ALBERT

Description: Key Points Connective tissue growth factor regulates adipogenic differentiation of MSCs. Connective tissue growth factor regulates leukemia engraftment.

Description: Notch1-mutated T-ALL is an aggressive hematologic malignancy lacking targeted therapeutic options. Genomic alterations in Notch1-gene and its activated downstream pathways are associated with metabolic stress response and heightened glutamine (Gln) utilization to fuel oxidative phosphorylation (OxPhos) (Kishton at al., Cell Metabolism 2016, 23:649, Herranz at al., Nat Med, 2015, 21(10): 1182-1189). Hence, targeting NOTCH1-associated OxPhos and/or Gln dependency could constitute a plausible therapeutic strategy for T-ALL. In this study we examined metabolic vulnerabilities of NOTCH1-driven T-ALL and tested pre-clinical efficacy of novel mitochondrial complex I (OxPhosi) IACS-010759 and of glutaminase inhibitor CB-839 (GLSi) in T-ALL models including Notch1-mutated T-ALL cell lines, patient-derived xenograft (PDX) and primary T-ALL cells. We have previously reported and confirmed in this expanded study the anti-leukemia efficacy of IACS-010759 (EC50s 0.1-15 nM) (Molina at al., Nat Med, 2018, 24: 1036; Baran at al., Blood, 2018, 132:4020). Metabolic characterization demonstrated that OxPhosi caused striking dose-dependent decrease in basal and maximal oxygen consumption rate (OCR), ATP and NADH generation in T-ALL cell lines and primary T-ALL samples (p

Description: The main therapeutic challenge in the treatment of Acute Lymphocytic Leukemia (ALL) is the development of strategies aimed at overcoming resistance to chemotherapy. Interactions between leukemia cells and microenvironment promote leukemia cell survival and confer resistance to drugs commonly used to treat ALL. Recent reports indicate that the endosteum at the murine bone-bone marrow (BM) interface is hypoxic, and data in a rat model demonstrate that leukemic cells infiltrating bone marrow were markedly hypoxic compared to cells in bone marrow of healthy rats. Hypoxia-inducible factor 1α (HIF-1α) is a key regulator of the cellular response to hypoxia. To characterize expression and function of HIF-1α in the bone marrow from ALL patients, HIF-1α expression was analyzed by immunohistochemistry in the bone marrow specimens from 16 newly diagnosed patients with pre-B ALL. HIF-1α was found to be expressed in 10/16 samples tested (62.5%). Of the 16 patients, 5 patients subsequently relapsed, all of which have expressed HIF-1α at diagnosis. No relapses were seen in the 6 patients with negative HIF-1α levels at presentation. To examine the molecular mechanisms of survival of leukemic cells growing under hypoxic conditions of bone marrow microenvironment, we established a co-culture system of pre-B ALL cells with BM-derived mesenchymal stem cells (MSC). Culture of REH cells under hypoxia (1% O2) resulted in induction of HIF-1α protein which was further increased in leukemia/stroma co-culture. Exposure of cells to hypoxia resulted in robust activation of AKT phosphorylation in leukemic cells. We have recently demonstrated that rapamycin analogs inhibit AKT signaling in AML cells via inhibition of mTORC2 formation (Zeng et al., Blood 109:3509-12, 2007). Likewise, mTOR inhibition by RAD001 completely blocked HIF-1α and pAKT in REH cells. Importantly, REH cells co-cultured with MSC under hypoxia/high glucose environment exhibited significantly lower apoptotic rates (p=0.02) and growth inhibition (p=0.002) in response to vincristine, and these effects were reversed by mTOR blockade with RAD001. We have further demonstrated that inhibition of mTOR signaling reduced expression of the glucose transporter Glut-1 and diminished glucose flux, decreased glycolytic rate and ATP production, both in leukemic cell lines and in primary ALL blasts (n=8). This was associated with decreased mitochondrial membrane potential and inhibition of the hypoxia-induced hexokinase (HKII) in the mitochondrial fraction of ALL cells. In summary, data suggest that mTOR/AKT signaling critically controls HIF-1α expression and function in ALL cells studied under the hypoxic conditions characteristic of bone marrow microenvironment. Hence, mTOR inhibition or blockade of HIF-1α-mediated pro-survival signaling events may reverse microenvironment-mediated chemoresistance and improve clinical outcomes in ALL.

Description: The bone marrow microenvironment provides functional and structural support for both normal and leukemic hematopoietic stem cells. Importantly, the marrow microenvironment is known to be hypoxic. Given the many critical functions of HIF-1α, we investigated HIF-1α levels in leukemic cell lines and primary AML blasts. Surprisingly, HIF-1α expression was non-detectable in AML cells grown in suspension cultures under normoxic (21% O2) or hypoxic (1% O2) conditions. Normoxic co-cultures with bone marrow-derived mesenchymal stromal (MSC) or MS5 cells likewise did not induce HIF-1α, but hypoxic co-culture conditions induced HIF-1α protein without changes in HIF-1α mRNA levels, suggesting post-transcriptional regulation. Functionality of HIF-1α was confirmed by concomitant increase in the levels of glucose transporter glut-1, the HIF-1α downstream target. Inhibition of stroma-leukemia cell interactions with the small molecule CXCR4 inhibitor AMD3465 (Genzyme/Anormed) at 100 nM completely abrogated the induction of HIF-1α in HL-60 and MOLM13 AML cells. While SDF-1 was unable to induced HIF-1α under normoxic conditions, it did so under both physical (1% O2) and chemical (COCl2) hypoxic conditions, in two different cell lines. Inhibition of P13K (with LY294002) or MEK/ERK signaling (with CI-1040) abrogated HIF-1α induction under hypoxic conditions. Immunohistochemical staining of bone marrow samples from primary AML confirmed the presence of HIF-1α in leukemic cells localized adjacent to bone-lining stromal elements. Results suggest that the bone marrow microenvironment of AML is hypoxic in vivo; in leukemia cells HIF-1α induction under low oxygen tension depends on the presence of stromal cells; HIF-1α induction is dependent on SDF-1/CXCR4 and is mediated by activation of P13K and MAPK signaling. Altogether these findings suggest that SDF-1α/CXCR4 interactions contribute to the survival of leukemic cells via specific induction of HIF-1α signaling by the bone marrow microenvironment. Disruption of these interactions via CXCR4 inhibition strategies may suppress multiple pro-survival HIF-1α targets in leukemic cells.

Description: Abstract 3311 Bcl-2 family proteins are key regulators of apoptosis. Aberrations in Bcl2 levels are known to promote tumorigenesis and chemoresistance. Thus strategies to target Bcl2 will likely provide effective therapies for malignancies such as acute myeloid leukemia (AML). ABT-737 is a small molecule BH3 mimetic that binds tightly to a hydrophobic cleft on Bcl-2 and Bcl-XL and exerts its proapoptotic function by preventing antiapoptotic Bcl-2 family members from sequestering activating BH3 proteins (Oltersdorf et al., Nature 2005). We have reported that ABT-737 effectively kills acute myeloid leukemia blast, progenitor, and stem cells without affecting normal hematopoietic cells. ABT-737 induces the disruption of the BCL-2/BAX complex and BAK-dependent but BIM-independent activation of the intrinsic apoptotic pathway (Konopleva et al., Cancer Cell 2006). The ABT-737 related clinical compound, ABT-263, is undergoing Phase I/II studies in chronic lymphocytic leukemia, with initial signs of clinical activity. However, the main side effect is thrombocytopenia resulting from inhibition of Bcl-XL. Hence, combinations of ABT-737 with non-cytotoxic agents are desirable to take full advantage of ABT236's unique spectrum of biophysical and preclinical activities. In this project, we studied pharmacologic interactions between ABT-737 and the DNA methyltransferase inhibitor 5-azacytidine (5-azaC). 5-azaC is a cytidine analog with clinical activity in myelodysplastic syndromes (MDS) and in AML. Since recent studies indicate that 5-azaC induce DNA damage in p53-dependent fashion, we tested the hypothesis that the pro-apoptotic effects of 5-azaC/ABT-737 combination are related to non-redundant activation of BH3-only proteins and mitochondrial apoptosis in AML cells with wt p53. In vitro, 5-azaC and ABT-737 in combination for 72 hrs induced growth inhibition and apoptosis in AML cell lines OCI-AML3, MOLM-13 and U937 in a highly synergistic, dose-dependent fashion, with combination indices (CIs) ranging from 0.1 to 0.22. These effects were observed even at low concentrations (5-azaC 100nM and ABT-737 10nM, at 10:1 ratio). In contrast, no synergistic apoptosis was seen in p53-null HL-60 cells. Likewise, ABT-737/5-azaC induced apoptosis in a synergistic fashion in OCI-AML3 cells infected with vector control shRNA (CI=0.04) but failed to induce cell death in OCI-AML3 p53 shRNA cells, indicating critical p53-dependent mechanisms of cell death. In primary AML samples sensitive to ABT-737 alone (n=3), synergistic and additive effects were seen. The combined effects of 5-azaC and ABT-737 were further investigated in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice injected with cells from a patient with primary refractory AML. Seven days after leukemia transplantation, mice were treated with vehicle, ABT-737 (intraperitoneally (IP), 75mg/kg/day for 10 days), 5-azacytidine (IP, 4mg/kg/day for 5 days) or with the combination. Engraftment of patient leukemia cells was analyzed by the immunohistochemical detection of CD45-positive cells in bone marrow and spleen seven weeks after transplantation. Both, ABT-737 and 5azaC each exerted anti-leukemic effects, as evidenced by significant reduction in leukemia cells in bone marrow and spleen (10% and 3% CD45+ cells detectable in 1/3 mice in ABT-737 and 5-azaC groups, respectively), no CD45+ AML cells were detected in organs of 3/3 mice treated with the combination. No overt hemorrhage was detected in the animals. In summary, the combination of 5-azaC and ABT-737 induces synergistic cells death in AML cell lines and in a subset of primary AML samples in a p53-dependent fashion. The mechanisms of this pharmacologic interactions including the p53-dependent upregulation of BH3-only proteins, described by us for ABT-737/MDM2 inhibitor combinations, are currently under investigation. Results suggest that this therapeutic strategy can be successfully utilized in AML patients with low mutation rate and unimpaired signaling of p53. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2040 Poster Board II-17 The main challenge in the treatment of acute lymphocytic leukemia (ALL) is overcoming resistance to chemotherapy. Recent studies indicate that interactions between leukemia cells and bone marrow (BM) microenvironment promote leukemia cell survival and confer resistance to drugs commonly used to treat ALL. We investigated whether hypoxia was a contributing factor in the protective role of the BM microenvironment. We found that the Hypoxia-Inducible-Factor 1a (HIF-1a; a marker normally expressed by only a few hematopoietic cells) was expressed in 68% of BM biopsies from patients with B-lineage ALL (n=53). Expression of HIF-1α detected either histochemically (n=53, p=0.023) or by Reverse Phase Protein Arrays (RPPA, n=116, p=0.0013) inversely correlated with survival of patients with newly diagnosed B-lineage ALL. Silencing of HIF-1α with siRNA, or blockade of mTOR signaling with rapamycin derivatives, reduced expression of the glucose transporter Glut-1, diminished glucose flux, decreased glycolytic rate and ATP production and sensitized leukemic cells to the pro-apoptotic effects of chemotherapeutic agents under hypoxic conditions. In line with this findings, we observed a marked expansion of hypoxic BM areas in immunodeficient mice engrafted with the ALL cell line Nalm6 or with primary ALL cells, as detected by administration of the reductive 2-nitroimidazole compound pimonidazole (PIM), which forms stable adducts in hypoxic regions. Altogether, these findings provided a rationale for examining the effects of hypoxia-activated pro-drugs or HIF-1a inhibitors to eliminate ALL progenitor cells within hypoxic niches. To this end, we tested PR-104, a hypoxia-activated dinitrobenzamide mustard currently undergoing Phase II trials in solid tumors. Under hypoxic conditions, this agent is reduced to hydroxylamine and amine metabolites that in turn induce DNA cross-links and cell death (Patterson et al., Clin Can Res 2007). In vitro, 25μM PR-104 induced hypoxia-selective cell death in Nalm6 ALL cells with 80% Annexin V-positivity at 0.1% O2, 46% at 1%O2 compared to 13% at 21%O2. The anti-leukemic efficacy of PR-104 was next examined in the in vivo leukemia models. Administration of PR-104 (250 mg/kg IP TIW for two weeks) prolonged survival of NOD/Scid/IL2Rg-KO (NOG) mice injected with cells from primary refractory FLT3-mutated AML, and decreased leukemia burden as indicated by histopathological analyses of CD45 positive cells in the BM, spleen, lung and liver. Notably, analysis of PIM distribution indicated clearance of the hypoxic leukemic niches. In NOG mice injected with leukemic cells from an infant with MLL-rearranged B-lineage ALL, PR-104 at 200 mg/kg IP on days 1, 2 and 6 resulted in a dramatic decrease in the percentage of circulating leukemic CD45+ cells on day 15 (control, 92%±6%; treated, 9%±4%; n=7 mice/group). The therapeutic effect of the drug was also tested in a Nalm6-luciferase ALL model where PR-104 administration resulted in decreased tumor burden as determined by luciferase activity and prolonged survival of the PR-104 treated as compared to control mice (p=0.006). Similar to the models of human leukemia, analysis of BM sections of control mice showed extensive areas of hypoxia (PIM-positive) in close proximity to GFP-positive leukemia cells in contrast to the treated mice in which only discrete areas of PIM positivity were detectable. Altogether, these findings strongly suggest that targeting hypoxia is feasible and may increase the sensitivity of ALL cells to chemotherapy. If successful, this approach of targeting hypoxic microenvironment, alone or in combination with other chemotherapeutic or targeted agents, may significantly impact ALL therapy and ultimately improve patient survival. Figure 1. Co-localization of hypoxic PIM(+) areas with GFP positive HALMG tumor areas in bone marrow of control but not of PR-104 treated mice. Figure 1. Co-localization of hypoxic PIM(+) areas with GFP positive HALMG tumor areas in bone marrow of control but not of PR-104 treated mice. Disclosures: No relevant conflicts of interest to declare.

Description: Adult T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy characterized by limited therapeutic options and a high rate of treatment failure due to chemoresistance. T-ALL is largely driven by activating NOTCH1 mutations, where oncogenic NOTCH1 facilitates glutamine oxidation, induces metabolic stress, and facilitates reliance on oxidative phosphorylation (OXPHOS)1. In other malignancies, the shift toward OXPHOS-dependent high-energy status is associated with acquired chemoresistance. In this study, we found that the novel inhibitor of mitochondrial complex I (OXPHOSi) IACS-0107592 has preclinical activity in NOTCH1-mutated T-ALL; we also characterize the cellular and metabolic responses to OXPHOS inhibition and propose that an OXPHOSi be incorporated into standard-of-care therapy to improve outcomes in patients harboring NOTCH1-mutated T-ALL. Exposure to IACS-010759 (0-370 nM) in vitro drastically reduced T-ALL viability, with EC50 ranging from 0.1-10 nM for cell lines (n=7) and from 13-60 nM for patient-derived xenograft (PDX)-derived and primary T-ALL cells (n=10) (Fig.1). Oral administration of IACS-010759 (7.5 mg/kg/day) significantly reduced leukemia burden and extended overall survival (p

Description: Background: Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) is a rare, distinct myeloid malignancy with historically poor survival, a high transformation rate to acute myeloid leukemia (AML), and no standard of care therapeutic approach. It is characterized by the overexpression of CD123+/IL3Rα, BCL-2 overexpression, and splicing factor abnormalities. BPDCN most commonly affects: skin, bone marrow (BM), and lymph node (LN). We sought to determine the outcomes of pts with BPDCN with focus on compartment of disease [(skin vs BM) at presentation. Patient Characteristics: We included in this analysis all pts age ≥ 18 years with a confirmed diagnosis of BPDCN treated at our institution. During October 1998-July 2018, a total of 70 pts were identified. At presentation to our institution: n=46 untreated; n=24 had prior therapy (median number therapies = 1 [1-6]. 61 (87%) were male, in line with historical expectation for BPDCN. Median age was 64 years [range 20-86 years]. BM was involved in 46 (66%), skin-only in 23 (33%), LN-only 1(1%). In addition to CD4+ and CD56+, tumor immune-phenotype demonstrated: TCL-1+ (44/44) and CD123+ (42/42). Conventional cytogenetics among 57 pts with available data showed: diploid in 40 (57%), complex in 13 (19%), deletion (12p13) in 2 (3%), miscellaneous in 2 (3%). Median BM blast count was 14% [0-95]. Historically, as there has been no standard of care, a variety of frontline chemotherapy regimens have been administered over time: HCVAD (n=24); clinical trial/targeted therapy (n=23); other/miscellaneous regimens (n=10); CHOP (n=7); hypomethylator (n=2); bortezomib-based (n=2); AML-based induction (n=2). Results, overall cohort (n=70): The median follow-up time in our series has now exceeded the one-year mark: 16.8 months [0.6 - 53.8 mo]. First complete remission (CR1) (by standard AML criteria) was achieved in 43 pts (61%); median CR1 duration: 6.7 mo [1.5-54.3 mo]. Median overall survival (OS) was 21.7 mo [0.6-158.0 mo]. 45 (64%) pts died, with the most common cause of death being multi-organ failure. Skin-only pts with BPDCN: Among 24 (34%) pts without BM involvement at diagnosis, 23 had skin-only involvement (1 pt with LN-only involvement at presentation). Comparison of pts with BM involvement versus those with skin-only demonstrated no statistically significant differences in outcomes: for pts with BM disease, the median OS, and median CR1 duration were 22.8 mo [0.6-65.8 mo], 6.0 mo [1.8-54.3 mo], respectively. For pts with skin-only disease, the median OS and median CR1 duration were 13.4 mo [2.4-158.0+ mo], 7.6 mo [1.5-21.4 mo], respectively, p =0.7 (OS), p =0.7 (CR1) [Figure] Next-generation Sequencing: A molecular gene panel has been performed prospectively in the BM specimens as part of standard of care in n=34; 29 (85%) have expressed some form of TET2 abnormality (standard mutation=14, variants=11, standard + variant=4). There was no statistically significant difference in terms of response rates in pts with known TET2 mutations/variants vs all others/not done. The second most common mutation was ASXL1 (n=17), followed by RAS mutations (n=9) (no statistically significant differences in OS or CRD for pts with ASXL1 or RAS vs others). Unexpectedly, there were pts with BPDCN with skin-only involvement who were noted to have occurrence of molecular mutations in the BM on their baseline gene panel; among these skin-only pts with material available for mutational analysis, the most common molecular abnormality was TET2 abnormality in the vast majority: 10/11 (91%). Conclusions: Even with longer-term follow-up, and despite various on- and off-protocol approaches implemented over the years for pts with BPDCN, it is notable that in this large series of pts treated at our institution, that the overall poor outcomes in this rare disease persist even in pts with skin-only disease (no marrow involvement at baseline), and that many of these skin-only pts harbor a genetic mutation, most commonly TET2 abnormality, even in the absence of marrow disease. These findings highlight the stem cell/clonal disease origins of BPDCN, and emphasize the aggressive nature of this hematologic malignancy even in the absence of overt marrow disease at diagnosis, highlighting the continued need for the development of novel targeted therapy approaches for pts with BPDCN including agents directed at CD123, BCL-2, and splicing pathways. Figure. Figure. Disclosures Pemmaraju: Affymetrix: Research Funding; stemline: Consultancy, Honoraria, Research Funding; cellectis: Research Funding; novartis: Research Funding; SagerStrong Foundation: Research Funding; abbvie: Research Funding; plexxikon: Research Funding; celgene: Consultancy, Honoraria; samus: Research Funding; daiichi sankyo: Research Funding. Khoury:Stemline Therapeutics: Research Funding. O'Brien:Vaniam Group LLC: Consultancy; Regeneron: Research Funding; TG Therapeutics: Consultancy, Research Funding; Aptose Biosciences Inc.: Consultancy; GlaxoSmithKline: Consultancy; Pharmacyclics: Consultancy, Research Funding; Abbvie: Consultancy; Astellas: Consultancy; Pfizer: Consultancy, Research Funding; Amgen: Consultancy; Janssen: Consultancy; Gilead: Consultancy, Research Funding; Alexion: Consultancy; Celgene: Consultancy; Sunesis: Consultancy, Research Funding; Kite Pharma: Research Funding; Acerta: Research Funding. Cortes:novartis: Research Funding. Jabbour:novartis: Research Funding. Jain:Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; Pfizer: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cellectis: Research Funding; BMS: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Cellectis: Research Funding; BMS: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Infinity: Research Funding; Servier: Research Funding; Verastem: Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologioes: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologioes: Research Funding; Verastem: Research Funding; Abbvie: Research Funding; Incyte: Research Funding; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; Incyte: Research Funding; Infinity: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Research Funding; Celgene: Research Funding; Astra Zeneca: Research Funding; ADC Therapeutics: Research Funding; Pfizer: Research Funding; Servier: Research Funding; ADC Therapeutics: Research Funding; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Genentech: Research Funding; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Aung:Stemline Therapeutics: Research Funding. Konopleva:Immunogen: Research Funding; abbvie: Research Funding; Stemline Therapeutics: Research Funding; cellectis: Research Funding.

Description: Purpose – To determine the value of Minimal Residual Disease (MRD) assessed by Multi-parameter Flow Cytometry (MFC) after achieving initial response to therapy, in predicting outcome in patients with acute myeloid leukemia (AML) Methods – We investigated the predictive value of MRD assessment by MFC in 191 patients with newly diagnosed AML treated between February 2010 and April 2014 at our institution who had available MRD assessment. MRD by MFC was assessed using an 8-color panel containing 19 distinct markers, on bone marrow specimens obtained at the time of achievement of CR and at approximately 30 days and 90 days after achieving CR. Residual leukemic blasts were identified based on phenotypic differences from normal myelomonocytic precursors. Sensitivity was estimated at 0.1% in most cases, with maximum achievable sensitivity of 0.01%, depending on the leukemic phenotype. Results – Of the 191 patients, 167 (87%) achieved complete remission (CR) or CR without platelet recovery (CRp). Their median age was 58 years (Range, 17-85). 84 (44%) were older than 60 years. Median WBC at presentation was 3.2 x 109/L(Range, 0.5-100.2 x 109/L) and median bone marrow blast percentage was 43% (Range, 11-96%). Cytogenetics was favorable risk in 4 (2%), intermediate risk in130 (68%) and adverse risk in 57 (30%). Treatment included cytarabine plus anthracycline in 170 (89%) and hypomethylating agents-based strategies in 21 (11%). 48 patients had available samples at 30 days post CR and 32 (67%) became MRD negative. Achieving MRD negative status was associated with a statistically significant improvement in CR duration (p=0.02) and overall survival (OS) (p=0.0005). 56 patients were evaluated for MRD status at 90 days and 45 (80%) were negative. Again, achieving MRD negative status was associated with a significant improvement in CR duration (p=0.002) and OS (p=0.0009). Conclusion – Achieving MRD negative status by MFC at 30 and 90 days post CR is associated with an improved outcome in patients with AML Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.