Transplantable Acute Myeloid Leukemia Research Articles

PU.1-Dependent Enhancer Inhibition Separates Tet2-Deficient Hematopoiesis from Malignant Transformation.

We identify moderately impaired PU.1 mRNA expression as a biological modality predisposing Tet2-deficient hematopoietic stem and progenitor cells to malignant transformation. Our study furthermore uncovers a methylation-sensitive PU.1 gene network as a common feature of myeloid leukemia potentially allowing for the identification of patients at risk for malignant transformation. See related commentary by Schleicher and Pietras, p. 378. This article is highlighted in the In This Issue feature, p. 369.

Abstract 3977: Pharmacological inhibition of Nrf2 overcomes acquired resistance to arsenic trioxide in FLT3 mutated acute myeloid leukemia

Abstract De novo acute myeloid leukemia (AML) patients with fms-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) have worse treatment outcomes. Arsenic trioxide (ATO) used in the treatment of acute promyelocytic leukemia (APL) had been reported to be effective in degrading the FLT3 protein in AML cell lines and sensitizing non-APL AML patient samples in-vitro (Liu et al. 2020). We have previously reported that primary cells from FLT3-ITD mutated AML patients were sensitive to ATO in-vitro compared to other non-M3 AML (Abraham et al, 2014) and molecular/pharmacological inhibition of NF-E2 related factor 2 (Nrf2), a master regulator of antioxidant response mechanism improved the chemosensitivity to ATO and daunorubicin (Karathedath et al. 2017). Here we assessed the role of pharmacological inhibition of Nrf2 in modulating acquired resistance to ATO in an FLT3-ITD mutated AML cell line (MV4-11 ATO-R).MV4-11 cell line containing FLT3-ITD was made resistant to ATO (MV4-11 ATO-R) using incremental method and showed 2-fold increase in IC50 to ATO. The basal expression of Nrf2 transcript and protein levels were compared between MV4-11 (naïve) and MV4-11 ATO-R (resistant) cells using quantitative RTPCR, immunoblotting, and immunofluorescence respectively. Mutations in the Keap1 gene were screened in naive and resistant cells by Sanger sequencing. The effect of known pharmacological inhibitor of Nrf2 (Digoxin (DIG)) in overcoming acquired resistance to ATO was tested by in-vitro cytotoxicity and apoptosis assays. Further, to identify the upstream activator of Nrf2, expression of Akt protein with and without DIG treatment was assessed by immunoblotting. Finally, the efficacy of Nrf2 inhibition by DIG in resistant cells were tested in a transplantable AML mice model.There was no significant increase in NRF2 RNA expression (<2-fold) while there was a higher nuclear Nrf2 expression and localization as well as increased Akt protein expression in the resistant cells compared to naïve cells. DIG treatment reduced expression of Nrf2 resulting in improved sensitivity (IC50 ATO-3.5µM vs ATO+DIG-2.05µM) and increased apoptosis (≈2 fold in apoptotic rates) to ATO in resistant cells. There was a reduction in Akt protein expression in DIG treated cells confirming that Nrf2 activation in the resistant cells is mediated by Akt. Treating AML mice with either DIG alone or in combination with ATO showed reduction in leukemic load and significant increase in overall median survival (26 & 23 days, p<0.001) when compared to untreated and ATO alone (20 & 16 days). Our study suggests that Nrf2 mediates acquired resistance to ATO in a FLT3-ITD mutated AML cell line. Inhibition of Nrf2 using a drug routinely used in the clinic was able to re-sensitize the resistant cells to ATO in-vitro and reduced leukemic burden in-vivo. Citation Format: Daniel Zechariah Jebanesan, Raveen Stephen Illangeswaran, Saswati Das, Rakhi Thalayattu Vidhyadharan, Nayanthara Karpillymoola Bijukumar, Bharathi M. Rajamani, Balaji Balakrishnan, Vikram Mathews, Shaji Ramachandran Velayudhan, Poonkuzhali Balasubramanian. Pharmacological inhibition of Nrf2 overcomes acquired resistance to arsenic trioxide in FLT3 mutated acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3977.

Multi-Recombinase Mouse Models of Flt3-Driven Leukemia Identifies Distinct Trajectories of Mutational Cooperativity and Leukemic Transformation

Genomic studies in acute myeloid leukemia (AML) have generated a near complete catalogue of genes mutated at varying frequencies both across patients and in individual leukemias. The high variability of mutation burden within a given leukemia is suggestive of a stepwise evolutionary process composed of early, clonal, mutations and subsequent subclonal events. The receptor tyrosine kinase, FLT3, is the most commonly mutated gene in AML, with mutations frequently manifesting as internal tandem duplications (ITDs) in the juxtamembrane domain leading to constitutive kinase activation. Although FLT3 is commonly a subclonal mutational event, FLT3 ITD mutations portend a poor prognosis particularly when combined with DNMT3A and NPM1, earlier mutations that drive clonal expansion. Notwithstanding its role as a subclonal driver, previous preclinical FLT3 models have utilized retroviral overexpression or germline mutant expression at the endogenous locus precluding accurate temporal modeling of disease. These efforts have prohibited evaluation of FLT3 mutational acquisition in the context observed in AML patients.Here, we report the development of an endogenously targeted, Flp inducible, Flt3 ITD mouse allele which can be somatically activated subsequent to cooperating disease alleles. When activated with a tamoxifen inducible FlpoER, Flt3 mutant mice developed rapid leukocytosis peaking at 4-6 weeks post activation and resolving by 8-10 weeks, a finding not previously observed in constitutive models. This leukocytosis was disproportionately monocytic and accompanied by pronounced anemia and thrombocytopenia. Long term, these mice develop a myeloproliferative disease , reminiscent of previously reported constitutive alleles. In competitive transplantation studies, Flt3 mutant cells initiated disease and outcompeted wild-type cells. Despite this competitive advantage, disease was incapable of transplanting into secondary recipients. We further observed a non-cell autonomous depletion of SLAM+ LSKs suggesting the Flt3 mutant cells cannot propagate disease in self-renewing stem cells.To evaluate how this allele influenced leukemic evolution we crossed this Flt3 ITD allele to a Flp inducible Npm1 c mouse where a pulse of tamoxifen simultaneously activated both alleles. The combination of mutant Npm1 and Flt3 resulted in progressive leukocytosis which did not resolve. Within 6 weeks of mutational activation, these mice developed a lethal AML with robust anemia, thrombocytopenia, leukocytosis and expanded cKIT+ blasts in the blood. RNA-sequencing and immunophenotyping by CyTOF revealed distinct patterns of differentiation, gene-expression and downstream signaling.In an effort to model sequential mutational acquisition, we crossed the Flp Flt3 ITD allele to a Cre-inducible Dnmt3a R878H. Cre mRNA was electroporated into lineage negative bone marrow cells to activate the Dnmt3a R878H allele and transplanted into lethally irradiated recipients. Four weeks post engraftment, Flt3 ITD was activated with a pulse of tamoxifen. In contrast to the Flt3-Npm1 model, we observed an increase and subsequent decrease in WBC similar to the kinetics observed in Flt3 ITD only mice. However, by 20 weeks we observed a robust and consistent increase in WBC accompanied by an emergence of cKIT+ cells in the blood. Histopathology indicated that >50% of mice expressing both alleles in sequence developed AML marked by increased blasts in the marrow, with moderate anemia and thrombocytopenia compared to the Flt3-Npm1 models. Critically, in contrast to Flt3 ITD only mice, acquisition of the Flt3 ITD in Npm1 or Dnmt3a mutant HPSCs induced fully transplantable AML with immunophenotypic characteristics seen in human AML with these same genotypes. Collectively these results demonstrate that different co-occurring mutations are capable of transforming Flt3 ITD mutant cells, albeit with distinct latencies and mechanisms of cooperativity.In summary, our studies utilizing novel multi-recombinase models of leukemogenesis reveal new insights into the early phase of oncogene activation, and how cooperating alleles influence this response. This inducible Flt3 ITD allele represents a significant advance in modeling clonal evolution in myeloid malignancies and provides a critical isogenic platform for preclinical development of novel leukemia therapeutic regimens. [Display omitted] DisclosuresBowman: Mission Bio: Honoraria, Speakers Bureau. Xiao: Stemline Therapeutics: Research Funding. Miles: Mission Bio: Honoraria, Speakers Bureau. Trowbridge: Fate Therapeutics: Patents & Royalties; H3 Biomedicine: Research Funding. Levine: Amgen: Honoraria; Lilly: Honoraria; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Imago: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Ajax: Membership on an entity's Board of Directors or advisory committees; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Zentalis: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Research Funding; Janssen: Consultancy; Astellas: Consultancy; Morphosys: Consultancy; Incyte: Consultancy; Auron: Membership on an entity's Board of Directors or advisory committees; Prelude: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Myeloproliferative Neoplasms in Pediatrics and Young Adults: Genetic Lesions, Diagnostic Features, and Clinical Outcomes

Introduction: Because Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are rare in pediatric and young adults (PAYA), the genomic landscape, clinical features, management, and prognosis for younger patients remain obscure. MPN are clonal hematopoietic stem cell (HSC) disorders characterized by overproduction of mature blood cells. While both polycythemia vera (PV ) and essential thrombocytosis (ET) present as indolent, chronic diseases, ~25% of older adults will progress to myelofibrosis (MF) and acute myeloid leukemia (AML) with abysmal outcomes. To help inform management of younger patients, we sought to elucidate molecular features, clinical findings, and outcomes in this unique population.Methods: The Hopkins MPN cohort enrolled 630 patients with ET, PV or MF between 2005-2015, all of whom had quantitative JAK2V617F, CALR, and MPL genotyping. Eighty-two (13%) patients were diagnosed at <30 years (PAYA), whereas 548 were diagnosed at >31 years (Adult). We compared clinical features, complications, and survival in both groups.Results:Epidemiology: The mean age of diagnosis was 25 years (range 1 week - 30 years) in PAYA versus (v.) XX years in Adults. Diagnosis at presentation in PAYA included: ET (n=53; 65%), PV (n=29; 35%), and, MF (n=4; 5%), contrasting with the Adult cohort, which included a great percentage with PV (42%) and MF (15%). Females comprised the majority in both cohorts (72% v. 59% in PAYA and Adults, respectively).Genomic landscape: The relative distribution of JAK2, CALR or MPL mutations were identical in both cohorts, with 50% of ET and MF positive for JAK2V617F, and 100% of PV patients positive for either JAK2V617F or JAK2 exon 12 mutations. CALR mutations comprised 11/38 (29%) ET and 5/12 (42%) MF in PAYA patients with similar frequencies in Adults. Only 1 PAYA patient had an MPL mutation. JAK2V617F allele burden increased with advancing age, although at a significantly lower rate in PAYA compared to the Adult cohort. Family history of MPN was similar in both cohorts (~9%). One PAYA patient had congenital ET (diagnosed at 1 week old); his mother, father and uncle also had ET.Complications: The most frequent complications were venous thromboses, followed by arterial events and migraines. The most common sites for venous thromboses included: abdominal (splenic, mesenteric, hepatic vein, portal vein, n=11 ), pulmonary emboli (n=X), leg (n=5), and cerebral sinus thrombosis (n=4). Arterial events included MI (n=4), TIA (n=2), and stroke (n=1). Three PAYA patients received orthotopic liver transplants for Budd Chiari Syndrome.Progression: Median follow-up was longer for PAYA compared to Adults (12 v. 8 years). MPN progressed in 21/82 (26%) of PAYA as follows: 1) PV to MF (n=6, median age 51 years, median time to progression 23 years), 2) ET to MF (n=4, median age 50 years, median time to progression 27 years), 3) ET to PV (n=10, median age 33 years, median time to progression 8 years). One PAYA patient developed AML 28 years after PV diagnosis and 2 years after progressing to MF. Time to transformation for all phases was longer, although age at transformation was lower, in PAYA compared to Adults.Survival: Deaths occurred in fewer (n=8; 9.7%) PAYA v. Adult patients (n=167; 30%). Overall median survival was longer in PAYA (32 v. 12 years), yet the median age of death in PAYA was lower (58.5 v. 72 years) compared to the Adult cohort. Cause of death in PAYA was attributed to post transplant complications (n-2), pulmonary hypertension (n=1), AML (n=1) and bone marrow failure (n=4).Conclusions: While JAK2V617F is a common acquired somatic mutation in HSC associated with aging and male sex, it also occurs in the PAYA population, and paradoxically, affects more females than males. Younger age at diagnosis is associated with lower risk disease (ET or PV v. MF) and a longer latency before progression to MF or AML, suggesting that older age, rather than disease duration, is a primary risk factor for progression.The genomic landscape of driver lesions was similar in PAYA and Adult cohorts. Despite presentation with lower risk disease, PAYA patients had a high rate of progression to MF. The high frequency of thromboses suggests that management of PAYA patients should include measures to prevent this complication, while the prolonged overall survival in PAYA underscores the need to control systemic symptoms while avoiding exposure to therapies that increase the risk of leukemia. DisclosuresNo relevant conflicts of interest to declare.

Acute Myeloid Leukemia (AML) Treated with Azacitidine: Survival Outcomes for Patients Who Complete More Than Six Cycles Are Similar to High-Risk Myelodysplastic Syndrome/Low Blast Count AML

INTRODUCTION: Despite accumulating evidence supporting the efficacy of hypomethylating agents in patients with AML and &gt; 30% bone marrow blasts as well as in relapsed/refractory AML, this therapy is not yet funded by National Health Plans / Healthcare Funding Agencies in a number of countries including Canada. The assistance of an industry-sponsored compassionate program has enabled provision of azacitidine for this group of patients at The Ottawa Hospital. We report here our local "real-world" experience of azacitidine efficacy in this diverse group of AML patients and identify a sub-group whose outcomes are equivalent to that of patients with higher-risk Myelodysplastic Syndrome (MDS) and AML with 20-30% blasts for whom azacitidine therapy has funding approval in Canada. METHODS: All patients who received azacitidine at The Ottawa Hospital between 2009 and 2016 were included in this single-center, retrospective analysis. Azacitidine was administered at a dose of 75mg/m2 subcutaneously daily for 7 consecutive days every 28 days. Response was evaluated with a repeat bone marrow aspirate and trephine biopsy after the 6th cycle. In those patients confirmed to have stable or responsive disease, azacitidine was continued until progression of disease, intolerable side-effects of the drug or the patient chose to discontinue therapy. Overall survival curves were generated using the Kaplan-Meier method and log-rank tests were used to compare subgroups of patients. Actuarial median survival months were calculated with 95% confidence intervals (CI). P-values less than 0.05 were considered statistically significant. RESULTS: During the study period, 109 patients received azacitidine: 54 had MDS /AML with 20-30% blasts (the 'funded' group) and 55 had either AML with &gt; 30 % blasts (n=23), AML relapsed post-intensive chemotherapy (n=14), AML relapsed post-allogeneic stem cell transplant (n=10) or primary refractory AML (n=8) (the 'unfunded' group). Median survival of the 'funded' group was 12.2 months while median survival of the 'unfunded' group was 5.6 months (95% CI 3.3-7.7; p=0.0058). Of the AML patients in the 'unfunded' group, 24% completed more than 6 cycles of azacitidine compared to 52% of patients in the 'funded' group. In both the 'funded' and 'unfunded' groups, patients who completed more than 6 cycles of azacitidine had similar survival outcomes (p=0.7277): the 'funded' group had a median survival of 19 months (95% CI 14.4-25.3) while the median survival of this sub-population of the 'unfunded' AML group was 22 months (95% CI 11.7-24.9). Patients in both groups who failed to complete more than 6 cycles of azacitidine also had a similar outcome (p=0.39), with a median survival of 5.7 months (95% CI 4.0-6.3) for patients with MDS/AML 20-30% blasts and 3.6 months (95% CI 2.2-5.1) for AML patients with &gt; 30% blasts or relapsed/refractory disease. Reasons for patients not completing at least 6 cycles of azacitidine included progression of disease (25%), bacterial infections most commonly pneumonia (53%) and patient preference (7%). CONCLUSION: A significant sub-population of AML patients with &gt; 30% blasts or refractory/relapsed AML can achieve a meaningful survival benefit with the hypomethylating agent, azacitidine. A higher proportion of this AML patient population discontinued azacitidine as a result of infective complications. The provision of routine prophylactic antibiotics may enable more patients with AML to receive an adequate amount of azacitidine to achieve therapeutic benefit and warrants further investigation. Our results add to the growing body of 'real-world' evidence that supports healthcare funding agencies to provide coverage of azacitidine for patients with AML who in some countries at present do not fulfill government funding criteria. Disclosures Bredeson: Otsuka: Research Funding. Maze:Pfizer Inc: Consultancy; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sabloff:Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; ASTX: Membership on an entity's Board of Directors or advisory committees, Research Funding; Actinium Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas Pharma Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi Canada: Research Funding.

Transforming activities of the NUP98-KMT2A fusion gene associated with myelodysplasia and acute myeloid leukemia.

Inv(11)(p15q23), found in myelodysplastic syndromes and acute myeloid leukemia, leads to expression of a fusion protein consisting of the N-terminal of nucleoporin 98 (NUP98) and the majority of the lysine methyltransferase 2A (KMT2A). To explore the transforming potential of this fusion we established inducible iNUP98-KMT2A transgenic mice. After a median latency of 80 weeks, over 90% of these mice developed signs of disease, with anemia and reduced bone marrow cellularity, increased white blood cell numbers, extramedullary hematopoiesis, and multilineage dysplasia. Additionally, induction of iNUP98-KMT2A led to elevated lineage marker-negative Sca-1+ c-Kit+ cell numbers in the bone marrow, which outcompeted wildtype cells in repopulation assays. Six iNUP98-KMT2A mice developed transplantable acute myeloid leukemia with leukemic blasts infiltrating multiple organs. Notably, as reported for patients, iNUP98-KMT2A leukemic blasts did not express increased levels of the HoxA-B-C gene cluster, and in contrast to KMT2A-AF9 leukemic cells, the cells were resistant to pharmacological targeting of menin and BET family proteins by MI-2-2 or JQ1, respectively. Expression of iNUP98-KMT2A in mouse embryonic fibroblasts led to an accumulation of cells in G1 phase, and abrogated replicative senescence. In bone marrow-derived hematopoietic progenitors, iNUP98-KMT2A expression similarly resulted in increased cell numbers in the G1 phase of the cell cycle, with aberrant gene expression of Sirt1, Tert, Rbl2, Twist1, Vim, and Prkcd, mimicking that seen in mouse embryonic fibroblasts. In summary, we demonstrate that iNUP98-KMT2A has in vivo transforming activity and interferes with cell cycle progression rather than primarily blocking differentiation.

PF531 TRANSFORMING ACTIVITIES OF THE NUP98-KMT2A FUSION GENE ASSOCIATED WITH MYELODYSPLASIA AND ACUTE MYELOID LEUKEMIA

Background: Inv (11)(p15q23) found in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) was previously shown to lead to the expression of a fusion consisting of the N-terminal part of nucleoporin 98 (NUP98) and almost the entire open reading frame (ORF) of the lysine methyltransferase 2A (KMT2A, aka MLL1). Aims: To address the role of the NUP98-KMT2A fusion as a potential leukemogenic oncogene. Methods: To overcome the limitations related to the large size of the fusion ORF, we generated DOX-inducible transgenic iNUP98-KMT2A mice. We studied the effects of iNUP98-KMT2A expression in hematopoietic cells ex vivo in clonogenic assays; and in vivo by assessing blood counts, histology, and immunophenotyping as well as by competitive repopulation assays. In addition, we established primary mouse embryonic fibroblast (MEF) cultures to address the effects of iNUP98-KMT2A on cell cycle progression and cellular senescence. Results: In contrast to other KMT2A fusion such as MLL-AF9, induction of iNUP98-KMT2A did not alter differentiation but impaired growth of bone marrow (BM)-derived cells in vitro. Induction of iNUP98-KMT2A in vivo resulted in signs of disease after a highly variable latency of 13–106 weeks (median latency = 80 weeks, n = 22). Some symptomatic mice presented with increased white blood counts with morphological signs of dysplasia, reduced red blood cells, and increased apoptosis in the BM with signs of extramedullary hematopoiesis. In addition, we observed a 3-fold expansion of Lin−Sca-1+c-Kit+ (LSK) hematopoietic stem and progenitor cells (HSPC) in the BM of these mice. BM cells from symptomatic iNUP98-KMT2A mice had a competitive advantage in reconstitution assays. About 20% (5/22) of the mice on DOX presented with transplantable AML with elevated WBC counts and leukemic blasts in the peripheral blood with infiltration of multiple organs. Expression of iNUP98-KMT2A was associated with aberrant cell cycle progression in LSK cells from primary-induced mice. Similarly, iNUP98-KMT2A expression affected cell cycle progression and abrogated cellular senescence in MEF associated with aberrant expression of Sirt1, Tert, Rbl2, Twist1, Vim, and Prkcd that was also observed in BM cells. Notably, similar to patients with NUP98-KMT2A, and in contrast to other KMT2A fusions, primary AML cells from diseased iNUP98-KMT2A mice expressed very low levels of the Hox-A-B-C gene cluster. In addition, iNUP98-KMT2A leukemic blasts were resistant to the effects of pharmacological targeting of Menin/KMT2A interface or BET-family proteins. Summary/Conclusion: Our work shows that the NUP98-KMT2A fusion has oncogenic potential in mice leading to an MDS-like disease or AML. In contrast to other KMT2A fusions, NUP98-KMT2A does not primarily block differentiation and provide aberrant self-renewal potential, but alters cell cycle progression leading to dysplasia and impairs cellular senescence.

Leveraging Single-Cell RNA Sequencing Experiments to Model Intratumor Heterogeneity.

PURPOSEMany cancers can be treated with targeted therapy. Almost inevitably, tumors develop resistance to targeted therapy, either from pre-existence or by evolving new genotypes and traits. Intratumor heterogeneity serves as a reservoir for resistance, which often occurs as a result of the selection of minor cellular subclones. On the level of gene expression, clonal heterogeneity can only be revealed using high-dimensional single-cell methods. We propose using a general diversity index (GDI) to quantify heterogeneity on multiple scales and relate it to disease evolution.MATERIALS AND METHODSWe focused on individual patient samples that were probed with single-cell RNA (scRNA) sequencing to describe heterogeneity. We developed a pipeline to analyze single-cell data via sample normalization, clustering, and mathematical interpretation using a generalized diversity measure, as well as to exemplify the utility of this platform using single-cell data.RESULTSWe focused on three sources of patient scRNA sequencing data: two healthy bone marrow (BM) donors, two patients with acute myeloid leukemia—each sampled before and after BM transplantation, four samples of presorted lineages—and six patients with lung carcinoma with multiregion sampling. While healthy/normal samples scored low in diversity overall, GDI further quantified the ways in which these samples differed. Whereas a widely used Shannon diversity index sometimes reveals fewer differences, GDI exhibits differences in the number of potential key drivers or clonal richness. Comparison of pre– and post–BM transplantation acute myeloid leukemia samples did not reveal differences in heterogeneity, although biological differences can exist.CONCLUSIONGDI can quantify cellular heterogeneity changes across a wide spectrum, even when standard measures, such as the Shannon index, do not. Our approach can be widely applied to quantify heterogeneity across samples and conditions.

Prognostic significance of microRNA-99a in acute myeloid leukemia patients undergoing allogeneic hematopoietic stem cell transplantation.

Overexpression of microRNA-99a (miR-99a) have been associated with adverse prognosis in acute myeloid leukemia (AML). Nevertheless, whether it also predicts poor outcome in post-allogeneic hematopoietic stem cell transplantation (allo-HSCT) AML patients remains unclear. To further elucidate the prognostic value of miR-99a, 74 AML patients with miR-99a expression report who underwent allo-HSCT from The Cancer Genome Atlas database were identified and grouped into either miR-99ahigh or miR-99alow based on their miR-99a expression levels relative to the median. Two groups had similar clinical and molecular characteristics except that miR-99ahigh group had fewer patients of the French-American-British M4 subtype (P = 0.018) and more frequent CEBPA mutations (P = 0.005). Univariate analysis indicated that high miR-99a expression was unfavorable for both event-free survival (EFS) and overall survival (OS; P = 0.029; P = 0.012, respectively). Multivariate analysis suggested that high miR-99a expression was an independent risk factor for both EFS and OS in AML patients who underwent allo-HSCT [hazard ratio (HR) 1.909, 95% confidence interval (CI) 1.043-3.494, P = 0.036 and HR 2.179, 95% CI 1.192-3.982, P = 0.011, respectively]. Our results further proved that high miR-99a expression could predict worse outcome in AML patients, even in those who underwent intensive post-remission therapy such as allo-HCST.

Abstract A003: Developing an AML mouse syngeneic model for combinatory chemotherapy and immunotherapy

Abstract Little progress has been made in treatment of acute myeloid leukemia (AML) for years. Following the success of immune checkpoint inhibitors in solid tumors, immunotherapy also offers huge promise in many hematologic malignancies, including AML. Anti-PD1 antibody is currently being investigated in combination with azacitidine in patients with relapsed AML. Other emerging immuno-oncology (I/O) therapeutics are also at different stages of preclinical research. However, we are lacking relevant animal models with intact immune system for the preclinical in vivo pharmacologic studies and mechanism-of-action analysis of I/O agents. To meet this need, we set out to develop a series of transplantable mouse AML models carrying Nras mutations and/or a set of clinical relevant fusion proteins, like AML1/ETO. Using a similar approach reported by Scott Lowe and colleagues (1), we constructed retroviral vectors expressing human AML1/ETO9a IRES GFP and NrasG12D, generated retroviruses, co-transduced the fetal liver cells, and performed syngeneic intravenous injections in C57BL/6 mice. The mice developed AML in the following months as the GFP-positive cells enriched as high as around 90% in PBMC. Leukemia cells also dominatde the spleen and bone marrow in moribund mice. The splenocytes from moribund AML1/ETO9a mice were banked and transplanted into new C57BL/6 mice for several rounds. The later passages were more malignant than the early passages. To validate the immune profiling of this model, FACS analysis was applied using mouse PD-L1, Gr-1, CD45, Mac-1, c-kit, and sca-1 antibodies for GFP-positive cells. In PBMC of moribund AML1/ETO9a mice, PD-L1 and c-kit were highly expressed, while the expression levels of Gr-1, CD45, Mac-1, and sca-1 were low. For efficacy test, we performed standard-of-care (SOC) assay with cytarabine, doxorubicin, sorafenib, and anti-PD1 antibody. The dosing started around 10 days post inoculation of 1 x 106 leukemia cells, when GFP-positive cells were 2-10%. While doxorubicin showed good response, cytarabine and sorafenib resulted in modest tumor growth inhibition, and anti-PD1 antibody failed to show any meaningful efficacy. We are currently testing more combinatory treatments, including anti-TIM3 and anti-CTLA4 antibodies. Taken together, we have developed a mouse AML model with clinical relevant mutations for preclinical efficacy assessment of combinatory chemotherapies, target therapies, and immunotherapies. Reference: 1. Zuber J, Radtke I, Pardee TS, et al. Mouse models of human AML accurately predict chemotherapy response. Genes Dev 2009. Citation Format: Xin Tang, Lily Tong, Annie Xiaoyu An, Likun Zhang, Jie Cai, Qian Shi, Jean Pierre Wery, Davy Xuesong Ouyang. Developing an AML mouse syngeneic model for combinatory chemotherapy and immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A003.

An Unusual Case of Acute Myeloid Leukemia Cell Infiltration of the Renal Allograft: A Case Report and Review of Literature

Kidney transplant recipients are at a high risk for developing malignancies with dermatologic cancers and lymphomas making up the bulk of these. Leukemia is rare in this cohort of patients. Kidney infiltration by leukemic cells is seen in about 34% of native kidneys based on autopsy studies. But leukemia infiltration of the kidney allograft has not been described much in the literature. Here we discuss the case of a 59-year-old kidney transplant recipient with acute myeloid leukemia (AML) who presented with acute kidney injury and his transplant kidney biopsy showed leukemic cell infiltration of the renal cortex. We also present a comprehensive literature review of post–kidney transplantation AML.

The target cell of transformation is distinct from the leukemia stem cell in murine CALM/AF10 leukemia models

The CALM/AF10 fusion gene is found in various hematological malignancies including acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia and malignant lymphoma. We have previously identified the leukemia stem cell (LSC) in a CALM/AF10-driven murine bone marrow transplant AML model as B220+ lymphoid cells with B-cell characteristics. To identify the target cell for leukemic transformation or 'cell of origin of leukemia' (COL) in non-disturbed steady-state hematopoiesis, we inserted the CALM/AF10 fusion gene preceded by a loxP-flanked transcriptional stop cassette into the Rosa26 locus. Vav-Cre-induced panhematopoietic expression of the CALM/AF10 fusion gene led to acute leukemia with a median latency of 12 months. Mice expressing CALM/AF10 in the B-lymphoid compartment using Mb1-Cre or CD19-Cre inducer lines did not develop leukemia. Leukemias had a predominantly myeloid phenotype but showed coexpression of the B-cell marker B220, and had clonal B-cell receptor rearrangements. Using whole-exome sequencing, we identified an average of two to three additional mutations per leukemia, including activating mutations in known oncogenes such as FLT3 and PTPN11. Our results show that the COL for CALM/AF10 leukemia is a stem or early progenitor cell and not a cell of B-cell lineage with a phenotype similar to that of the LSC in CALM/AF10+ leukemia.

Acute Myeloid Leukemia Alters Natural Killer Cell Maturation and Functional Activation

Acute myeloid leukemia (AML) is a devastating disease primarily affecting adults. Immune evasion is a major mechanism of AML persistence, and represents a barrier for long-term clinical success. Natural killer (NK) cells are a key component of the innate immune system and hold promise as a tool for effective anti-leukemia therapy. However, to date, the clinical success of NK therapy has been disappointing, indicating additional immune evasion strategies may affect the ability of NK cells to function in patients. We hypothesized that AML may evade the innate immune system by inhibiting NK cell maturation.To evaluate NK function and maturation during AML progression, our lab utilized a novel knock-in model of AML that expresses both Flt3-ITD and Mll-PTD mutations (PTD/ITD), and develops AML with 100% penetrance and recapitulates human disease. For these studies, both primary PTD/ITD mice and transplanted AML blasts were used. For transplant studies, AML or wild-type (WT) control cells (CD45.2+) were transplanted into irradiated C57BL/6 (CD45.1+) naïve recipients. Normal (CD45.1+) NK cells were defined as NK1.1+/CD3- by flow cytometry, and expression of receptors was determined within this population. NK cells in leukemic mice exhibited a reduction in the activating receptors Ly49D and Ly49H in the spleen, blood, LN, and bone marrow. There was also an increase in the inhibitory receptor Ly49C/I and NKG2C/A/E (both p<0.05). However, these alterations were not consistent with all activating and inhibitory receptors, as CD69 was elevated, while NKp46 expression was decreased (both p<0.05). NK function was determined by ELISA measurement of soluble interferon-gamma (IFN-g) production. While there was no change in IFN-g production at baseline, there was surprisingly a significant elevation in IFN-g detected upon stimulation with interleukin-12 and interleukin-18 in the NK cells isolated from leukemic mice as compared to WT control mice (p=0.0004).NK maturation was then evaluated by examining CD11b and CD27 protein expression on NK1.1+/CD3- cells in the spleen, bone marrow, blood, and LN. CD11b+/CD27+ (DP) NK cells were reduced in the spleen as well as bone marrow, blood and LN (p<0.02 for all organs). AML splenocytes had a corresponding increase in NK cells negative for both proteins (DN) when compared to WT control splenocytes (p=0.0004). Previous studies indicate the DP population of NK cells have the highest cytotoxic and cytokine producing functions, while DN NK cells are functionally immature. These alterations were more distinct as AML burden increased in the mice (p=0.0004). Interestingly, there was no significant difference in the most mature CD11b+/CD27- NK population in the spleen, but it there was a significant increase in the CD11b+/CD27- population in the blood (p=0.002). Additional analysis of two transcription factors known to regulate NK development, T-bet and Eomes, was also performed. A reduction in expression of both transcription factors was seen in total NK cells of leukemic mice, as well as NK subsets in the spleen (p<0.02). These data suggest that NK maturation is altered in the presence of AML, and that this alteration becomes more pronounced as leukemic burden increases. Studies evaluating the mechanistic link between AML and NK maturation are ongoing. Future studies will evaluate the efficacy of combinatorial therapies addressing both this imbalance in maturation as well as enhancing recognition to improve NK killing of AML cells. DisclosuresNo relevant conflicts of interest to declare.

AG-221, a Small Molecule Mutant IDH2 Inhibitor, Remodels the Epigenetic State of IDH2-Mutant Cells and Induces Alterations in Self-Renewal/Differentiation in IDH2-Mutant AML Model in Vivo

Somatic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are observed in patients with acute myeloid leukemia (AML). Leukemia-associated IDH1/2 mutations result in aberrant accumulation of the oncometabolite 2-hydroxyglutarate (2-HG). The observation that IDH1/2 mutations are mutually exclusive with TET2 mutations led to the finding that IDH1/2-mutant production of 2-HG inhibits TET2 function and induces changes in DNA methylation. These data suggested that small molecule inhibition of mutant IDH enzymes might reverse the aberrant epigenetic remodeling of IDH-mutant leukemia cells and restore normal hematopoietic differentiation.We therefore investigated the in vivo efficacy of AG-221, a potent and selective mutant IDH2 inhibitor in early-phase clinical trials, in murine models of IDH2-mutant leukemia. We first assessed the impact of AG-221 on 2-HG production in hematopoietic cells expressing mutant IDH2-R140Q. AG-221 treatment (10mg/kg or 100mg/kg bid) led to a reduction in 2-HG in vivo (96.7% below pre-treatment levels). Moreover, AG-221 treatment restored megakaryocyte-erythroid progenitor (MEP) differentiation that is suppressed by mutant IDH2 expression in vivo (mean MEP% mean, 39% Veh vs 50% AG-221).We next investigated the impact of mutant IDH2 inhibition with AG-221 on DNA methylation in vivo. We used eRRBS, a bisulfite-based next-generation sequencing platform, to assess the effect of AG-221 therapy on DNA methylation. AG-221 or vehicle therapy treated LSK stem cells (lin- Sca+ c-Kit+) were sorted from mice expressing IDH2-R140Q and evaluated by eRRBS. AG-221 therapy reversed the effects of mutant IDH2; we observed a significant reduction in DNA methylation, including 180 genes that had 20 or more hypomethylated differentially methylated cytosines (DMCs) following treatment. 84 of these genes had reduced methylation at 10 or more DMCs in the gene promoter with AG-221 therapy compared to vehicle. Mutant IDH2 inhibition with AG-221 reversed aberrant methylation at many genes with a known role in hematopoietic proliferation and differentiation, including the master transcriptional factor RUNX1.We next assessed in vivo effects of the small-molecule IDH2-R140Q inhibitor in a mouse model of IDH2-mutant leukemia. We generated mice that simultaneously expressed a constitutive Flt3ITD knock-in allele and a conditional mutant IDH2R140Q knock-in allele. As reported recently using retroviral/transgenic models, Mx1-Cre IDH2R140QFlt3ITD developed fully penetrant, transplantable AML with expansion of c-Kit+ positive blasts in the peripheral blood, and widespread leukemic infiltration. AG-221 inhibited the serial replating capacity of IDH2R140QFlt3ITD expressing cells in vitro. We competitively transplanted IDH2R140QFlt3ITD AML cells and normal bone marrow cells into secondary recipients, and then assessed the effect of AG-221 therapy on leukemia in vivo and on disease burden. AG-221 (100mg/kg bid) treatment of mice engrafted with Mx1-Cre IDH2R140QFlt3ITD AML cells markedly reduced 2HG levels consistent with on target inhibition in vivo. AG-221 therapy induced differentiation of leukemic cells, with an increase in the CD11b+ population and a decrease in the c-Kit+ population in the peripheral blood at 2wks. We next assessed the impact of treatment with both AG-221 therapy with AC220, a potent, specific Flt3 inhibitor in late phase clinical trials. Combined IDH2R140Q and Flt3ITD inhibition resulted in a marked decrease in leukemic burden to vehicle-treated mice, with a significant reduction in leukemic cell chimerism in vivo in the setting of combined inhibition at 2 wks, (mean 45.2 fraction 88% veh, 73% AG-221, p<.01).These data demonstrate that AG-221 inhibits mutant IDH2-mediated 2-HG production in vivo and reverses the effects of mutant IDH2 on DNA methylation in mutant stem/progenitor cells. AG-221 induces differentiation and impairs self-renewal of IDH2-mutant leukemia cells, effects that are further enhanced by simultaneous inhibition of Flt3ITD. Clinical trials combining IDH2 inhibitors with other targeted AML therapies are warranted in order to increase therapeutic efficacy. DisclosuresIntlekofer:Foundation Medicine, Inc: Consultancy. Thompson:Agios: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Travins:Agios Pharmaceuticals: Employment, Stockholder Other. Straley:Agios: Employment, Equity Ownership. Gliser:Agios Pharmaceuticals: Employment, Stockholder Other. Yen:Agios: Employment, Equity Ownership. Levine:Agios: Consultancy, Membership on an entity’s Board of Directors or advisory committees.

Modeling de novo leukemogenesis from human cord blood with MN1 and NUP98HOXD13

AbstractLeukemic transformation of human cells is a complex process. Here we show that forced expression of MN1 in primitive human cord blood cells maintained on stromal cells in vitro induces a transient, but not serially transplantable, myeloproliferation in engrafted mice. However, cotransduction of an activated HOX gene (NUP98HOXD13) with MN1 induces a serially transplantable acute myeloid leukemia (AML). Further characterization of the leukemic cells generated from the dually transduced cells showed the activation of stem cell gene expression signatures also found in primary human AML. These findings show a new forward genetic model of human leukemogenesis and further highlight the relevance of homeobox transcription factors in the transformation process.

Functional Dissection and Targeting Of Bmi1 Independence Of MN1 Leukemia

While PcG protein, Bmi1, plays a critical role in development of leukemic stem cells (LSCs), we have recently shown a differential Bmi1 dependency for LSCs initiated by different oncogenic transcription factors associated with distinct prognostic outcomes. PML-RARA and AML1-ETO leukemias associated with good prognosis are dependent on Bmi1, whereas poor prognostic leukemia induced by MLL fusion that is capable of activating multiple Hox genes is Bmi1 independent. However the role of Bmi1 and the mechanisms of over-coming Bmi1 dependency in other acute myeloid leukemia (AML) subtypes are still largely unknown. Aberrations of the MN1 gene mostly as an over-expression or rarely as a fusion partner of TEL in patients carrying the translocation t(12;22)(p13;q11) are frequently found in AML and myelodysplastic syndrome (MDS). Forced expression of MN1 induces aggressive AML with a 100% penetrance in mouse models within a few weeks, a latency significantly shorter than myeloid malignancy induced by MN1-TEL. Consistently, MN1 over-expression correlates with reduced drug response and poor prognosis of AML patients as well as high level of Bmi1 expression. In spite of these striking experimental and clinical features, the molecular mechanisms underlying MN1 leukemia are still largely unknown, and little progress has been made in targeting this leukemia. In the current study, we sought out to investigate the role of Bmi1 for MN1 mediated leukemic transformation and the mechanisms underlying this aggressive leukemia. In contrast to MN1-TEL that is dependent on Bmi1 for transformation of murine primary hematopoietic cells, MN1 over-expression could transform Bmi1-/- hematopoietic progenitor cells (HPCs) and induced serially transplantable AML in mice with the same latency as MN1 transformed wild type (wt) HPCs. Further molecular analyses revealed a significant up-regulation of p16Ink4a and cellular senescence in MN1-TEL Bmi1-/- HPCs, which was absent in MN1 transformed cells. Senescence in MN1-TEL Bmi1-/- cells could be rescued by re-expression of Bmi1 or suppression of p16 respectively, consistently suggesting an inherent functional difference between MN1 and MN1-TEL reflected by contrasting Bmi1 dependences. To identify the functional domain critical for the Bmi1 independency, structure/function analysis revealed that the removal of DNA binding domain (DBD) in the TEL moiety conferred Bmi1 independent transformation to MN1-TEL (MN1-TELΔDBD). MN1-TELΔDBD was able to induce transplantable leukemia in both the wild type and Bmi1 deficient cells. Conversely, fusion of TEL DBD to full length MN1 abolished its Bmi1 independence, and failed to transform Bmi1 deficient cells. These results strongly suggest that MN1 regulated molecules/pathways critical for Bmi1 independent transformation are misguided by the TEL DBD in MN1-TEL leukemia, providing a unique platform to dissect the pathways for Bmi1 independent transformation in AML. By performing global gene expression analyses on MN1, MN1-TEL and MN1-TELΔDBD transformed cells, we identified 1727 genes differentially expressed in MN1 transformed cells compared with MN1-TEL transformed cells; whereas only 44 genes were differentially expressed in MN1-TEL versus MN1-TELΔDBD transformed cells. When we overlapped these two gene sets together, we generated a unique gene set containing 34 genes associated with Bmi1 independence, including metabolic enzymes, signaling molecules and transcription factors such as Hoxa gene that has previously implicated in Bmi1 independent leukemic transformation. To assess the functional significance and the potential of targeting the candidates in this gene list in overcoming Bmi1 independent transformation, we performed functional analyses using shRNA approaches with a focus on those pharmacologically tractable candidates to suppress Bmi1 independent leukemic transformation. As a result, we were able to identify and demonstrate two different classes of enzymes with rigid catalytic domains that are required for Bmi1 independent transformation by MN1. Together, we dissect the mechanisms underlying Bmi1-independent leukemic transformation, and provide promising novel targets for MN1 leukemia. Disclosures: No relevant conflicts of interest to declare.

IGF binding protein 2 is a cell-autonomous factor supporting survival and migration of acute leukemia cells

BackgroundThe role of IGF binding protein 2 (IGFBP2) in cancer development is intriguing. Previously we identified IGFBP2 as an extrinsic factor that supports the activity of hematopoietic stem cells (HSCs).Methods and resultsHere we investigated the role of IGFBP2 in in human leukemia cells and in the retroviral AML1-ETO9a transplantation acute myeloid leukemia (AML) mouse model.ResultsIGFBP2 is highly expressed in certain human AML and acute lymphoblastic leukemia (ALL) cells. Inhibition of expression of endogenous IGFBP2 in human leukemia cells led to elevated apoptosis and decreased migration and, consistently, to decreased activation of AKT and other signaling molecules. We also studied the effects of IGFBP2 knockout in the retroviral AML1-ETO9a transplantation AML mouse model. The deletion of IGFBP2 in donor AML cells significantly decreased leukemia development in transplanted mice. Lack of IGFBP2 resulted in upregulation of PTEN expression and downregulation of AKT activation, in the mouse AML cells. The treatment of IGFBP2 deficient AML cells with a PTEN inhibitor restored the wild-type colony forming ability. The deletion of IGFBP2 also led to decreased AML infiltration into peripheral organs and tissues, suggesting that IGFBP2 is required for the migration of AML cells out of bone marrow.ConclusionIGFBP2 is a critical cell-autonomous factor that promotes the survival and migration of acute leukemia cells.

Distinct classes of c-Kit–activating mutations differ in their ability to promote RUNX1-ETO–associated acute myeloid leukemia

AbstractThe t(8;21) RUNX1-ETO translocation is one of the most frequent cytogenetic abnormalities in acute myeloid leukemia (AML). In RUNX1-ETO+ patient samples, differing classes of activating c-KIT receptor tyrosine kinase mutations have been observed. The most common (12%-48%) involves mutations, such as D816V, which occur in the tyrosine kinase domain, whereas another involves mutations within exon 8 in a region mediating receptor dimerization (2%-13% of cases). To test whether distinct subtypes of activating c-KIT mutations differ in their leukemogenic potential in association with RUNX1-ETO, we used a retroviral transduction/transplantation model to coexpress RUNX1-ETO with either c-KitD814V or c-KitT417IΔ418-419 in murine hematopoietic stem/progenitor cells used to reconstitute lethally irradiated mice. Analysis of reconstituted animals showed that RUNX1-ETO;c-KitD814V coexpression resulted in 3 nonoverlapping phenotypes. In 45% of animals, a transplantable AML of relatively short latency and frequent granulocytic sarcoma was noted. Other mice exhibited a rapidly fatal myeloproliferative phenotype (35%) or a lethal, short-latency pre-B-cell leukemia (20%). In contrast, RUNX1-ETO;c-KitT417IΔ418-419 coexpression promoted exclusively AML in a fraction (51%) of reconstituted mice. These observations indicate that c-KitD814V promotes a more varied and aggressive leukemic phenotype than c-KitT417IΔ418-419, which may be the result of differing potencies of the activating c-Kit alleles.

Prognostic Significance of Detection of Minimal Residual Disease by Multiparametric Flow Cytometry Before Allogeneic Stem Cell Transplantation in Patients with Acute Myeloid Leukemia,

Abstract 4116The main prognostic determinant of the outcome of allogeneic stem cell transplantation (allo-SCT) for acute myeloid leukemia (AML) is disease phase at transplant while the relevance of the assessment of minimal residual disease (MRD) at transplant has not been elucidated. Although molecular markers are highly sensitive for the detection of MRD, they are available in only half of AML cases to date. On the other hand, multiparametric flow cytometry (MFC) offers the feasibility of MRD assessment in virtually all patients. We retrospectively studied the influence of pre-transplant MRD status by MFC on the outcomes of patients with AML in relation to other known prognostic factors.From 01/2003 to 12/2010, 102 AML patients (male/female; 58/44), aged 15–64 (median; 42) years, received allo-SCT in our center at first (CR1, N=69) or subsequent (CR≥2, N=33) morphologic complete remission. Donors were HLA-compatible siblings (N=45), matched unrelated (N=34) or alternative (haploidentical relatives or unrelated cord blood; N=6 and 17, respectively). The conditioning regimen was myeloablative in 85 (83.33%), and reduced-intensity in 17 (16.67%) cases. Data on MRD assessment by four- or five-color MFC before transplant were available in 92 patients. MRD was defined as any percentage of cells with an aberrant antigen expression pattern compared to normal or regenerating marrow. Transplant outcomes associated with MRD in univariate and multivariate analysis were overall (OS) and disease-free (DFS) survival, relapse incidence, and non-relapse-related mortality (NRM). Prognostic parameters included in the statistical model were the following: age and gender of recipient or donor, interval from diagnosis to transplant, de novo versus secondary AML, CR1 versus CR≥2 at transplant, cytogenetic risk group (according to Intergroup ECOG/SWOG definition), recipient and donor cytomegalovirus (CMV) serostatus, donor type, conditioning regimen (myeloablative versus reduced-intensity), degree of HLA match, method of graft-versus-host disease (GVHD) prophylaxis, and the modified European Blood and Marrow Transplantation Group (EBMT) risk score (Hemmati et al, 2011).With a median follow-up of 47 (range, 6–98) months, OS and DFS were 54.2% and 51.72%, respectively. The cumulative incidence of relapse was 24.45%, and of NRM 23.82%. Eighteen out of 92 (19.57%) patients had MRD by MFC before allo-SCT. According to univariate analysis, the presence of MRD showed a negative association with OS (29.63% versus 63.17% at 4 years in patients with or without MRD, respectively, P=0.053) and DFS (28.52% in MRD-positive versus 59.74% in MRD-negative cases at 4 years, P=0.025, Figure 1). Presence of MRD correlated with higher incidence of relapse compared to MRD negativity (52.7% versus 15.8%, respectively, P=0.001) (Figure 2), but did not influence NRM. In multivariate analysis (Cox proportional hazards, backward stepwise selection), the parameters that remained independent adverse risk factors in terms of specific outcomes were: a) For OS: age and male gender of patient, alternative donor, unfavorable karyotype, CR≥2, and secondary AML, b) For DFS: presence of MRD (HR=3.2, P=0.009), CR≥2 (ÇR=3.1, P=0.005), reduced-intensity conditioning (HR=2.6, P=0.034), and unfavorable karyotype (HR=2.06, P=0.074), c) For relapse: presence of MRD (ÇR=3.83, P=0.003), and unfavorable karyotype (HR=3.55, P=0.007), and d) For NRM: age of recipient, CR≥2, and alternative donor. [Display omitted] [Display omitted] In conclusion, the presence of MRD by MFC before allo-SCT for AML is associated with a significantly higher risk of relapse and an inferior DFS, despite morphologic complete remission at transplant. Moreover, pre-transplantation MRD status has independent prognostic significance in addition to the disease phase, cytogenetic risk group, and intensity of the conditioning regimen. Prospective studies are warranted to examine if the adverse impact of MRD on the outcomes of allo-SCT can be reversed by immunologic manipulations aiming at augmenting graft-versus-leukemia (GVL) effect. Disclosures:No relevant conflicts of interest to declare.

Prognostic Factors of Reducing Relapse in Patients Undergoing Allogeneic Hematopoietic Cell Transplantation From Related Donor for Acute Myeloid Leukemia: The Japan Society for Hematopoietic Cell Transplantation (JSHCT) AML Working Group

Abstract 3085 Background:A llogeneic hematopoietic cell transplantation (allo-HCT) is a curative treatment option for patients with acute myeloid leukemia (AML), however a major obstacle to success is represented by the relapse of the disease. We retrospectively analyzed prognostic factors to determine whether there are clinical features that predict relapse after transplantation. Patients and Methods:Clinical data were collected from the Transplant Registry Unified Management Program (TRUMP) in Japan. An individual patient data-based analysis was performed on patients with AML who achieved or continued complete remission (CR) after related allo-HCT between 1991 and 2009. A logistic regression model was used to analyze associations between relapse and clinical factors. Patients with CR1 or CR2 were defined as having a standard-risk disease, and patients with ≤CR3 or non-CR were defined as having a high-risk disease. Conditioning regimens included ≤ 8Gy in fractionated doses of total body irradiation, busulfan dose < 9mg/kg, melphalan dose ≤ 140 mg/m2 defined as a reduced-intensity conditioning (RIC) regimen. Results:We analyzed a cohort of 1, 960 patients with AML. The median age at allo-HCT was 42 years (range, 16 to 74 years). Among the 1, 960 patients, 778 patients (38%) relapsed after allo-HCT. The probabilities of survival at 5-year for all patients, standard-risk patients, and high-risk patients were 49%, 61%, and 31%, respectively. In a multivariate analysis, significant factors for no relapse were standard-risk disease at the time of allo-HCT (HR 0.366, 95%CI 0.279–0.480, p<0.0001), presence of chronic GVHD (HR 0.442, 95%CI 0.338–0.579, p <0.0001), and CMV infection (HR 0.452, 95%CI 0.230–0.886, p =0.021). Intensity of conditioning regimen (RIC vs myeloablative), disease subtype of AML, HLA matching (matched donor vs mismatched donor), source of stem cells (peripheral blood vs bone marrow), and presence of acute GVHD did not affect relapse rates. Conclusion:These results indicate that in patients with standard-risk AML at the time of HCT, chronic GVHD or CMV infection after HCT were crucial for reducing relapse. Immune modulation mediated by chronic GVHD or CMV infection may have played a role in preventing disease relapse. The use of donor lymphocyte infusion before relapse may reduce the recurrence for selected patients who do not develop chronic GVHD. Disclosures:No relevant conflicts of interest to declare.