetically normal AML (occurring in more than half of these cases) and may potentially serve as a stable target for MRD monitoring in these patients.
  2. Leukemia-associated immunophenotypes (LAIPs) consisting of aberrant patterns of antigen expression can be identified in the leukemic blasts of most patients with AML at diagnosis. They include:
    1. Asynchronous expression of antigens (ie, aberrant epression of antigens across maturation stages, such as coexpression of early and late antigens)
    2. Cross-lineage expression of lymphoid markers
    3. Overpression or lack of expression of lineage-appropriate markers
    4. All of the above
  3. MRD monitoring will increasingly be an important aspect of AML treatment; however, it currently has some limitations for routine clinical use. All of the following are limitations EXCEPT:
    1. Lack of standardization of techniques to compare the results of studies
    2. Limited number of studies of MRD in AML have been published.
    3. Inability to detect residual leukemia after therapy and use it to predict outcome
    4. Few studies of MRD in AML incorporate results in the treatment decision-making process.

Correspondence: Farhad Ravandi, MD, Department of Leukemia, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 428, Houston, TX 77030. E-mail: fravandi@mdanderson.org

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