Minimal residual disease (MRD) in AML; coming of age Dr. Mehmet Yılmaz Gaziantep University Medical School Sahinbey Education and Research hospital
1. The logistics of MRD assessment in AML 2. The clinical significance of MRD 3. MPFC for MRD detection 4. The choice of tissue for MRD determination in remission 5. Clinical trials aimed at eradicating or preventing MRD 6. Perspectives
1. The logistics of MRD assessment in AML The achievement of (CR) is a prerequisite for cure in AML. CR, based on the morphologic recognition of 5% of leukemic blasts in the BM, does not provide sufficient insight into the quality of the response. Despite CR rates of 50%-80% (depending on age), relapse within 3-5 years from diagnosis. Therefore, there is great need of more sensitive prognostic factors that can predict relapse
MRD MRD defined as any measurable disease or leukemia detectable above a certain threshold (defined by the methodology applied), predicts failure to maintain a morphologic CR and affects survival negatively
The MRD concept. x-axis represents time; y-axis represents tumor burden. The MRD concept. x-axis represents time; y-axis represents tumor burden. The horizontal black line represents the sensitivity of standard morphologic analyses, such as light microscopy using immunohistochemical methods. I, II, and III indicate 3 different courses of disease documented by MRD measurements. Patient course I (early relapse) can be distinguished from II and III (late relapse and complete cytogenetic response, respectively) by MRD measurements during therapy (period A). Relapse (patient course I and II) can in some cases be identified several months before clinical symptoms by MRD measurements after discontinuation of therapy (period B). 2011 by American Society of Hematology Hokland P, and Ommen H B Blood 2011;117:2577-2584
The detection of residual leukemic cells with techniques more sensitive and less subjective than morphology has added a new prognostic dimension to the treatment of AML. With few exceptions, 1- the integration of MRD has superseded standard risk classifications based on age, WBC count, and cytogenetic and genotypic features. 2. To date, MRD has rarely been used in the prospective risk stratification of AML patients, given that testing the clinical utility of MRD monitoring has been marred by inconsistencies in MRD thresholds, 3. uncertainty on the choice of the most informative MRD time points, 4. and the lack of standardized MRD assays. Therefore, clinical proof for improved outcome after MRD-directed therapy remains scarce.
The interactions between MRD and presenting prognostic features continue to reveal an unforeseen complexity. Whereas genetic features such as unfavorable cytogenetics and poor-risk gene mutations are among the factors associated with an increased frequency of MRD after therapy, MRD status adds independent prognostic information within these established risk categories.
2. The clinical significance of MRD Although rapid tumor clearance after therapy initiation is of critical value, some investigators have found that post consolidation MRD levels carry superior prognostic power. One relies on MRD detection early on, after induction therapy, to refine risk stratification, which otherwise relies mostly on pretreatment parameters.
Relapse rates in the 4 AML risk categories according to number of LAP cells in the first BM in mcr. High risk (MRD: greater than 10 2 LAP + cells); intermediate risk (MRD: 10 3 to 10 2 LAP + cells); low risk (MRD: fewer than 10 3 LAP + cells); and very low risk (fewer than 10 4 LAP + cells; none have had relapses San Miguel JFBlood. 2001;98:1746-1751.
Overall survival of patients with AML according to MRD levels San Miguel JFBlood. 2001;98:1746-1751.
Relapse rates of APL and non-apl patients with AML according to the MRD levels.high risk (MRD: greater than 10 2 LAP+ cells), intermediate risk (MRD: 10 3 to 10 2LAP+ cells), low risk (MRD: fewer than 10 3LAP+ cells); and very low risk (fewer than 10 4 LAP+... San Miguel J F et al. Blood 2001;98:1746-1751 2001 by American Society of Hematology
Results of the AML02 multicentre trial. 232 patients with de-novo AML (n=206), therapy-related or MDSrelated AML (n=12), or MLL (n=14) were enrolled at eight centres. The primary aim of the study was to compare the incidence of MRD positivity of the high-dose group and the low-dose group at day 22 of induction 1. Induction 2 consisted of ADE with or without gemtuzumab ozogamicin (GO) consolidation therapy included three additional courses of chemotherapy or haematopoietic stem-cell transplantation (HSCT). Levels of MRD were used to allocate GO and to determine the timing of induction 2.
Cumulative incidence (CI) of relapse or induction failure according to minimal residual disease (MRD)(A) CI for patients who were positive or negative for MRD after Induction I. (B) CI for patients with negative, low, or high MRD after of Induction I. (C) CI for patients who were positive or negative for MRD after Induction II. (D) CI for patients with negative, low, or high MRD after Induction II.
According to these studies early evaluation of disease response may also be of particular interest in the context of novel, investigational agents. The other approach uses serial monitoring of MRD during hematologic remission in an effort to prevent the development of clinical relapse by reacting to increasing MRD levels with preemptive therapy.
The introduction of high-dose versus low-dose cytarabine did not significantly lower the rate of MRDpositivity after induction 1 (34%vs 42%,p=0.17). 80% (155 of 193) of patients achieved MRD of less than 0.1% after induction 2, and the cumulative incidence of relapse for this group was 17% (SE 3). MRD of 1% or higher after induction 1 was the only significant independent adverse prognostic factor for both event-free and overall survival Rubnitz J, Lancet Oncol. 2010;11:543
It is still a matter of debate what is the best method to use to measure of MRD. The various methods are compared based on their sensitivity and specificity, The chosen threshold determines both sensitivity and specificity of an MRD assay. MRD thresholds with prognostic relevance are highly dependent on methodology (molecular or immunophenotypic)
Using multi-color antibody (Ab) combinations, the limit of detection with multiparameter flow cytometry (MPFC) can reach 10-4 (0.01%) depending upon the leukemiaassociated immunophenotype (LAIP). used and sample quality, a level comparable to that achieved by RT-PCR methodology for measuring molecular targets. The few direct comparisons of the 2 methodologies published in AML have demonstrated that quantitation of molecular targets and MPFC were equally effective in identifying patients at high risk of relapse. Rossi G et al.leuk Res. 2012 Apr;36(4):401-6. Perea G, Leukemia. 2006; 20(1):87-94.
Prognostic value of a 0.1% cutoff value of MRD assessed by FC in AML with t(8;21) and inv(16). Cumulative incidence of relapse according to MRD detected by FC at the end of chemotherapy Prognostic value of a 410 copies cutoff value of MRD assessed by RQ-PCR in AML with t(8;21) and inv(16). Cumulative incidence of relapse according to MRD detected by RQ-PCR at the end of treatment. Perea C et al Leukemia (2006) 20, 87 94
German-Austrian AML Study Group treatment trials To evaluate the prognostic impact of MRD in patients with AML expressing the CBFB- MYH11 fusion transcript. PCR was performed on 684 bone marrow (BM; n = 331) and/or peripheral blood (PB; n = 353) samples from 53 younger adult (16 to 60 years old) patients with AML treated in prospectively.
Graphical view of the minimal residual disease (MRD) checkpoints I, II, and III that allow the identification of patients at high risk of relapse. (B) Recommendation for sample collection with regard to the different checkpoints.
Kaplan-Meier survival estimates for patients with inv(16) according to their real-time quantitative reverse transcriptase polymerase chain reaction (PCR) status at checkpoints I (PCR negativity in bone marrow during consolidation) and II (at least two PCR-negative specimens in bone marrow and/or peripheral blood during consolidation and early * 3 months] follow-up). (A) Relapse-free survival (RFS) at checkpoint I. (B) RFS at checkpoint II. (C) Overall survival at checkpoint II. Using the standardized Europe Against Cancer quantitative RT-PCR protocol, CBF/ MYH11 copy ratios at diagnosis and after induction therapy did not affect outcome; however, achievement of MRD in at least 2 specimens during or up to 3 months after consolidation predicted for long-term remission.
Monitoring of CBFB-MYH11 transcript levels should be incorporated into future clinical trials to guide therapeutic decisions. Corbacıoglu A et al. J Clin Oncol. 2010;28(23):3724-3729.
Impact of additional karyotypic markers at diagnosis and after induction RT-PCR positively on relapse free survival.. (A) RFS in entire cohort. (B) CIR of patients who were RT-PCR-positive or -negative at the end of induction. (C) Event-free survival of patients who were RT-PCR positive or negative at the end of induction. (D) CIR among good risk cases (WBC < 5 10 9 /Lt and platelet count > 20 10 9 /Lt at diagnosis) of those who were RT-PCR positive or negative at the end of induction. (E) CIR among highrisk group (not fulfilling good risk criteria) who were RT-PCR positive or negative at the end of induction. (F) RFS among those who had and those who did not have an additional cytogenetic 2012 by American Society of Hematology Chendamarai E et al. Blood 2012;119:3413-3419
After induction with single-agent ATO, any molecular MRD is associated with a risk of relapse. Therefore, the prognostic value of MRD needs to be assessed in the context of each therapy. Blood. 2012;119(15):3413-3419.
Subgroup analysis of RFS and CIR of 143 AML patients stratified according to pretreatment karyotype or FLT3 status and levels of MRD after consolidation. Subgroup analysis of RFS and CIR of 143 AML patients stratified according to pretreatment karyotype or FLT3 status and levels of MRD after consolidation. (A-B) Those with a level of residual leukemic cells < 0.035% are referred to as intermediate karyotype-mrd, favorable karyotype-mrd, or FLT-wt MRD, whereas those with levels 0.035% are categorized as intermediate karyotype-mrd +, favorable karyotype-mrd +, or FLT-wt MRD +. Survival outcomes of these subsets and of U-RK and FLT3-ITD category are shown (P <.001 for all comparisons). (C-D) FLT-wt patients achieving a MRD-negative status show a better outcome than those who remained MRDpositive after consolidation (P <.001). 2012 by American Society of Hematology Buccisano F et al. Blood 2012;119:332-341
Sequential study of 138 patients during the clinical course showed that DNMT3A mutations were stable during AML evolution the DNMT3A mutation may be a potential biomarker for monitoring of MRD was an independent poor prognostic factor for OS and RFS (P <.001). Hou H et al. Blood 2012;119:559-568
OS and RFS in total patients and in younger patients with CN-AML. 2012 by American Society of Hematology Hou H et al. Blood 2012;119:559-568
Changes in mutation status between presentation and relapse may limit the clinical applicability of these markers and prompts the search for new, more stable mutations, such as of DNMT3A. Clonal instability implies that gene mutations should be reestablished at the time of relapse, not only for the purpose of MRD determination but especially before continuing therapy targeting those mutations.
Next-generation sequencing (NGS) might be particularly useful for MRD targets like FLT3-ITD, which is plagued by mutational shifts between diagnosis and relapse By multi clonality at presentation leading to the outgrowth of a clone at relapse different from that dominant at diagnosis, and by variable insertion sites and lengths among patients. Chromosomes Cancer. 2012;51(7):689-695.
Increased sensitivity for MRD monitoring is particularly important post allogeneic hematopoietic stem cell transplantation (HSCT), when presence or rise in MRD is likely to be predictive of relapse. Especially in patients who lack specific molecular markers, quantitative analysis of chimerism (ratio of donor- to recipient-derived hematopoiesis) serves as a reliable indicator of imminent relapse, particularly if mixed chimerism is assessed in CD34 cells. Curr Hematol Malig Rep. 2010;5(3):157-162.
Chimerism-based pre-emptive intervention with azacitidine has been shown to substantially delay relapse, allowing for donor lymphocyte infusions or a second HSCT.
Variations in the kinetics of leukemic cells affect the usefulness of MRD monitoring for the detection of relapse and determine the frequency of optimal MRD assessments during CR. Due to variable, but generally faster, doubling times of the PML/RARA clone, more frequent sampling is necessary.
The growth rate of NPM1MUT, FLT3- ITD leukemic cells is double that of NPM1MUT/FLT3 wild-type blasts, suggesting that relapse kinetics in AML depend on the complex genetic makeup of individual patients.
3. MPFC for MRD detection The advantage of MPFC-based MRD-assays is that they determine accurately the number of leukemic majority of AML patients. With common methodologies, only approximately 50% of AML patients have a suitable molecular target. Levels chosen to distinguish MPFC MRD+ from MRD- patients range from 0.035%-1%, with most studies using 0.1%. Semin Oncol. 2008;35(4):388-400. Blood. 2001;98(6):1746-1751.. Lancet Oncol. 2010;11(6):543-552. Leuk Res. 2012;36(4):401-406.
The potentially higher prognostic MRD threshold in AML has important implications: (1) sensitivities of current MPFC MRD assays suffice and (2) reducing MRD levels in AML to those significant prognostically in ALL may not be necessary for improved clinical outcome. One significant drawback of most prognostic MRD levels to date is that they were derived retrospectively without validation in prospective clinical trials. Blood. 2012;120(2):468-472.
The analysis of immunophenotypic aberrancies with MPFC as a measure of MRD presents with its own challenges 1. Comparisons of paired presentation/relapse samples often show selective LAIP changes. 2. Such immunophenotypic shifts will not affect the utility of MPFC for MRD detection, provided that as many independent LAIPs as possible are monitored per patient. This approach also reduces the likelihood of falsepositive MRD results due to the potential presence of LAIP Ag combinations at low frequencies in normal BM, BM after chemotherapy, or after growth factor administration.
Granted, depending on the expertise of the flow cytometry operator, Ag expression patterns may be misinterpreted. In this regard, standardized protocols and automated data file analyses may be particularly useful for MRD detection in AML., With 200 000 cells acquired, a 20-cell cluster represents a sensitivity of 1 in 104 (0.01%). As a result, if the sample quantity is limited, the sensitivity of the MRD assay will be lowered. This stresses the importance of sample quality for accurate MRD evaluation.
Abs suitable for MRD detection (1) distinguish leukemic blasts from normal myeloid precursors; (2) detect lineage-foreign markers (eg, lymphoidaffiliated Ags such as CD7 and CD19), (3) detect altered density of myeloid or lineageuncommitted Ags compared with normal myeloid precursors (eg, CD33 and CD11a), or (4) detect asynchronous expression of Ags, (eg, CD123 and CD34).
Characteristic immunophenotypes that are associated with recurrent genetic lesions, also termed surrogate marker profiles allow a highly focused approach to MRD assessment (eg, CD19,CD11a,CD56/ AML1/ETO AML or CD2 CBF/MYH11 AML). Unfortunately, to date, reliable surrogate profiles are rare and do not comprise common gene mutations in AML, such as FLT3-ITD. NPM1MUT AML typically lacks CD34 and CD133 expression; this relationship between genotype and immunophenotype is useful for MRD testing, even if it is not found in all cases. The disappearance of characteristic Ag profiles at relapse could signify the loss of the defined molecular clones. Leukemia. 2012;26(6):1313-1320.
The persistence of LSCs after therapy and outgrow that relapse may explain treatment failure in MPFC-MRD AML, because routine MRD Ab panels fail to detect LSCs. Contained within the CD34 CD38 cell compartment, LSCs in AML, but not APL, differ from normal hematopoietic stem cells by high expression of several Ags, including CD123 and C- type lectin-like molecule-1 (CLL-1). Best Pract Res Clin Haematol. 2010;23(3):391-401.
4. The choice of tissue for MRD determination in remission Hemodilute BM aspirates yield lower MRD levels if the contaminating PB contains fewer residual blast cells. In APL, MRD conversion occurs earlier in the BM than in the PB.
For molecular MRD targets with slow relapse kinetics, such as CBF/MYH11 and NPM1MUT (only when FLT3- ITD), PB testing may suffice provided that MRD sampling is done frequently for the period in which most relapses occur.
Real-time quantitative reverse transcriptase polymerase chain reaction (RQ-PCR) analysis in paired bone marrow (BM) and peripheral blood (PB) samples obtained after induction therapy, during consolidation therapy, and during follow-up. Corbacioglu A et al. JCO 2010;28:3724-3729 2010 by American Society of Clinical Oncology
In NPM1 MUT AML, MRD- results in PB despite continued MRD+ BM tests increase with prolonged therapy. With MPFC, the PB contains distinctly fewer residual blasts than BM at all time points. However, MRD information from the PB can have prognostic significance. Leukemia. 2010;24(9):1599-1606. Blood. 2012;119(2):332-341.
Despite lower sensitivity, MRD MPFC assays in PB have higher specificity due to the absence of normal immature myeloid cells, which can complicate MRD analysis in BM. With gene over expression as the MRD target, the normal physiologic background is lower in the PB than in the BM, thus interfering less with MRD interpretation. Blood. 2011;117(9): 2577-2584.
The relationship between expression of an overexpressed MRD marker (eg, WT1) in PB and BM and MRD sensitivity in PB and BM. In the example in this figure, (BM MRD level/pb MRD level) = (BM physiologic level/pb physiologic level), making the 2 tissues equally useful. Hokland P, and Ommen H B Blood 2011;117:2577-2584 2011 by American Society of Hematology
5. Clinical trials aimed at eradicating or preventing MRD In the pediatric AML02 trial, MRD levels did not differ whether induction included high- or low-dose cytarabine, being paralleled by similar relapse and survival parameters. In each treatment arm, the presence of high MRD ( 1%) was an independent adverse prognostic factor for survival Lancet Oncol. 2010;11(6):543-
The benefits of allogeneic HSCT in MRD+ pediatric patients or adult AML patients are questionable, even though a St Jude study suggested that improvements in HSCT over time have diminished the negative effects of MRD. Further reduction of MRD before transplantation in that study did not impact outcome, a finding that contrasts with observations in ALL Leung W et al. Blood 2012;120:468-472
Survival and cumulative incidence of relapse after HCT stratified by MRD level. Leung W et al. Blood 2012;120:468-472 2012 by American Society of Hematology
Current strategies in eliminating MRD are disappointingly limited. Clofarabine and cytarabine have been tested in pediatric AML patients in CR with MRD 0.1% (NCT01158885). This drug combination was used previously in adult AML (NCT00863434), although data on this are not yet available.
Cumulative incidence (CI) of relapse or induction failure according to minimal residual disease (MRD)(A) CI for patients who were positive or negative for MRD after Induction I. (B) CI for patients with negative, low, or high MRD after of Induction I. (C) CI for patients who were positive or negative for MRD after Induction II. (D) CI for patients with negative, low, or high MRD after Induction II. Lancet Oncol. 2010;11(6):543-
Novel therapies are needed to treat MRD AML after chemotherapy more successfully; for example, in the form of immunotherapy. Clin Dev Immunol. 2011;2011:104926
Two ongoing trials in adult AML are testing this hypothesis: 1. NCT01347996 is assessing the effects of maintenance therapy with histamine dihydrochloride (Ceplene) and 2. low-dose IL-2 on MRD and NCT01041040 (LAM07), which is being conducted by the PETHEMA Foundation, stratifies patients before first consolidation according to karyotype and molecular findings at presentation and 0.1% MRD at the end of induction. Similar in design to the pediatric AML02 trial, this is the first trial in adult AML that responds to MRD-positivity with targeted immunotherapy. Lancet Oncol. 2010;11(6):543-552
Therapeutic vaccination with dendritic cells, which is intended to generate anti leukemic T- cell responses, is another investigational approach with great potential for eradicating MRD. Bull Cancer. 2012;99(6):635-642.
A randomized trial of epigenetic priming with the hypomethylating drug decitabine (NCT01177540) also holds promise. Preemptive therapy with the DNA-methylation inhibitor 5-azacytidine in NPM1MUT AML patients in molecular relapse yielded promising results. Haematologica. 2011;96(10):1568-1570.
MRD course of 7 patients with molecular response to single agent azacitidine. MRD course of 7 patients with molecular response to single agent azacitidine. Tx: date of allogeneic HSCT; R: hematologic relapse. MRD monitoring in bone marrow samples is represented by the continuous lines; dashed lines reflect MRD monitoring from peripheral blood samples. 2011 by Ferrata Storti Foundation Sockel K et al. Haematologica 2011;96:1568-1570
6. Perspectives In pediatric AML, MRD has been added to conventional risk stratifications, but not so in adult AML (with the exception of APL). Randomized trials assigning patients in morphologic CR to MRD-adapted treatment arms are a rarity rather than the normal. Recently, the National Cancer Institute (NCI) has initiated an effort to standardize MPFC-MRD measurements among the leukemia reference laboratories of the 3 adult cooperative groups. Although seemingly small, this is a step of the utmost importance in the right direction.
Theoretically, we could be ready to treat every patient with AML individually according to risk allocation, which must include MRD levels. This approach would intensify treatment or switch chemotherapy to an MRD-targeting strategy in MRD+ patients and reduce treatment intensity in MRDLOW/- patients.