Measurable Residual Disease Levels Research Articles

Impact of Measurable Residual Disease (MRD) By Multiparameter Flow Cytometry (MFC): A Real-World Study in 1,076 Patients with Acute Myeloid Leukemia (AML)

Impact of Measurable Residual Disease (MRD) By Multiparameter Flow Cytometry (MFC): A Real-World Study in 1,076 Patients with Acute Myeloid Leukemia (AML)

DNA-Based Methods for Measurable Residual Disease Detection in NPM1-Mutated Acute Myeloid Leukemia; Establishment of Cut-Offs for qPCR, Digital Droplet PCR and Targeted Deep Sequencing

For detection of measurable residual disease (MRD) in acute myeloid leukemia with NPM1 mutations, RT-qPCR with quantification of leukemic transcripts is currently considered the method of choice; however, MRD can also be determined with DNA-based methods, offering certain advantages. For example, digital droplet PCR (ddPCR) and targeted deep sequencing (deep seq) do neither require standard curves nor reference genes and are thus less labor intense than RT-qPCR. Also, deep seq allows for quantification independently of type of NPM1 mutation. In addition, DNA-based techniques enable MRD assessment of other mutations, beyond the reach of RT-qPCR, which is limited to analyses of highly expressed genes or fusion transcripts (e.g. core binding factor leukemias). With the rapid development of highly sensitive DNA-based techniques for MRD detection, there is a need to establish clinically relevant cut-offs for accurate interpretation of MRD results and risk stratification. Here, we compare and provide MRD cut-offs for three different DNA-based MRD methods for NPM1 mutations: quantitative PCR (qPCR), ddPCR and deep seq. To compare the DNA-based methods with RT-qPCR, we analyzed 110 follow-up peripheral blood (PB) or bone marrow (BM) samples from 32 AML patients harboring NPM1 mutation type A. First, we compared the mere detectability of leukemic signals (without reference to specific MRD cut-off points). We found a high correlation between results from RT-qPCR and the three DNA-based methods (Rs=0.936 for RT-qPCR vs qPCR, Rs=0.774 for RT-qPCR vs ddPCR and Rs=0.743 for RT-qPCR vs deep seq, p<0.001). As expected, RT-qPCR was the most sensitive method. Among the DNA-based methods, qPCR was the most sensitive, detecting leukemic DNA in 95% (55/58) of the RT-qPCR positive samples, compared to 72% (42/58) and 62% (36/58) for ddPCR and deep seq, respectively. Interestingly, the transcript level for a given amount of measurable leukemic DNA (RNA copy number per leukemic DNA molecule) fluctuated considerably between different follow-up samples for certain patients. In some cases, the RNA/DNA ratio exceeded a hundredfold difference between different follow-up time points in both PB and BM. Hence, transcript analysis may be more complex than just a simple measurement of leukemic cell burden, which in turn may influence accurate risk stratification and treatment decisions, if relying on RT-qPCR measurements alone. To select adequate DNA MRD cut-offs, we performed ROC curve analyses for each method at various DNA cut-offs, comparing them with the gold standard RT-qPCR cut-off. In BM, this cut-off can be defined as a less than 3 log reduction of mutated NPM1 transcripts vs diagnosis, separating MRDhigh from MRDlow/undetectable (sometimes inaccurately termed "MRD-positivity" and "MRD-negativity", respectively). In PB, the mere detectability of mutated NPM1 transcripts is considered the relevant cut-off. DNA cut-offs were chosen based on the area under the curve (AUC) for the ROC analyses (Table 1), and influenced by available literature including recommendations of prognostically relevant MRD levels. For qPCR, a cut-off at 0.1% leukemic DNA was judged relevant in BM. For ddPCR and deep seq, 0.05% was chosen to adjust for measuring allelic ratio (variant allele frequency (VAF)) rather than mutant DNA alone. In PB, the selected cut-off was detectable leukemic signal irrespective of DNA method. We next determined the accuracy of the selected cut-offs, for identification of samples with clinically relevant MRD, by comparing them with the gold standard RT-qPCR. In general, the selected DNA cut-off values generated high specificity as well as high positive and negative predictive values (Table 1). The vast majority of all MRD analyses (93% (368/395)) showed concordant results irrespective of MRD method. In BM samples, MRD assessment by the DNA based methods agreed with MRD status as determined by RT-qPCR (MRDhigh high vs MRDlow/undetectable) in 93% (62/67) of the analyses for qPCR, 96% (64/67) for ddPCR, and 97% (65/67) for deep seq. In PB, the agreement was 95% (41/43), 88% (38/43) and 86% (37/43), respectively. In summary, we found strong agreement between different MRD methods and based on this could provide clinically relevant cut-offs for risk stratification. Thus, in BM follow-up samples from AML patients with NPM1 mutation, we propose 0.1% leukemic DNA as cut-off for qPCR and 0.05% VAF for ddPCR and deep seq. Disclosures No relevant conflicts of interest to declare.

Evaluating Blinatumomab for the Treatment of Relapsed/Refractory ALL: Design, Development, and Place in Therapy.

Although adults with B-cell acute lymphoblastic leukemia (B-ALL) achieve high complete remission (CR) rates following treatment with intensive multi-agent chemotherapy regimens, up to two-thirds of these patients eventually relapse. Unfortunately, adults with relapsed or refractory (R/R) B-ALL have a poor prognosis, with variable responses to salvage chemotherapy regimens and allogeneic stem cell transplant. As such, the need to develop effective and well-tolerated treatments for this patient population has been of paramount importance over the past decade. In this regard, treatment options for R/R B-ALL patients have expanded considerably over a relatively short period of time, with the approvals of blinatumomab, inotuzumab ozogamicin and tisagenlecleucel occurring within only the past six years. Blinatumomab, a CD19 x CD3 bispecific T-cell engager (BiTE) was the first of these immune therapies to receive approval, and for many patients, is used as first-line salvage therapy. A number of large clinical trials have demonstrated improved progression-free survival and overall survival for R/R B-ALL patients receiving blinatumomab as compared to those receiving conventional salvage chemotherapy. In addition to being approved for both Philadelphia chromosome-negative and Philadelphia chromosome-positive R/R B-ALL, blinatumomab is also the only ALL therapy that carries approval for the treatment of measurable residual disease (MRD). Although blinatumomab has changed the therapeutic landscape for adults with R/R B-ALL, a number of important clinical considerations and questions remain, including the potential role of blinatumomab in the frontline setting, mechanisms of resistance, optimal goal MRD level, the role of transplant following MRD clearance, the optimal place for blinatumomab in the context of other recently approved immune-mediated therapies, and real world outcomes for patients treated outside the context of clinical trials. These issues are the focus of ongoing studies, which will hopefully inform future clinical practice regarding the utility of blinatumomab in the treatment of B-ALL patients.

Minimal residual disease in Acute Lymphoblastic Leukemia in the context of hematopoetic stem cell transplantation

Minimal or measurable residual disease (MRD) is considered the most important independent prognostic factor in acute lymphoblastic leukemia (ALL). MRD status after clinical remission has been used to establish the risk of relapse and therapeutic stratification, identifying patients who can benefit from therapeutic intensification, including allogeneic stem cell transplantation (alloSCT). The pre alloSCT MRD also identifies patients eligible for transplant and those with low or high risk of relapse after transplantation, according to the level of MRD detected. However, MRD status post-alloSCT has been shown to be a more powerful predictor of relapse than pre-transplant MRD. In addition, it is important to take into account that there are some factors to be considered to better interpret MRD information, these include: the method used for MRD assessment and its sensitivity and specificity, which may vary according to each specific time point of evaluation; the treatment regimen used; and the identification of genetic lesions that combined with MRD information can further improve the management of patients with ALL.

Monitoring CAR-T cell kinetics in clinical trials by multiparametric flow cytometry: Benefits and challenges.

Exceptional clinical responses produced by the first chimeric antigen receptor T [CAR-T] cell therapies, and their entry into commercial markets prompted a logarithmic increase in the number of next generation CAR-T clinical trials. As a result, there is a growing interest in understanding the analytical approaches utilized for reliable monitoring of these "living" drugs, and the challenges encountered during their clinical development. Multiparametric flow cytometry (MFC) assays have played a crucial role in understanding the phenotype and function of first approved CAR-T therapies. Herein, three main areas for monitoring CAR-T therapies in clinical trials are discussed: (1) analytical considerations critical for development of MFC assays for the reliable enumeration of CAR-T levels, (2) operational challenges associated with clinical trial sampling and transportation, and (3) differential cellular kinetics observed by MFC and qPCR analyses and their relationship with efficacy (measurable residual disease levels). Initial experiences described here may enable design of fit-for-purpose tools and help to more rapidly advance the development of next generation CAR-T therapies.

Multiparametric Flow-Cytometry Is a Reliable Tool for Measurable Residual Disease Assessment and Risk-Stratification of FLT3-Mutated AML Patients

Background: Mutations of the gene encoding Fms Related Tyrosine Kinase 3 (FLT3), at the juxta-membrane level (ITD), represent the most common lesions found in Acute Myeloid Leukemia (AML), identifying a subgroup of patients (pts) with unfavorable prognosis. FLT3-ITD mutations are considered an unreliable tool for measurable residual disease (MRD) monitoring, due to their intraclonal heterogeneity and instability during the course of disease. Instead, multiparametric flow cytometry (MFC) may represent an alternative to monitor MRD in this molecular subset. In fact, through the recognition and monitoring of leukemia associated immunophenotypes, MFC is applicable to > 90% of AML patients with a sensitivity of 10-4. Aims: The aim of our study was to investigate the reliability of MFC in MRD assessment of 72 FLT3-ITD positive pts whose treatment allocation was prospectively decided according to the genetic/cytogenetic profile at diagnosis and post consolidation MRD. FLT3-ITD pts were to receive, after induction and consolidation, allogeneic stem cell transplant (ASCT), whatever the source of stem cells. In this subgroup analysis, we investigated if FLT3-ITD mutated pts have a different propensity to achieve high quality (e.g. MRD negative) complete remission as compared to FLT3 wildtype ones. Furthermore, we seek for a correlation between different levels of MRD and overall (OS) and disease-free survival (DFS). Methods: We included in the analysis 72 pts with de novo AML carrying FLT3-ITD mutations whose MRD assessment at the post-consolidation timepoint was available. Pts were defined as MRD-negative, when obtaining a residual leukemic cells count below the threshold of 3.5x10-4 (0.035%). MRD positive pts (with MRD ≥ 3.5x10-4 RLC) were stratified into 3 classes according to the levels of MRD (0.035%-0.1%; >0.1%-1%; >1%). We compared the MRD status and clinical outcome with a matched group of FLT3 wildtype AML (n = 203) treated in the same protocol. Results: Overall median age was 49 (range 18-60.9). The 2 cohorts were balanced in terms of age and sex distribution. In the FLT3-ITD group, 80/126 (64%) cases carried a concomitant NPM1 mutation vs 107/374 (28.6%) of FLT3 wildtype ones (p <0.001). Furthermore, FLT3 mutated pts had a median WBC count of 35x109/L vs 9.5x109/L of those FLT3 wildtype (p < 0.001). MRD determination after consolidation cycle was available in 72/126 FLT3-ITD pts (57%) and in 203/374 FLT3 wildtypeones (54.3%), respectively. After having received induction and consolidation course, 47/72 FLT3-ITD pts (65,2%) were submitted to allogenic stem cells transplantation (ASCT). At the post-consolidation time-point, MRD negativity rate was significantly lower in FTL3-ITD pts (27/72, 37.5%) as compared to those FLT3 wildtype (94/203, 46.3%). Furthermore, 38/72 (52.8%) and 10/72 (13.9%) FLT3-ITD pts had a level of MRD > 0.1% and > 1%, respectively as compared to 65/203 (33.0%) and 15/203 (7.4%) of FLT3 wildtypeones, respectively (p=0.017). When considering the different MRD stratification levels of FLT3-ITD pts, OS probability at 24 months was 57.2% (27 pts), 71.4% (7 pts), 53.6% (28 pts) and 20% (10 pts), for the MRD categories <0.035%, 0.035%-0.1%, >0.1%-1%, >1%, respectively (p=0.028). DFS probability at 24 months was 53.8% (27 pts), 71.4% (7 pts), 34.9% (27 pts) and 20% (10 pts), for the MRD categories <0.035%, 0.035%-0.1%, >0.1%-1%, >1%, respectively (p=0.038). Summary/Conclusion: We demonstrated that MRD determination by MFC is a reliable tool to assess remission quality and prognosis in FLT-ITD positive patients. This subpopulation shows a lower propensity to obtain a MRD negative CR, with the majority of pts maintaining an amount of MRD > 0.1% after standard treatment. Even though most of these pts were addressed to ASCT, post-consolidation MRD maintained its negative impact on OS and DFS, particularly for those pts with MRD >1%. In the attempt to improve the quality of response, prevent leukemia recurrence and pursue a durable remission, delivery of FLT3 inhibitors as a maintenance after transplant may represent a promising option. Disclosures Venditti: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees. Buccisano:Janssen: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Achievement of Complete Remission (CR) with Measurable Residual Disease (MRD) Negativity Is Highly Prognostic in Patients (pts) with Relapsed or Refractory (R/R) Acute Myeloid Leukemia (AML) Receiving First Salvage Chemotherapy

Achievement of Complete Remission (CR) with Measurable Residual Disease (MRD) Negativity Is Highly Prognostic in Patients (pts) with Relapsed or Refractory (R/R) Acute Myeloid Leukemia (AML) Receiving First Salvage Chemotherapy

The Sequential Flamsa-Bu Conditioning Regimen Does Not Improve Outcome in Patients Allografted for High Risk Acute Myeloid and Myelodysplasia Irrespective of Pre-Transplant MRD Status: Results of the UK NCRI Figaro Trial

INTRODUCTION: Allogeneic stem cell transplantation (allo-SCT) is an important curative strategy in adults with high risk acute myeloid leukemia (AML) and myelodysplasia (MDS). Disease relapse represents the major cause of treatment failure and whilst retrospective analyses have identified that pre-transplant measurable residual disease (MRD) is an important predictor of transplant outcome this has never been examined prospectively. The advent of reduced intensity conditioning (RIC) regimens has substantially increased the number of older adults eligible for allo-SCT but the optimal RIC regimen in high risk AML remains unknown. Registry data have demonstrated improved outcomes using a sequential transplant regimen utilizing cytosine arabinoside (araC)/amsacrine (AMSA) cytoreduction followed by a fludarabine (Flu)/busulfan (Bu) based RIC regimen (FLAMSA-Bu). However, although the FLAMSA-Bu regimen is now widely used in adults with high risk AML and MDS its benefit has not been evaluated in a randomized trial. We report the results of a randomized trial evaluating the FLAMSA-Bu regimen compared with standard RIC regimens which also represents the first prospective evaluation of the impact of pre-transplant MRD levels on transplant outcome. PATIENTS AND METHODS: 244 patients (median age 59 yrs) with high risk AML (n=164) or high risk myelodysplasia (n=80) were randomized 1:1 to a control arm determined by investigator's choice of either Flu/B2/ATG (Flu, Bu 3.2 mg/kg x 2 days, ATG 2.5 mg/kg x 2 days); Flu/Mel/Alemtuzumab (A) (Flu, Mel 140 mg/m2, A 50 mg) or Flu/Bu2/A (Flu, Bu 3.2 mg/kg x 2 days, A 50 mg) versus an experimental arm of FLAMSA-Bu (Flu, araC 2g/m2 x 4 days, AMSA 100mg/m2 x 4 days, Bu -total dose 11.2 mg/kg). Patients over the age of 60 received an adjusted FLAMSA-Bu regimen utilising a reduced dose of araC (1mg/m2 x 4 days) and a total Bu dose of 6.4 mg/kg. Patients were transplanted using either an HLA identical sibling (n=49) or matched (10/10 or 9/10) unrelated donor (n=195). All patients received cyclosporine GVHD prophylaxis. 155 patients with AML were in CR1 or CR2 at the time of transplant and 9 had primary refractory disease. MRD was monitored by flow cytometry (applying different-from-normal analysis when no diagnostic/relapse leukemic aberrant immunophenotype was available). Pre-transplant MRD levels were measured up to four weeks prior to transplantation in 201 patients (MRD positive = 80 (40%), MRD negative = 94 (47%), inadequate sample = 27 (13%)). MRD results were not reported to clinicians. The primary outcome was overall survival. RESULTS: Baseline characteristics including CR1/CR2 status, adverse cytogenetics and MRD levels were similar between regimens. Median follow up was 35 months. Transplant outcomes were comparable between patients allografted in the control and FLAMSA-Bu arms. 2 yr overall survival (OS) and cumulative incidence of relapse (CIR) were 61% and 30% respectively in the control arm vs 62% and 26% for the FLAMSA-Bu arm. Transplant related mortality at 100 days was 3.0% in patients allografted using the control regimen vs 14% in patients allografted using the FLAMSA-Bu regimen and 17% vs 21% at 1 year. In the study cohort pre-transplant MRD positivity was associated with both an increased CIR compared to patients testing MRD negative (2 yr CIR 42% vs 19%, p=0.009) and decreased OS (2 yr OS 52% vs 71%, p=0.048). The FLAMSA-Bu regimen failed to improve OS or reduce CIR in either MRD positive or MRD negative patients. CONCLUSIONS: This trial, the largest randomized trial of RIC regimens in AML to date, did not detect any benefit of intensification of the conditioning regimen in adults with high risk AML or MDS. Specifically, the FLAMSA-Bu regimen was not associated with improved transplant outcome in patients who were MRD positive pre-transplant. These data include the first demonstration in a prospective analysis that the presence of pre-transplant MRD measured in real time is associated with reduced OS consequent upon an increased risk of disease relapse. Further randomized studies of novel conditioning regimens in adult AML, crucially with integrated MRD studies, are now required but these results support exploration of alternative strategies, such as pre or post-transplant pharmacological intervention, as the most promising strategy to reduce the risk of disease relapse post allograft. Disclosures Russell: Jazz: Consultancy, Honoraria, Speakers Bureau; DSI: Consultancy, Honoraria, Speakers Bureau; Pfizer Inc: Consultancy, Honoraria, Speakers Bureau; Astellas: Consultancy, Honoraria, Speakers Bureau. Freeman:Jazz Pharmaceuticals: Speakers Bureau. OffLabel Disclosure: We report data using the combination of fludarabine, busulphan, amsacrine and cytosine arabinsoide as a conditioning regimen in patients allografted for high risk acute myeloid leukemia

Comparison of MALDI-TOF Mass Spectrometry Analysis of Peripheral Blood and Bone Marrow Based Flow Cytometry for Tracking Measurable Residual Disease (MRD) in Patients with Multiple Myeloma

Comparison of MALDI-TOF Mass Spectrometry Analysis of Peripheral Blood and Bone Marrow Based Flow Cytometry for Tracking Measurable Residual Disease (MRD) in Patients with Multiple Myeloma

Flowsom: An R-Based Evaluation Strategy for Flow Cytometry-Based Measurable Residual Disease (MRD) Diagnostics in Acute Myeloid Leukemia (AML)

Patients with Acute Myeloid Leukemia (AML) frequently relapse due to chemorefractory AML cells persisting after intensive chemotherapy at levels below the 5% morphological detection threshold (measurable residual disease, MRD). MRD has been established as an important prognostic factor for relapse-free and overall survival, making it highly relevant for post-remission treatment stratification. In contrast to MRD assessment by molecular techniques, multiparameter flow cytometry (MFC)-based MRD measurements are applicable in more than 95% of AML patients, while still offering a sensitivity of 10-4 to 10-5. Current MFC MRD assessment strategies measure 8-10 fluorochromes in parallel, resulting in a high-dimensional data set. However, evaluation of this data is usually performed by scatterplot-based manual, two-dimensional analysis. This leads to loss of information and significant inter-observer variability in MRD diagnostics. We therefore established a computational data analysis strategy for MFC MRD diagnostics, based on the unsupervised FlowSOM algorithm. By comparison with healthy bone marrow (HBM) data, FlowSOM analysis can identify aberrant (sub-)populations of cells, clustered in nodes (according to similarity of their antigen profile). These nodes can be denoted as "nodes of interest" (NOI) to simplify MRD analysis after clustering. Aim of the project was to establish FlowSOM analysis protocols and retrospectively evaluate their prognostic significance in a cohort of 46 patients with known outcomes. Bone marrow samples of these patients were analyzed at aplasia (day 16 after initiation of induction chemotherapy). Only patients with morphological blast clearance at aplasia were included. Healthy reference FlowSOM trees were established by merging flow data of 17 HBM. Analysis protocols were developed to report individual ("any node" approach) and cumulative ("sum node" approach) differences in NOI percentages when comparing HBM and MRD samples. We then performed FlowSOM MRD analyses in a patient subcohort of 19 AML patients. Importantly, for these analyses, we excluded patients who underwent allogeneic stem cell transplantation in first remission (non-HSCT subcohort). Median follow-up time was 8.3 (range 2-40) months for this subcohort. Receiver operating characteristic (ROC) analyses were used to determine optimal threshold values to differentiate relapse (n=5) and non-relapse (n=14) patients within the cohort. For "sum node" analysis strategies (defining MRD levels as cumulative difference of NOI percentages) a threshold of -2.44% was identified, optimized for Youden (Y) index and diagnostic odds ratio (DOR). For the "any node" strategy (defining MRD levels by the maximum difference of any NOI), a threshold of 0.04%, also optimized for the Y-index and DOR, discriminated best between relapse and non-relapse patients. Relapse-free survival (RFS) was significantly shorter for MRD-positive (MRDpos) patients identified by "sum node" analysis (median 8 months vs. not reached, p=0.016) and tended to be shorter for MRDpos patients by "any node" analysis (median 8 months vs. not reached, p=0.1). When applying the thresholds identified in the non-HSCT cohort to the full set of 46 patients (median follow-up interval 10.6 months, range 2-40), median RFS was not reached for the MRD-negative group (both for "sum node" and "any node" analysis), and was 14 ("sum node", p=0.098) and 14 months ("any node", p=0.360) for the MRDpos patients. Median overall survival for MRDpos patients by "sum node" analysis was 27 months, whereas it was not reached for MRD-negative patients. However, this difference did not reach statistical significance (p=0.335), probably due to the small sample size. Taken together, FlowSOM-based analysis strategies seem well suited to identify patients with MRD positivity after intensive induction chemotherapy. MFC MRD positivity at aplasia, defined by FlowSOM-based analysis, is associated with inferior RFS in retrospective analyses of small patient cohorts. Due to the underlying computational, unsupervised data analysis, FlowSOM-based assessment can be a means to harmonize MFC MRD evaluation. These promising results need to be verified in larger cohorts, with inclusion of post-induction assessments, and should be followed by prospective analyses to delineate the diagnostic validity of FlowSOM for AML MRD diagnostics in clinical trials. Disclosures Subklewe: Janssen: Consultancy; Morphosys: Research Funding; Celgene: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Miltenyi: Research Funding; Oxford Biotherapeutics: Research Funding; Pfizer: Consultancy, Honoraria; AMGEN: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Research Funding.

The Role of Measurable Residual Disease (MRD) in Hematopoietic Stem Cell Transplantation for Hematological Malignancies Focusing on Acute Leukemia.

The significance of measurable residual disease (MRD) in hematopoietic stem cell transplantation (HSCT) is well recognized in different hematological malignancies, but the evidence indicate that pre-transplant MRD status is of particular importance in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). In ALL, inadequate response at the level of MRD is a commonly accepted risk factor for relapse and thus an indication for allogeneic HSCT. Similarly, growing evidence from the literature strongly suggest that MRD detected by multiparameter flow cytometry or molecular techniques should be also used for risk stratification in AML at the time of HSCT. Despite the well-defined association of MRD and outcomes of HSCT in acute leukemias, there are still many open issues such as the role of additional pre-transplant consolidation for MRD eradication, the ability of HSCT to overcome negative influence of MRD positivity on survival, the impact of conditioning regimen intensity on MRD clearance post HSCT, and transplantation outcomes or the selection of optimal donor with regards to MRD status. In addition, the role of MRD assessment in guiding post-transplant maintenance treatment should also be addressed in prospective trials. These open issues mostly awaiting further clinical studies will be discussed in our current review.

PB1770 RARE VARIANTS OF CBFB‐MYH11 TRANSCRIPTS IN ACUTE MYELOID LEUKEMIA: CONTRIBUTION WITH 3 CASES

Background:Acute myeloid leukemia (AML) with inv(16)(p13.1q22)/t(16;16)(p13.1;q22) and/or the respective CBFB‐MYH11 rearrangements is a distinct subtype of AML characterized by the presence of abnormal eosinophil population in the bone marrow and “favorable” prognosis. At molecular level, more than 10 different CBFB‐MYH11 fusion genes and transcripts have been reported so far. The most frequently detected variant is type A transcripts, which accounts for approximately 85% of the CBFB‐MYH11‐positive cases. The remaining variants are rare and therefore their incidence, association with clinical and laboratory features and response to therapy are largely unknown.Aims:To determine the incidence and main characteristics of CBFB‐MYH11‐positive Bulgarian AML patients (pts) with rare types of fusion transcriptsMethods:During a 15‐years period bone marrow aspirates of 856 AML pts, both adults and children, were tested for the main fusion transcripts in the Laboratory of Cytogenetics and Molecular Biology of the National Specialized Hospital for Active Treatment of Hematological diseases – Sofia, Bulgaria. Screening for CBFb‐MYH11 fusion transcripts was carried‐out by Reverse Transcription Polymerase Chain Reaction (RT‐PCR) using a protocol recommended by BIOMED‐1 Concerted Action.Results:Overall, CBFb‐MYH11 fusion transcripts were found in 49 (5.7%) pts, including 47 adults (95.9%) [42 with de novo AML (dnAML); 3 with therapy‐related AML, and 2 CBFb‐MYH11‐positive myeloid blast crisis of BCR‐ABL1‐positive chronic myeloid leukemia (BC CML)] and 2 children (4.1%). Typical type A CBFb‐MYH11 transcripts were found in 46 pts (93.9%). In the remaining 3 (6.1%) cases, rare variants of transcripts (2 type D; 1 type B) were seen: (1) in a 41‐year old woman with dnAML; (2) in a 8‐year old girl with dnAML, and (3) in a 33‐year old male patient with primary BC CML. All pts presented with leukocytosis (32/29/474x109/l); and anemia (Hb 77/104/111 g/l). In addition, in dnAML cases (#1,#2) was observed thrombocytopenia (8/53x109/l), while in BC CML patient was found mild thrombocytosis (474x109/l). No hepatosplenomegaly was found in dnAML cases. Blast cell bone marrow involvement varied from >90% (#1) to 53%/32% (#2;#3). In 2 pts (#1,#2), blast cells were with myelomonocytic characteristics, while the BC CML (#3) corresponded to AML with maturation. Immunophenotyping revealed CD34‐expression in all cases, with aberrant co‐expression of CD19 in 2 of them (#1,#2). Molecular screening for D816 c‐KIT mutation was negative in both tested cases (#1;#3). Complete remission with a measurable residual disease level below 0.1% was achieved in both dnAML pts after standard chemotherapy (7+3) (#1) or induction according to the AML BFM protocol (#2). Hoewever, a relapse was registered 1 year later in patient #1, treated with FLAG‐IDA that resulted in heavy aplasia, complicated with a fatal hemorrhagic shock. The BC CML patient (#3) received AML chemotherapy in combination with imatinib, and the BCR‐ABL1/ABL1 level was 3.6% (IS), with 0.1% CBFb‐MYH11 transcripts at the 4th month.Summary/Conclusion:Rare CBFb‐MYH11 transcripts variants are very rare and account for 0.35% of AML Bulgarian patients. Presentation of these cases is highly variable, including de novo AML both in adults and children, as well as a primary BC of CML. However, they seem to be responsive to routine therapeutic protocols.

Clinical Use of Measurable Residual Disease in Acute Myeloid Leukemia.

Treatment of acute myeloid leukemia (AML) remains a high-risk venture for the patient suffering from the disease. There is a real risk of succumbing to the treatment rather than the disease, and even so, cure is much less than certain. Since the establishment of complete remission as a prerequisite for cure in the 1960s, a number of years passed before advanced techniques for detecting minute amounts of disease matured sufficiently for clinical implementation. The two main techniques for detection of measurable residual disease (MRD) remain qPCR and multicolor flow cytometry. When performed in expert laboratories, both these modalities offer treating physicians excellent opportunity to follow the amount of residual disease upon treatment and offer unparalleled prognostication. In some AML and age group subsets, evidence now exist to support the choice of both proceeding to allogeneic transplant and not doing so. In other AML subgroups, MRD has sufficient discriminative power to identify patients likely to benefit from allogeneic transplant and patients likely not to. After treatment or transplantation, follow-up by molecular techniques can, with high certainty, predict relapse months before bone marrow function deterioration. On the other hand, options upon so-called molecular relapse are less well tested but recent evidence supports the use of azacitidine both in transplanted patients and patients consolidated with chemotherapy. In conclusion, MRD testing during treatment is a superb prognosticator and a major tool when choosing whether a patient should be transplanted or not. The exact use of MRD testing after treatment is less well defined but evidence is mounting for the instigation of treatment upon rising MRD levels (pre-emptive treatment) before morphologically detectable relapse.

Strategies for minimal residual disease detection: current perspectives.

Currently, the post-remission treatment in acute leukemia is based on the genetic profile of leukemic cells at diagnosis (ie, FLT3 ITD positivity) and on the level of measurable residual disease (MRD) after induction and consolidation chemotherapy. Two methods are currently preferred for MRD evaluation in many centers: multiparameter flow cytometry and real-time quantitative PCR. Additional methods such as next-generation sequencing and digital PCR are under investigation, in an attempt to increase the sensitivity and thus allowing the detection of small clones. Many studies suggest that MRD positivity after chemotherapy is associated with negative prognosis, and the reappearance of MRD during follow-up allows impending relapse to be identified and consequently enables early intervention. Finally, MRD positivity before hematopoietic stem cell transplantation is predictive of the outcome. Although the significance of MRD in acute leukemia has been widely explored, the assessment of molecular MRD is not yet a routine practice. In this review, we describe the significance of MRD in different settings and the main markers and methods used for MRD detection.

Development of a Novel Next-Generation Sequencing (NGS)-Based Assay for Measurable Residual Disease (MRD) in FLT3-ITD AML and Its Potential Clinical Application in Patients Treated with Chemotherapy Plus FLT3 Inhibitors

The presence of MRD in patients with acute myeloid leukemia (AML) who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) confer a negative prognostic impact by increasing the risk of relapse. However, the ability to detect these mutations in remission bone marrow specimens is hampered by the limited sensitivity of conventional polymerase chain reaction (PCR)-based assays, which detect approximately only 1 of every 100 (1%) mutant cells. To address this problem, we have developed a novel NGS-based MRD assay for the detection of FLT3-ITD mutations.Using isolated genomic DNA from bone marrow aspirates or whole-blood samples, PCR primers flanking exons 14 and 15 of the FLT3 gene were designed and highly diverse NGS libraries were generated. These libraries were then sequenced by Illumina's sequencing-by-synthesis method. The bioinformatics approach we used identifies unique FLT3-ITD mutations of varying length along with wild type sequences and calculates a mutant allelic frequency. The assay was validated using clinical samples to assess accuracy and reproducibility. DNA samples from selected mutant cell lines representing different FLT3-ITD lengths were spiked into normal DNA to evaluate assay sensitivity and linearity. The assay was linear (R2 = 0.958) down to FLT3-ITD allele frequency levels of 0.035% but was capable of detecting FLT3-ITD mutations at a level as low as 0.003%.We next validated the assay using clinical samples from patients with FLT3-ITD AML. The negative prognostic impact of FLT3-ITD mutations can be mitigated in part when an FLT3 inhibitor is administered in combination with induction chemotherapy, as demonstrated in CALGB10603/RATIFY (N Engl J Med. 2017;377:454). It was reported in this study that patients treated with an FLT3 inhibitor combined with chemotherapy followed by allogeneic transplant in first remission had better overall survival than their counterparts in the control arm. One hypothesis for this outcome is that the FLT3-inhibitor-treated patients had a lower leukemic burden prior to transplant. As a pilot test of this concept, we used our MRD assay on a series of bone marrow aspirate samples collected from 10 patients with newly diagnosed FLT3-ITD AML. The patients were selected to be as uniform as possible. All patients had intermediate-risk karyotype, a detectable FLT3-ITD mutation by conventional PCR, and mutated NPM1. All patients received cytarabine-based intensive induction and achieved morphologic first remission with a single course of chemotherapy. Finally, all patients underwent allogeneic transplant in first remission. The sample analyzed for MRD was the first collected after remission induction, 5-8 weeks after the start of therapy. The investigators performing the MRD assay were blinded to the clinical data. Four patients received chemotherapy alone, while 6 were treated with chemotherapy (7+3) plus an FLT3 inhibitor. In all patients' remission samples, the MRD assay identified the FLT3-ITD mutation that precisely matched the one observed in the original diagnostic specimen. This demonstrates the sensitivity of the assay (all samples had a detectable mutation), and the unique length of each patient's mutation confers a degree of specificity not achievable with MRD detection methods that focus on other AML-associated mutations. Supporting our hypothesis was the observation that patients treated with FLT3 inhibitors had MRD levels lower than those in patients treated with chemotherapy alone (Figure).Our results help establish the role of NGS-based MRD assays for the clinical management of FLT3-ITD AML. This assay could be used to define the depth of remission, identify persistent disease, and help guide decision making in the use of FLT3 inhibitors as continuation therapy. This study provides validation of the clinical utility of our MRD assay, which will be used to analyze the remission samples from patients in the ongoing phase 3, randomized, double-blind, placebo-controlled QuANTUM-First clinical trial, in which patients with newly diagnosed FLT3-ITD AML are randomized to receive either the highly potent and selective FLT3 inhibitor quizartinib or placebo in combination with chemotherapy, followed by single-agent quizartinib as continuation therapy. [Display omitted] DisclosuresShi:Novartis: Employment, Equity Ownership; Daiichi Sankyo: Other: Provide clinical trial testing services. Chang:Daiichi Sankyo: Employment. Laing:Novartis: Employment; Daiichi Sankyo: Other: Provide clinical trial testing services. Berisha:Daiichi Sankyo: Employment. Adams:Johns Hopkins University: Employment. Ding:Navigate BP: Employment; Daiichi Sankyo: Other: Provide clinical trial testing services. Nakamaru:Daiichi Sankyo: Employment. Lameh:Navigate BioPharma Inc,: Employment; Daiichi Sankyo: Other: Provide clinical trial testing services. Pollner:Navigate BioPharma Inc.: Employment. Kobayashi:Daiichi Sankyo: Employment.

Minimal/Measurable Residual Disease Detection in Acute Leukemias by Multiparameter Flow Cytometry.

Minimal or measurable residual disease (MRD) detected by multiparameter flow cytometry (MFC) is an independent prognostic indicator in acute leukemia. However, the predictive value of MFC MRD is affected by technical challenges, interpretive complexities, and inadequate standardization, particularly in acute myeloid leukemia (AML). Here, we critically review the methodological principles of the MFC MRD assay and discuss clinical implications of MRD. Key components of MFC MRD assays to be discussed include the principles of MFC, panel selection, analysis approaches, level of quantifiable MRD and calculation, reporting, and areas of improvements. Key components of clinical implications include context-dependent detection threshold and the integral role of MRD assessment by MFC in the era of ever-expanding molecular testing. With advancements in technology and standardization, MFC along with molecular assays will continue to play an important role in MRD assessment to evaluate treatment response and risk stratification.