Leukemia-associated Immunophenotypes Research Articles

Prognostic Impact of Persistent Measurable Residual Disease in Patients with Acute Myeloid Leukemia FLT3+ Treated with Intensive Chemotherapy Plus Midostaurin - a Retrospective Study of the Polish Adult Leukemia Group (PALG)

Background: Measurable residual disease (MRD) is detected during morphological remission after standard chemotherapy in acute myeloid leukemia (AML) patients (pts). It is a strong prognostic factor for relapse and shorter overall survival (OS). Immunophenotyping by multiparameter flow cytometry (MFC) is currently the most commonly used method in AML pts. There is still a lack of studies evaluating MRD in specific AML populations in the era of targeted therapies, including thyrosine kinase inhibitors. A retrospective study in adult pts with a newly diagnosed AML FLT3+ treated with intensive chemotherapy plus midostaurin has been performed to evaluate the prognostic impact of positive MRD (MRD+) on RFS and OS. Objectives: To assess the prognostic impact of MRD status during the clinical course of AML FLT3+ on RFS and OS. Patients and methods: Seventy three pts with AML FLT3 were treated in Polish hematological centers between 2018 and 2021. Bone marrow aspiration was performed after induction and consolidation treatment to assess response and MRD by MFC. MRD+ was defined as ≥ 0.1% abnormal cells within mononucleated cells by MFC with diagnostic leukemia-associated immunophenotype (LAIP) and different from normal (DfN) aberrant immunophenotype approaches. Results: The median age of the pts was 55 (range: 18-74). Induction regimen was DA "3+7" in 50 (70%), DA-90 in 8 (11%), DAC in 11 (15%), other in 2 (4%) pts. Fifty seven (78%) pts achieved first composite complete remission (CRc=CR+CRi) after 1 or 2 courses of induction, 12 (17%) had primary refractory disease (PRD) and 2 (3%) pts died in aplasia. Median number of consolidation courses was 2 (range: 1-4). Fifty one (89%) pts with CR1 fulfill the criteria for MRD analysis after 1 or 2 courses of induction and 34 (48%) pts after consolidations. Thirty four pts had 2 timepoints evaluation: after induction and at the end of consolidation courses. MRD+ was reported in 35 (68%) of pts after induction (median 0.5; IQR 0.25-0.85) and in 19 (52%) of pts at the end of consolidations (median 0.64; IQR 0.22-1.99). Analysis of 4 pts groups was performed: Group 1.: MRD+ after induction and at the end of consolidations (persistent MRD+)(n=16; 47%); Group 2.: MRD+ after induction with conversion to MRD- at the end of consolidations (n=6; 18%); Group 3.: MRD- after induction with conversion to MRD+ at the end of consolidations (n=2; 6%); Group 4.: MRD- after induction and at the end of consolidations (persistent MRD-)(n=10; 29%); Baseline clinical characteristics were similar between 4 groups regarding age, FLT3-ITD and FLT3-TKD frequency, FLT3-ITD allelic ratio (AR), WBC, PLT, peripheral blood and bone marrow blast percentage, AML type: de novovs sAML vs tAML (p-values > 0.05). The probability of 1- year RFS was significantly lower in pts with persistent MRD+ (group 1.) vs group 2. vs 3. vs 4. (27% vs 83% vs 50% vs 78%; p=0.034, respectively). Relapse rate after 1 year was in group 1. vs 2. vs 3. vs 4.: 73% vs 16% vs 50% vs 20% (p=0.015), respectively. The shorter OS, but insignificant, was noted in group 1. vs 2. vs 3. vs 4. (p=0.49). A univariate analysis revealed that allo-HSCT (hazard ratio [HR] 0.345; 95% confidence interval (CI), 0.152-0.784; p=0.011) and male sex (HR, 0.433; 95% CI, 0.217-0.861; p=0.017) are predictors for longer OS. MRD- at the end of consolidations (HR, 0.211; 95% CI, 0.066-0.669; p=0.008), persistent MRD- (HR, 0.252; 95% CI, 0.070-0.909; p=0.035), allo-HSCT (HR, 0.416; 95% CI, 0.183-0.947; p=0.0368) are predictors for longer RFS. However, older age (HR, 1.049; 95% CI, 1.018-1.081; p=0.0017) and higher WBC (HR, 1.005; 95% CI, 1.002-1.008; p=0.0022) are predictors for shorter RFS. Conclusions: We observed high rate of CR1 MRD+ AML FLT3 pts after induction (68%) and at the end of consolidations (52%). Significantly lower RFS and higher relapse rate, but not OS, were noted in pts with persistent MRD+ in comparison to pts in other MRD groups. Longer follow-up period and larger studies are necessary to confirm our preliminary findings.

AML-487 Prognostic Significance of Residual Disease Detected by Flow Cytometry in Acute Myeloid Leukemia With Normal Karyotype and Negative FLT3-ITD/ D835

Complete remission in patients with AML is still cytomorphological, representing a highly heterogeneous state with a wide range of tumor burden and diverse clinical findings. To evaluate prognostic significance of residual disease detected by flow cytometry in acute myeloid leukemia with normal cytogenetics and negative FLT3-ITD/D835. This is a prospective study, spanning a period of five years from January 2017 to December 2021. Our patients were followed for 25 months. The study is carried out in the context of hospitalized care for better management of LAM. We followed 54 adult patients diagnosed with AML according to the 2016 WHO criteria in the hematology and pediatric oncology department of the August 20 Hospital in Casablanca, of which 30 patients achieved complete cytological remission after induction. Any patient presenting a therapeutic failure in post induction and salvage therapy was excluded from the study. After that, we selected patients with normal karyotype for FLT3 gene mutational analysis. Quantification of minimal residual disease (MRD) and leukemic stem cells (LSC) using a six-color Navios type flow cytometer and the cxp acquisition and analysis software. A large panel of monoclonal antibodies was used to determine leukemia-associated immunophenotypes in most patients. Molecular analysis of FLT3-ITD/D835 was performed by PCR/RFLP (restriction fragment length polymorphism). The hypothesis of our study was that the group of patients with favorable and intermediate prognosis will have a relatively better relapse-free survival (RFS) than those with a poor prognosis. The intermediate risk group was predominant with a rate of 83%, of which 67% (20/30) had a normal karyotype. MRD and LSC positivity in this group was predominant with considerable decrease in hematogone count frequency as a marker of good prognosis. Non-mutated FLT3 gene in this group conferred a more improved relapse-free survival compared to that of the negative MRD in all our patients studied. MRD, LSC and are FLT3 gene analysis are powerful prognostic factors for relapse that should be integrated routinely to guide our clinicians in risk stratification, especially in patients with normal karyotype.

Impact on Outcome of Minimal Residual Disease after Hematopoietic Stem Cell Transplantation with Fludarabine, Amsacrine, and Cytosine Arabinoside-Busulfan Conditioning: A Retrospective Monocentric Study

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) after conditioning with a sequential association of fludarabine, amsacrine, and cytosine arabinoside (FLAMSA) followed by a reduced-intensity conditioning regimen has emerged for patients with high-risk acute myeloid leukemia (AML), especially in refractory or relapsing patients. Here we aimed to address retrospectively the impact of pretransplantation minimal residual disease (MRD) by flow cytometry on the outcomes of high-risk AML patients who underwent allo-HSCT after sequential FLAMSA-busulfan (FLAMSA-Bu)-based conditioning regimens. We included 165 high-risk AML patients who underwent transplantation after FLAMSA-BU in this retrospective single-center "real life" study. All patients received in vivo T cell depletion with antithymocyte globulin (5 mg/kg). MRD detection was based on a leukemia-associated immunophenotype using the European LeukemiaNet recommendations, with a threshold of .1%. Univariate and multivariate analyses were performed using R version 4.1.1 (R Foundation for Statistical Computing, Vienna, Austria). With a median follow-up of 4.0 years post-transplantation, the median overall survival (OS) was 54.9 months. Overall, 41 patients (24.8%) relapsed post-transplantation, with a resulting cumulative incidence of relapse (CIR) of 26.7% at 2 years and 34.0% at 5 years. Detectable MRD preceding allo-HSCT and refractory status were associated with worse median OS and CIR rates compared with patients without detectable MRD; however, OS was not significantly different between pre-HSCT MRD-positive and refractory patients (median, .7 year versus 2.0 years; P=.3). Conversely, pre-HSCT MRD negativity was associated with a reduced 2-year CIR. Neither European LeukemiaNet risk stratification nor age had a significant influence on OS. In the multivariate analysis, only pre-HSCT MRD positivity and lower conditioning regimen intensity were significantly associated with a poorer OS. The cumulative incidence of extensive chronic graft-versus-host disease at 2 years was 26.15%. The estimated nonrelapse mortality (NRM) of the entire cohort at 2 years was 23.1%, with age and unrelated donor identified as risk factors for higher NRM. Our data ahow that FLAMSA-Bu conditioning did not reverse the pejorative effect of detectable pre-HSCT MRD, suggesting that such patients should be offered alternative strategies before HSCT to reach deeper remission.

Identification of Leukemia-Associated Immunophenotypes by Databaseguided Flow Cytometry Provides a Highly Sensitive and Reproducible Strategy for the Study of Measurable Residual Disease in Acute Myeloblastic Leukemia.

Simple SummaryThe complete immunophenotypic characterization of acute myeloid leukemia is essential for an accurate diagnosis and follow-up, which is determinant in the course of the disease. In many cases, the only option for the evaluation of minimal residual disease is flow cytometry, so the aim of this study is to develop an automatized multidimensional strategy to identify and characterize LAIPs as well as to detect new emerging aberrances in AML patients during the follow-up. The integrated DFN/LAIP strategy that we propose allows the identification of the most useful markers for minimal residual disease monitoring, improving the sensitivity and specificity of these studies. Furthermore, the use of databases and the automation of the analysis provide the basis for the generation of objective conclusions in minimal residual disease evaluations.Background: Multiparametric Flow Cytometry (MFC) is an essential tool to study the involved cell lineages, the aberrant differentiation/maturation patterns and the expression of aberrant antigens in acute myeloid leukemia (AML). The characterization of leukemia-associated immunophenotypes (LAIPs) at the moment of diagnosis is critical to establish reproducible strategies for the study of measurable residual disease using MFC (MFC-MRD). Methods: In this study, we identify and characterize LAIPs by comparing the leukemic populations of 145 AML patients, using the EuroFlow AML/ MDS MFC panel, with six databases of normal myeloid progenitors (MPCs). Principal component analysis was used to identify and characterize the LAIPs, which were then used to generate individual profiles for MFC-MRD monitoring. Furthermore, we investigated the relationship between the expression patterns of LAIPs and the different subtypes of AML. The MFC-MRD study was performed by identifying residual AML populations that matched with the LAIPs at diagnosis. To further validate this approach, the presence of MRD was also assessed by qPCR (qPCR-MRD). Finally, we studied the association between MFC-MRD and progression-free survival (PFS). Results: The strategy used in this study allowed us to describe more than 300 different LAIPs and facilitated the association of specific phenotypes with certain subtypes of AML. The MFC-MRD monitoring based on LAIPs with good/strong specificity was applicable to virtually all patients and showed a good correlation with qPCR-MRD and PFS. Conclusions: The described methodology provides an objective method to identify and characterize LAIPs. Furthermore, it provides a theoretical basis to develop highly sensitive MFC-MRD strategies.

PB1814: RARE PHENOTYPE WITH CD33 ABSENT IN ACUTE MYELOID LEUKEMIA MIMICKING MRD POSITIVITY: A CASE REPORT

Background: Treatment of acute myeloid leukemia (AML) remains difficult to this day, with high rates of relapse that lead to poor long-term prognosis and low cure rates and are usually driven by residual leukemic cells that can be identified as measurable residual disease (MRD). Multiparametric Flow Cytometry (MFC) has been a fast and effective tool for detection of MRD, but disease heterogeneity makes MRD assessment a clinical challenge, especially in cases with rare phenotype, needing clinical and technical expertise to be accurately performed. Aims: To show a rare case of AML with absent CD33 expression and the importance of accurately interpreting MDR in AML. Methods: We report a clinical case of AML with absent CD33 assessed for FLT3 and NPM1 mutation, RUNX1/RUNX1T1 and CBFB-MYH11 rearrangements, karyotype and an 8-color panel immunophenotyping (Table 1). Empty gating strategy was used to identify LAIPs (Leukemia Associated-Immunophenotype), different from normal and leukemic stem cells, compared to healthy BM from diagnosis and follow up. Assays were performed at the Hematology Laboratory of Ribeirão Preto the Medical School, University of São Paulo, in accordance to Local Ethical Boards. Results: A 21-year-old male with AML without maturation, presented anemia (7.9 g/dL), thrombocytopenia (26 000/µL) and a total white blood cell count of 5710/μL with 84% of blasts in BM. MFC revealed CD45dim and low side scatter, CD117+, CD34+, HLA-DR+, CD38+, CD123+, CD11c+, CD64+, CD15+, CD133+, CD14-, CD7-, CD11b-, CD4-, CD56-, CD19-, CD41a-, NG2- and, unexpectedly, completely negative CD33. No metaphases or molecular abnormality was found. Treatment consisted of 2 induction cycles (cycle 1: 3 days of Daunorubicin 60 mg/m2 and 7 days of cytarabine 200 mg/m2; cycle 2: 6 days of cytarabine 1 g/m2 twice a day). Consolidation consisted of 1 or 2 cycles of 6 days of cytarabine 1 g/m2 twice a day. BM were obtained at diagnosis, after 1st and 2nd induction, and 3, 9, 12 and 15 months after consolidation of chemotherapy. A specific phenotype (CD117+/CD34+/CD33wk/CD13+ ≥ 0.1%) was preserved from diagnosis to all follow up points: 0,506%, 0,681%, 0,402%, 0,101%, 0,158% and 0,122% (Figure 1). Complete remission was achieved by day 30 after 1st induction and no change in the outcome was reported, even though an altered maturational phenotype persisted with complete absent CD33 expression, during and after treatment, suggesting a very rare polymorphism of CD33 receptor that mimicked MRD positivity. Image:Summary/Conclusion: Post-analytical phase of MFC is the most vulnerable to wrong interpretations. CD33 is a myeloid antigen expressed on malignant blasts in AML and had been reported as a potential target therapy. CD33low blasts are associated to a more mature AML and its high expression is related to adverse landscapes in AML, highlighting the importance of its evaluation. Standard care for AML enrolls MRD monitoring and failure to achieve an MRD-negative, CR or detected MRD during or after therapy is associated with relapse. Still, MRD analysis must embrace a set of standards in order to prevent post-analytic errors. Here we present a case report of AML with persistent absent CD33 expression since diagnosis that we found to be mimicking MRD positivity. Our data emphasizes the importance of clinical and technical expertise in MFC analysis and correlation of diagnostic and follow-up interpretation, especially in a heterogeneous disease such as AML, bringing up the need of standardization and constant training of the team.

Characterization of myelodysplastic syndromes hematopoietic stem and progenitor cells using mass cytometry.

Myelodysplastic syndromes (MDS) at risk of transformation to acute myeloid leukemia (AML) are difficult to identify. The bone marrows of MDS patients harbor specific hematopoietic stem and progenitor cell (HSPC) abnormalities that may be associated with sub-types and risk-groups. Leukemia-associated characteristics of such cells may identify MDS patients at risk of progression to AML and provide insight in the pathobiology of MDS. Bone marrow samples from healthy donors (n =10), low risk (n =12) and high risk (n =13) MDS patients were collected, in addition, AML samples for 5 out of 6 MDS patients that progressed. Mass cytometry was applied to assess expression of stem cell subset and leukemia-associated immunophenotype markers. We analyzed the data using FlowSOM to cluster cells with similar expression of 10 commonly used stem cell markers. Metaclusters (n =20) of these clusters represented populations of cells with a related phenotype, largely resembling known stem cell subsets. Within specific subsets, intra-cellular expression levels of pCREB, IkBα, or pS6 differed significantly between healthy bone marrow (HBM) and MDS or consecutive secondary AML samples. CD34, CD44, and CD49f expression was significantly increased in high risk MDS and AML-associated metaclusters. We identified MDS/sAML cells with aberrant phenotypes when compared to HBM. Such cells were observed in clusters of both primary MDS and secondary AML samples. High-dimensional mass cytometry and computational data analyses enabled characterization of HSPC subsets in MDS and identification of leukemia stem cell populations based on their immunophenotype. Stem cells in MDS that display leukemia-associated features may predict the risk of developing AML.

Identifying Leukemia-associated Immunophenotypes in Acute Myeloid Leukemia Patients Using Multiparameter Flow Cytometry.

ObjectivesWe sought to identify leukemia-associated immunophenotypes (LAIPs) in 50 acute myeloid leukemia (AML) patients at diagnosis using an eight-color multiparameter flow cytometry (MFC) panel and to detect if they showed any alteration in relapsed/refractory cases.MethodsWe used the eight-color MFC panel with CD45/side scatter log gating strategy to analyze LAIPs in 50 AML patients presenting to Alexandria University Hospitals, Egypt at diagnosis and relapse and refractory cases. Twenty age and sex matched bone marrow samples from patients performing bone marrow aspirate for non-malignant hematological indications were included as controls.ResultsLAIPs were observed in 43 (86.0%) cases. Only one aberrant immunophenotype was identified in four cases (9.3%), while two to 12 aberrant immunophenotypes were found in the other 39 (90.7%) cases. Strong LAIPs were obtained by combining CD2, CD4, CD56, with either CD34 or CD117, in contrast to CD19, which has to be combined with CD117. Refractory cases showed the presence of the same LAIPs at both initial diagnosis and persistent disease. One case showed the acquisition of new LAIPs after relapse.ConclusionsThe good choice of LAIPs depends on their specificity rather than their frequency. The results of this study can help in increasing the sensitivity of LAIPs strategy in minimal residual disease using MFC in AML patients, which is considered an important post-diagnosis parameter associated with prognosis and clinical outcome.

Highly Multiplexed Cell Surface Immunophenotyping with Genotyping and Concurrent Transcriptomic Analysis of NPM1 mutated Acute Myeloid Leukemia

BACKGROUNDThe pathogenesis of acute myeloid leukemia (AML) is often attributed to the presence of somatic allelic variant(s) in hematopoietic stem/progenitor cells. However, malignant clones may have heterogenous cell-surface immunophenotypes including overlap with non-malignant cells. While leukemia-associated immunophenotypes and difference from normal approaches are used for flow cytometric assessment during and after treatment, such analysis may underrepresent true leukemia disease burden. Assessments of AML measurable residual disease (MRD) using flow cytometry and molecular methods have been reported as discrepant. Single-cell RNA sequencing experiments have recently attempted to distinguish malignant cells based on gene expression and/or immunophenotypic profiles alone. We hypothesized that single-cell genotyping of mutated transcript(s) coupled with broad surface proteome and transcriptome profiling could provide an integrated multimodal method for AML characterization. METHODSWe adapted the previously reported “genotyping of transcriptomes” (PMID: 31270458) to identify cells carrying the NPM1 type A mutation commonly seen, and typically stable throughout the disease course, in AML. Healthy human peripheral blood mononuclear cells (PBMC) were mixed with an AML cell line carrying NPM1 type A mutation (OCI-AML3) at 7:3 ratio and labelled with 163 oligo-tagged antibodies. Single cell 3'v3 gene expression- (GEX), antibody derived tag- (ADT) and genotyping of NPM1 (GNPM) -libraries (10X Genomics) were sequenced on the NovaSeq 6000 (Illumina). Results were processed using Seurat 4.0 toolkit. RESULTSIn total, 72% (n=1680) of barcoded cells could be genotyped for NPM1. Of the genotyped cells, 59% (n = 986) were not NPM1 mutated. Visualization using Uniform Manifold Approximation and Projection (UMAP) showed separation of healthy PBMCs and OCI-AML3 cells using protein data, confirmed by annotation using NPM1 genotyping (Figure 1). We found a significant positive correlation between mRNA and corresponding cell surface protein expressions in non-mutated (Pearson's coefficient, r = 0.502, p = 6.87e-11) and NPM1 mutated (r= 0.392, p= 7.5e-7) cells. Compared to non-mutated, NPM1 mutated cells showed nearly 14-fold higher NPM1 transcript levels. In addition, a total 63 proteins were highly expressed on the surface of NPM1 mutated cells (Figure 2). Among these, CD33 and CD36 showed maximum 8-fold increase in expression. Other highly expressed proteins with at least >2.5-fold change were cell adhesion molecules (including CD328, CD155, and CD56), extracellular matrix binding proteins (CD49a/b) and interleukin receptor (CD123). CONCLUSIONOverall, our results demonstrate proof of principle that high-throughput cell surface proteome, transcriptome and genotyping analysis can be simultaneously performed to comprehensively and confidently characterize individual AML cells. Patient-specific multiomics data with broad cell-surface proteomic screening may allow novel target identification for monitoring and/or therapeutic intervention. Ongoing work will now use this methodology to characterize a cohort of NPM1 mutated AML patient samples. [Display omitted] DisclosuresHourigan: Sellas: Research Funding.

Blast cells surviving acute myeloid leukemia induction therapy are in cycle with a signature of FOXM1 activity

BackgroundDisease relapse remains common following treatment of acute myeloid leukemia (AML) and is due to chemoresistance of leukemia cells with disease repopulating potential. To date, attempts to define the characteristics of in vivo resistant blasts have focused on comparisons between leukemic cells at presentation and relapse. However, further treatment responses are often seen following relapse, suggesting that most blasts remain chemosensitive. We sought to characterise in vivo chemoresistant blasts by studying the transcriptional and genetic features of blasts from before and shortly after induction chemotherapy using paired samples from six patients with primary refractory AML.MethodsLeukemic blasts were isolated by fluorescence-activated cell sorting. Fluorescence in situ hybridization (FISH), targeted genetic sequencing and detailed immunophenotypic analysis were used to confirm that sorted cells were leukemic. Sorted blasts were subjected to RNA sequencing. Lentiviral vectors expressing short hairpin RNAs were used to assess the effect of FOXM1 knockdown on colony forming capacity, proliferative capacity and apoptosis in cell lines, primary AML cells and CD34+ cells from healthy donors.ResultsMolecular genetic analysis revealed early clonal selection occurring after induction chemotherapy. Immunophenotypic characterisation found leukemia-associated immunophenotypes in all cases that persisted following treatment. Despite the genetic heterogeneity of the leukemias studied, transcriptional analysis found concerted changes in gene expression in resistant blasts. Remarkably, the gene expression signature suggested that post-chemotherapy blasts were more proliferative than those at presentation. Resistant blasts also appeared less differentiated and expressed leukemia stem cell (LSC) maintenance genes. However, the proportion of immunophenotypically defined LSCs appeared to decrease following treatment, with implications for the targeting of these cells on the basis of cell surface antigen expression. The refractory gene signature was highly enriched with targets of the transcription factor FOXM1. shRNA knockdown experiments demonstrated that the viability of primary AML cells, but not normal CD34+ cells, depended on FOXM1 expression.ConclusionsWe found that chemorefractory blasts from leukemias with varied genetic backgrounds expressed a common transcriptional program. In contrast to the notion that LSC quiescence confers resistance to chemotherapy we find that refractory blasts are both actively proliferating and enriched with LSC maintenance genes. Using primary patient material from a relevant clinical context we also provide further support for the role of FOXM1 in chemotherapy resistance, proliferation and stem cell function in AML.

Prognostic significance of DEK-NUP214 fusion gene in patients with acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation

Objective: To investigate the dynamic change and clinical impact of DEK-NUP214 fusion gene in patients with acute myeloid leukemia (AML) receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: Real-time quantitative polymerase chain reaction (RQ-PCR) and multicolor flow cytometry (FCM) were used to detect DEK-NUP214 gene expression and leukemia-associated immunophenotype (LAIP) in 15 newly diagnosed patients with positive DEK-NUP214 and receiving allo-HSCT from September 2012 to September 2017 at Peking University People's Hospital. The clinical outcome was analyzed using Kaplan-Meier survival curves. The impact of DEK-NUP214 expression was analyzed by log-rank test. Results: The subjects were followed-up with a median period of 657 (62-2 212) days. The median DEK-NUP214 expression level at diagnosis was 488% (274%-1 692%). Thirteen patients achieved complete remission before allo-HSCT. Thirteen patients had a residual DEK-NUP214 expression of 0.38% (0.029%-738.9%) before allo-HSCT. After allo-HSCT, DEK-NUP214 expression in 9/13 patients remained positive, which dropped by around 500 folds (5.7-5 663.0 folds) within a month post-transplant. Five patients died and 2 patients relapsed. The 3-year cumulative incidence of relapse in patients with positive DEK-NUP214 before transplant was 17.5%±11.3% and the 3-year overall survival was 60.5%±13.8%. After allo-HSCT, DEK-NUP214-negative patients had a better outcome. Conclusion: Quantitative monitor of DEK-NUP214 fusion gene could be a sensitive indicator of MRD status after allo-HSCT.

ABSENT CD33 IN AML PATIENT MIMICS MRD POSITIVE: A SINGLE CASE REPORT

Diagnosis and treatment of acute myeloid leukemia (AML) remains difficult to this day, cure rates remain low and measurable residual disease (MRD) assessment is still challenging. Early detection of residual leukemic cells is essential to predict therapeutic success and Multiparametric Flow Cytometry (MFC) has been an effective tool for it. Here, we focus on the importance in caution when interpreting MDR in AML. Bone marrow (BM) sample was assessed to FLT3 and NPM1 mutation, RUNX1/RUNX1T1 and CBFB-MYH11 rearrangements, karyotype (KT) and immunophenotyping. Using an 8-colour antibody panel (CD45, CD34, CD117, CD 33, HLA-DR, CD38, CD123, CD13, CD19, CD7, CD11b, CD4, CD56, CD64, CD11c, CD15, CD133, CD41a and NG2), we applied a gating strategy with fixed gates comparing normal to AML BM, settled to catch LAIPs (Leukemia Associated-Immunophenotypes), abnormal cell maturation pattern and Leukemic Stem Cell, that were detected on diagnosis and evaluated for MRD. Assays were performed at the Hematology Laboratory of the Medical School of Ribeirão Preto, University of São Paulo, in accordance to Local Ethical Boards. A 21-year-old male patient with AML without maturation, diagnosed after 60 days of symptoms, presented anemia (7.9 g/dL), thrombocytopenia (26 000/μL) and white blood cell count of 5710/μL with 73% blasts in peripheral blood and 84% in BM. MFC revealed CD45dim and low side scatter; positive staining for CD117, CD34, HLA-DR, CD38, CD123, CD11c, CD64, CD15 and CD133; negative staining for CD33, CD14, CD7, CD11b, CD4, CD56, CD19, CD41a and NG2. We found no metaphases (KT), no molecular abnormality and he failed risk stratification. Treatment consisted of 2 induction cycles (cycle 1: 3 days of Daunorubicin 60 mg/m2 and 7 days of cytarabine 200 mg/m2; cycle 2: 6 days of cytarabine 1 g/m2 twice a day) and 1 or 2 consolidation cycles (6 days of cytarabine 1 g/m2 twice a day). BM was obtained at diagnosis, 30 and 33 days after 1 st and 2 nd induction, and 3, 9, 12 and 15 months after consolidation of chemotherapy. The positivity of a specific phenotype (CD117+/CD34+/CD33wk/CD13+ ≥ 0.1%) was preserved at diagnosis and follow-ups: 0,506%, 0,681%, 0,402%, 0,101%, 0,158% and 0,122%. Complete remission was achieved by day 30 after 1st induction and no change in the outcome was reported. Absent CD33 was observed in diagnosis and follow-ups, during and after treatment. Post-analytical phase of MFC is the most vulnerable to wrong interpretations. CD33 is a common myeloid antigen expressed on malignant blasts in AML and had been reported as a potential target therapy. CD33 low blasts are associated to a more mature AML and its high expression is related to adverse landscapes, highlighting the importance of CD33 evaluation. Standard care for AML enrolls MRD monitoring and failure to achieve an MRD-negative, CR or detected MRD during or after therapy is associated with relapse or poor outcomes. Still, MRD analysis must embrace a set of standards in order to prevent post-analytic errors. Here we present a case report of AML with absent CD33 maintained from diagnosis to MRD follow-ups, mimicking MRD positivity. This data emphasizes the importance of caution in MFC analysis and correlation of diagnostic and follow-ups results interpretation, as well as constant training of the team.

Molecular Monitoring of NPM1 Is a Useful Tool for Detecting Early-Stage Relapse and to Guide Pre-Emptive Treatment Approaches in Patients with NPM1-Mutated Acute Myeloid Leukemia

Molecular Monitoring of NPM1 Is a Useful Tool for Detecting Early-Stage Relapse and to Guide Pre-Emptive Treatment Approaches in Patients with NPM1-Mutated Acute Myeloid Leukemia

Minimal Residual Disease (MRD) Detection By Flow Cytometry during Aplasia and Post-Induction Is an Independent Prognostic Parameter for Relapse-Free Survival in AML

The identification of patients at high risk of relapse is crucial for treatment stratification in acute myeloid leukemia (AML). Persistence and reoccurrence of AML cells with aberrant phenotypes can be assessed by flow cytometry at high sensitivity in more than 95% of patients. Therefore, assessment by flow cytometry enables determination of MRD status in patients without suitable molecular markers (e.g. NPM1). Previously, we demonstrated that early detection of MRD during aplasia is associated with shorter relapse-free survival (RFS) after intensive induction chemotherapy (Köhnke T et al., Leukemia 2015). Here, we aimed to validate these findings in an independent patient cohort. In addition, we assessed the prognostic value of a combined MRD determination during aplasia and post induction for improved prediction of relapse-free survival (RFS).Flow cytometry MRD was analyzed in patients with AML (excluding APL) diagnosed between 2012 and 2016 receiving intensive induction chemotherapy (sHAM or 7+3). MRD flow analysis was performed using a NAVIOS flow cytometer. Presence ≥0.1% Leukemia-associated immunophenotype (LAIP)-positive cells (identified by comparison with bone marrow from healthy donors) during aplasia or post-induction defined MRD positivity. Kaplan-Meier estimators and log-rank tests were used to retrospectively analyze survival data. Cox's proportional hazards regression model was used to determine the influence of individual factors in multivariate analyses. Pearson's chi square test was used to compare categorical variables between MRD groups.A total of 105 patients were included. In 3 cases (2.9% of all cases), no LAIP could be identified, and these patients were excluded from further analyses. MRD assessment during aplasia was available in 87 cases. Regarding flow MRD assessment post induction, patients refractory to induction therapy were excluded. Therefore, 72 patients had flow MRD assessments available post induction therapy. 59 patients achieving CR or CRi after induction therapy had flow MRD assessments available at both time points.MRD positivity during aplasia was associated with refractory disease (36.2% vs. 10.0% for MRD pos. vs. MRD neg., p=0.017) and shorter median event-free survival (EFS, 5.8 vs. 15.7 months, p=0.011). Importantly, early blast clearance by morphology had no prognostic value if flow MRD assessment was available (HR for EFS 2.0, p=0.03 for flow MRD vs. HR 1.6 and p=0.17 for Blast clearance by morphology). However, differences in median RFS for MRDpos vs. MRDneg patients (10.3 vs. 20.5 months) for assessment during aplasia have not reached significance yet (p=0.135). MRDpos patients post induction had significantly shorter median EFS (10.8 vs. 15.7 months, p=0.032) and RFS (7.7 months vs. not reached, p=0.007) than MRDneg patients. Using a combination strategy for aplasia and post induction assessments, we grouped patients with MRD positivity at both timepoints (confirmed MRD positivity) vs. all others (MRDneg or MRDuncertain). Confirmed MRD positivity was associated with significantly shorter median EFS (6.7 vs. 15.7 months, p=0.003) as well as RFS (4.4 months vs. not reached, p=0.002). Confirmed MRD positivity was an independent predictor of EFS and RFS (HR 2.7 and 2.8, p=0.017 and p=0.018, respectively).Taken together, patients with confirmed two-time flow MRD positivity showed lower EFS and RFS compared to MRDneg or MRDuncertain patients, respectively. Identification of patients with poor treatment response was improved in comparison with cytomorphologic assessment or independent flow MRD assessments at both timepoints alone. Using this approach, early identification of patients with adverse prognosis is possible in >95% of AML patients. MRD flow will be prospectively evaluated as a therapy stratification tool in patients with AML receiving intensive induction chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Identification of Relapse Risk Using a Multiparameter Flow Cytometry-Based Detection of Minimal Residual Disease in Adult Patients with Acute Myeloid Leukemia at ELN Intermediate-Risk

Background: Minimal Residual Disease (MRD) measurement is a useful tool to refine prognosis in adult Acute Myeloid Leukemia (AML) patients in morphological complete remission (CR). Multiparametric flow cytometry (MFC) may provide independent prognostic information particularly in intermediate (Int)-I and Int-II ELN 2010 risk groups.Patients and methods: Analysis was performed on bone marrow (BM) aspirates of 120 ELN Int-I/II patients (pts) from 2 independent institutions, separately analyzed in two MFC labs. Treatment consisted of one-two idarubicin-based induction courses followed by High-dose-Ara-C consolidation and by allogeneic transplantation (NILG, n: 18/51; GIMEMA, n: 20/69). The median age was 49yr (18-73yr), F/M ratio was 49/71; de-novo and secondary AML cases were 94.2% and 5.8%, respectively. FLT3 gene was mutated in 41.7% of the patients (50/120), being wild type in 56.6% of the cases (68/120). FLT3-ITD was detected in 35.0% of the patients (42/120) and FLT3-D835 was detected in 7.5% of the patients (9/120). NPM1 gene was mutated in 31.6% of the patients (38/120) being wild type in 65.8% of the cases (79/120). ELN 2010 int risk groups were distributed as follows: 79.2% Int-I (n: 95) and 20.8% Int-II (n: 25). At diagnosis, a panel of 22 monoclonal antibodies, combined in four 8-color tubes, was employed to detect leukemia-associated immunophenotypes (LAIPs). MRD was measured after the first cycle (TP1) and after the second cycle (TP2). MRD was considered positive if residual leukemic cells were equal or higher than 0.1% and 0.035% at TP1, and than 0.035% at TP2, respectively. Relapse-free survival (RFS) and overall survival (OS) were analyzed using Kaplan-Meier curves and the log-rank test.Results: One-hundred-eighteen and 116 patients were tested at TP1 and TP2, respectively. At TP1, MRD-positivity was detected in 72.9% (86/118) at a threshold of 0.1% and 83.9% pts (99/118) at a threshold of 0.035%. Both thresholds were significant in predicting relapse (P=0.0002 and P=0.0158 for the 0.1% and the 0.035% threshold, respectively). We observed a shorter RFS in MRD-positive versus MRD-negative patients: median RFS was 10.8 months (0.1% threshold) and 11.3 months (0.035% threshold) in MRD-positive pts as compared to 28.5 months (0.1% threshold) and 19.5 months (0.035% threshold) in MRD-negative patients. Furthermore, MRD-positive pts presented with a significantly shorter OS as compared to MRD-negative patients (0.1% threshold, P=0.0185), with a median OS of 18.2 months as compared to 33.7 months for MRD-negative pts. When the 0.035% threshold was used, OS was not different between the two groups of pts. At TP2, MRD-positivity was detected in 75.8% of the pts (88/116) and significantly predicted relapse (P=0.0023, 0.035% threshold): the median RFS was 10.8 months in MRD-positive pts vs 26.3 months in MRD-negative. Moreover, we observed a significant improvement in terms of OS in MRD-negative vs MRD-positive pts (P=0.0411), the median OS being 37.0 and 18.2 months, respectively. Notably, the proportion of MRD-positive and MRD-negative patients did not differ in terms of age, sex, ELN risk classification, FLT3 mutational status, and number of allogeneic transplanted patients.Conclusions: Overall, our findings demonstrate a correlation between MRD levels by MFC and relapse risk in ELN-intermediate risk patients. The use of an allogeneic transplantation approach rather than other consolidation strategies in ELN-intermediate risk AML patients is often debated. MFC-based MRD potentially allows for a more appropriate and tailored post-remission therapeutic strategy in these AML patients. Furthermore, harmonization of MRD analysis between different labs and different protocols sharing a common technical approach seems to be feasible. Future prospective studies are warranted for prospective evaluation of MRD in ELN-intermediate AML patients. DisclosuresRossi:AbbVie: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

A cross-standardized flow cytometry platform to assess phenotypic stability in precursor B-cell acute lymphoblastic leukemia (B-ALL) xenografts.

With the continued poor outcome of relapsed acute lymphoblastic leukemia (ALL), new patient‐specific approaches for disease progression monitoring and therapeutic intervention are urgently needed. Patient‐derived xenografts (PDX) of primary ALL in immune‐deficient mice have become a powerful tool for studying leukemia biology and therapy response. In PDX mice, the immunophenotype of the patient's leukemia is commonly believed to be stably propagated. In patients, however, the surface marker expression profile of the leukemic population often displays poorly understood immunophenotypic shifts during chemotherapy and ALL progression. We therefore developed a translational flow cytometry platform to study whether the patient‐specific immunophenotype is faithfully recapitulated in PDX mice. To enable valid assessment of immunophenotypic stability and subpopulation complexity of the patient's leukemia after xenotransplantation, we comprehensively immunophenotyped diagnostic B‐ALL from children and their matched PDX using identical, clinically standardized flow protocols and instrument settings. This cross‐standardized approach ensured longitudinal stability and cross‐platform comparability of marker expression intensity at high phenotyping depth. This analysis revealed readily detectable changes to the patient leukemia‐associated immunophenotype (LAIP) after xenotransplantation. To further investigate the mechanism underlying these complex immunophenotypic shifts, we applied an integrated analytical approach that combined clinical phenotyping depth and high analytical sensitivity with unbiased high‐dimensional algorithm‐based analysis. This high‐resolution analysis revealed that xenotransplantation achieves patient‐specific propagation of phenotypically stable B‐ALL subpopulations and that the immunophenotypic shifts observed at the level of bulk leukemia were consistent with changes in underlying subpopulation abundance. By incorporating the immunophenotypic complexity of leukemic populations, this novel cross‐standardized analytical platform could greatly expand the utility of PDX for investigating ALL progression biology and assessing therapies directed at eliminating relapse‐driving leukemic subpopulations.

Label-Free Analyses of Minimal Residual Disease in ALL Using Deep Learning and Imaging Flow Cytometry

MD/MC and PR/JI contributed equallyMinimal residual disease is an important response biomarker for risk stratification in acute lymphoblastic leukaemia (ALL) and is assessed by two methodologies, molecular analyses of patient-specific, clonal antigen receptor rearrangements or flow cytometric analyses of aberrant leukaemia-associated immunophenotypes. Some clinical regimens use both methodologies and sequential samples of bone marrow and/or peripheral blood are assessed during, at the end of induction therapy and at time points beyond. Both MRD methods are highly specialised, laborious and for resource poor countries, are not affordable. In addition, a proportion of patients are not evaluable by either method.Technological advances in cytometry have led to imaging flow cytometers (IFC) which deliver all standard flow cytometric parameters, as well as a high quality image of each cell. We have previously shown that machine learning of IFC data can classify cell cycle phases within a cell, using only data from bright field (BF) and dark field (DF)1 and we hypothesised that this might be possible in the MRD setting. Thus, paired diagnostic and follow up bone marrow aspirates taken during remission induction from children with ALL (n=23, 6 at day 8 or day 15 or day 28 following the start of treatment) were stained with CD19 APC, CD10PE, CD34Texas Red, CD45APCH7 and DAPI and ran on an imaging flow cytometer (Amnis, Imagestream X Mark II) at x40 magnification. A standard sequential gating technique identified ALL cells and levels (range 90-0.01%) correlated with that assessed by traditional flow cytometry. Next, 200 morphological features were extracted from BF and DF images of cells including pixel intensities, size, shapes, textures and subcellular components and both supervised machine and deep learning algorithms were developed to correctly classify ALL cells in clinical samples. The classifiers were trained initially using all fluorescent and BF and DF parameters but then with a sequential leave-one-parameter-out. In parallel, iteration of the training-testing sets on 23 datasets, with a leave-one-sample-out approach was performed, to ensure prediction accuracy across samples. Correct cell classifiers peaked at 98.21% when all parameters were considered but even without antibody-conjugated signals, relying only in DAPI, DF and BF signals, correct cell classification averaged 85.36%. Finally, DF/BF in combination and BF alone still gave rise to correct cell call rates of 81.62% and 71.26% respectively.Thus, we report a new MRD detection method that is label free, using only the morphological features extracted from BF and DF cell images. The method is cheap, quick, and highly applicable and with a technology that could be simplified to be laser-free and allow point of care testing. Transferability of this unique methodological approach to other types of leukaemia is also feasible and may be further developed to give functional information e.g. cell cycle status.

Pre-Transplant Exhausted Marrow Infiltrating T Lymphocytes in MRD Negative Patients with Acute Leukemia Predict Early Relapse Following Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplant (alloSCT) is curative for patients with high-risk acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Despite this, relapse remains the leading cause of treatment failure. The use of flow cytometry to identify aberrant leukemia-associated immunophenotypes is an established method of detecting minimal residual disease (MRD). MRD status immediately prior to transplant remains one of the single best predictors of post-transplant outcome. Unfortunately, not all MRD(-) patients maintain remission post-transplant. Therefore, we sought to find additional predictors of post-transplant relapse. T cell exhaustion occurs following continuous exposure to antigen and leads to sustained up-regulation of multiple inhibitory receptors, progressive loss of function, and expression of a distinct transcriptional state. Bachireddy et al recently reported that exhausted CD8+ marrow infiltrating lymphocytes (MILs) were a novel predictor of response to donor lymphocyte infusion (DLI) for patients with relapsed chronic myelogenous leukemia (CML). Based on this principle, we hypothesized that exhausted CD8+MILs may also be predictive of outcome for MRD(-) patients prior to alloSCT.We matched pre-transplant samples from 17 MRD(-) patients who maintained remission for greater than 2 years post-transplant to 11 MRD(-) patients who suffered relapse within 2 years of transplant. Patients had either ALL or AML and did not differ by demographic details. MILs were evaluated for exhaustion using 2 multicolor flow cytometry panels which collectively included a viability dye, T cell markers (CD3 and CD8) surface inhibitory receptors (PD-1, Tim-3, and TIGIT), transcription factors (T-bet and Eomes), and intracellular cytokine staining (IL-2, TNF-α, and IFN-γ) in response to CD3/CD28 bead stimulation.There was no difference in the percentage of total T cells (CD3), the CD8+ T cells (CD3+CD8+), or CD8+ T cell subsets between the 2 groups. While there was also no difference in the mean fluorescent intensity (MFI) of Tim-3, both PD-1 and TIGIT were significantly elevated on the CD8+ MILs of MRD(-) patients that eventually relapsed (p=0.01 and p=0.03, respectively). Among CD8+ MILs, cells with the highest PD-1 expression (PD-1High) were significantly elevated in the relapse group (p=0.006), as was co-expression of multiple inhibitory receptors on single cells (p=0.01) (Figure A,B). In contrast, the proportion of CD8+ MILs lacking all 3 of these inhibitory receptors was significantly higher among the MRD(-) group that maintained remission (p=0.01) (Figure C). Interestingly, CD8+ MILs in the remission group with dual PD-1+TIGIT+ expression or expressing PD-1High were more likely to be central memory cells (both p=0.05), while these same immunophenotypes were significantly more often effector memory cells in patients who went on to relapse (p=0.03 and p=0.02, respectively).As noted above, inhibitory receptor expression alone does not define exhaustion. We further assessed the CD8+ inhibitory receptor expressing MILs in the relapse group for other phenotypic evidence of exhaustion by evaluating their function and transcription factor expression. Cells with either PD-1+TIGIT+ dual expression or PD-1High were directly compared to cells that lacked inhibitory receptors. The dual PD-1+TIGIT+ cells had impaired ability to produce TNF-α and IFN-γ (both with p<0.0001), while PD-1High cells produced significantly less TNF-α (p<0.0001), IFN-γ (P<0.0001) and IL-2 (p=0.0001). Moreover, both sets of MILs had significantly decreased polyfunctional cytokine production compared to cells without these surface receptors (p < 0.0001) (Figure D). Furthermore, transcription factor expression was also altered among inhibitory receptor expressing cells. They exhibited both increased early exhaustion as evidenced by high T-bet expression in PD-1 expressing cells as well as terminal exhaustion indicated by significantly elevated Eomes expression on PD-1High expressing cells (Figure E,F).Collectively, this study suggests that the presence of exhaustion in CD8+ MILs in acute leukemia patients who are MRD(-) prior to transplant may be at higher risk for post-transplant relapse. If validated, this data may be used to further risk-stratify patients and/or direct pre- or post-transplant therapy, including the potential use of checkpoint blockade. [Display omitted] DisclosuresBachanova:Seattle-Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oxis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Zymogen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees. Wagner:Novartis: Research Funding; Magenta Therapeutics: Research Funding. Miller:Oxis Biotech: Consultancy; Fate Therapeutics: Consultancy, Research Funding; Celegene: Consultancy.

Prognostic Value of Minimal Residual Disease in Children with Acute Lymphoblastic Leukemia.

Purpose To assess the prognostic value of determination of minimal residual disease in the bone marrow after multiagent remission induction therapy in children with acute lymphoblastic leukemia. Methods From September 2001 to December 2004, 102 patients with newly diagnosed B-cell precursor acute lymphoblastic leukemia were enrolled on protocol ALL-XH-99. Minimal residual disease in the bone marrow at the end of remission induction therapy, before high-dose methotrexate, early intensification as well as 1 year, 2 year of maintains therapy was detected by multi-parameter flow cytometry with various combinations of monoclonal antibodies for leukemia-associated immunophenotype. The probability of event-free survival was estimated by Kaplan-Meier analysis and the distributions of probability of event-free survival (pEFS) were compared using the log-rank test. Chi-square analysis or Fisher exact test were used to compare differences in the distribution of biologic presenting features. A Cox proportional hazards model was used to identify independent prognostic factors. Results [circ1] The probability of 39-month event-free survival (EFS) was significantly worse for patients who achieved a minimal residual level ≥ 0.01% in the bone marrow on the remission date as compared to that of other patients with a minimal residual level < 0.01% (0.00% vs 83.00±9.90%, P=0.0000). [circ2]Univariate analysis indicated that there were no relationship between level of minimal residual disease at the date of complete remission and biologic presenting features (gender, age, white blood cells), but did Philadelphia chromosome, ALL-XH-99 risk groups as well as lymphoblasts in the bone marrow on day 19 (P<0.05). [circ3] Multivariate analysis suggests patients with high level minimal residual disease after the first induction course was an independent prognostic factor (hazard ratio, 5.381; 95% confidence interval 0.004 to 0.624; P=0.020). Conclusions Early treatment response as assessed by minimal residual leukemia determination by flow cytometry has important prognostic significance and can be performed in a resource-poor patient population. Patients with level of minimal residual disease ≥ 0.01% at end of remission induction ought to be used an novel and more intentive chemotherapy.

Measurable residual disease response in acute myeloid leukemia treated with venetoclax and azacitidine.

7018 Background: In the phase 3 VIALE-A trial, rates of composite complete remission (CRc; complete remission [CR] + CR with incomplete hematologic recovery [CRi]) and measurable residual disease response (MRD&lt;10-3) were higher in patients (pts) treated with venetoclax (Ven) + azacitidine (Aza) compared to Aza alone (23.4%/7.6%, p&lt;0.001). There is limited evidence of the clinical significance of MRD monitoring in pts receiving low-intensity chemotherapy. Herein, we explored the outcomes of pts treated with Ven+Aza who achieved both CRc and MRD&lt;10-3 in the VIALE-A trial (NCT02993523). Methods: Enrolled pts were ≥18 years and unfit for intensive chemotherapy. Pts received Ven 400 mg orally; days 1–28 and Aza 75 mg/m2; days 1-7/28-day cycle. Bone marrow aspirate samples for multiparametric flow cytometry assessments by integrated leukemia-associated immunophenotypes and different than normal procedures were collected for central analysis (Covance Central Laboratory Services) at baseline, end of cycle 1, and every 3 cycles thereafter. Assessments were performed independent of disease responses. MRD response was defined as &lt;1 residual blast /1000 leukocytes (&lt;10-3). CRc, DoR, OS, and EFS were assessed. Disease assessments were per modified International Working Group response criteria for AML. Results: 211/286 (74%) pts treated with Ven+Aza with at least one valid post-baseline MRD assessment were considered MRD evaluable; 78/211 (37%) achieved MRD&lt;10-3 and 133/211 (63%) had MRD≥10-3. Median age (MRD&lt;10-3/ MRD≥10-3) was 76 (range: 49-89)/77 (58-91) yrs. Pts (MRD&lt;10-3/ MRD≥10-3) received median of 14.5 (range: 1-28) /7.0 (1-30) cycles of Ven+Aza. At median follow-up of 22.0 (range: 20.1-23.0)/20.8 (19.8-22.3) months (mos), CRc + MRD&lt;10-3/ MRD≥10-3 was achieved by 67 (86%)/ 97 (73%); 20/67 (30%) achieved CRc + MRD&lt;10-3 by end of cycle 1. Median DoR, OS, and EFS were not reached in pts with CRc + MRD&lt;10-3 response (Table). The 12-mo estimates for DoR, OS, and EFS for pts with CRc + MRD&lt;10-3response were 81.2%, 94.0%, and 83.2%, respectively. Adverse events ≥grade 3 (MRD&lt;10-3/ MRD≥10-3) were febrile neutropenia (50%/43%), neutropenia (50%/35%), and thrombocytopenia (44%/44%), similar to the overall population. Conclusions: Pts with best response of CRc who achieved MRD&lt;10-3 response with Ven+Aza treatment had longer DoR, OS, and EFS than pts who were CRc and MRD positive. Clinical trial information: NCT02993523. [Table: see text]

Log reduction of leukemic cells and minimal residual disease by flow cytometry represent effective predictors of clinical outcome in elderly patients with acute myeloid leukemia.

Nowadays minimal residual disease (MRD) and log-reduction of leukemic cells are poorly investigated in elderly patients with acute myeloid leukemia (AML) treated with hypometilating agents (HMAs). Studies focusing on MRD in elderly AML patients who received HMAs are scant and devoid of rigorous criteria for both enrollment and monitoring. Log-reduction has never been investigated in these patients. Thus, the purpose of our study was to compare the prognostic impact of MRD and log-reduction of leukemic cells at the optimal time of assessment in older AML patients. Elderly patients who completed at least six cycles of HMAs and showed suitable leukemia-associated immunophenotypes (LAIPs) for the MRD and log-reduction assessment by flow cytometry were enrolled in the study. After comparing the times of assessment C4 (4-cycles) and C6 (6-cycles), C6 has been chosen as optimal. Patients who achieved MRD negativity or 2-log-reduction of leukemic cells at C6 had a significantly longer DFS. Particularly, results of 2-log-reduction were confirmed a multivariate analysis. Patients with MRD negativity or 2-log reduction of leukemic cells showed an improvement of their OS, although not significantly. Our data confirmed the predictive role of MRD and 2-log reduction also in older AML patients treated with HMAs.