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Introduction In acute myeloid leukemia (AML), FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is a common driver mutation associated with high tumor burden and poor prognosis. This mutation is common in normal karyotype AML and such patients have high leukocyte count. The presence of this mutation can be predicted by certain hematological and immunophenotypic characteristics in day-to-day practice. Objective This study was undertaken to assess the strength of association between FLT3 gene mutation and hematological and immunophenotypic characteristics. Materials and Methods Morphological, hematological, immunophenotypic, and cytogenetic characteristics of FLT3 mutations recorded in 424 patients of AML in adults and children between 2016 and 2019 in a tertiary care cancer center in Western India. Blasts were classified according to French-American-British method. Tumor burden was assessed by serum lactate dehydrogenase (LDH) levels, leucocyte count, and peripheral smear blast percentage. Results Out of 424 cases, FLT3-ITD and FLT3-tyrosine kinase domain mutation were found in 72 and 25 AML patients, respectively. Patients with FLT3 mutation had high tumor burden, characterized by high leukocyte count (p < 0.001), peripheral blood (p = 0.01) and bone marrow (p = 0.03) blast percentage, and high serum LDH (mean 777.8 vs. 586; p = 0.10) compared with FLT3-negative patients. They also featured high platelet count (p < 0.001). Morphological, immunophenotypic, and cytogenetic characteristics also have been presented in the study. Conclusion Observations of the study suggest the presence of definitive hematological and immunophenotypic characteristics along with raised serum LDH levels serve as surrogate markers and indicators of FLT3 mutation in AML patients.

DNA length polymorphisms are found in many serious diseases, and assessment of their length and abundance is often critical for accurate diagnosis. However, measuring their length and frequency in a mostly wild-type background, as occurs in many situations, remains challenging due to their variable and repetitive nature. To overcome these hurdles, we combined two powerful techniques, digital polymerase chain reaction (dPCR) and high-speed atomic force microscopy (HSAFM), to create a simple, rapid, and flexible method for quantifying both the size and proportion of DNA length polymorphisms. In our approach, individual amplicons from each dPCR partition are imaged and sized directly. We focused on internal tandem duplications (ITDs) located within the FLT3 gene, which are associated with acute myeloid leukemia and often indicative of a poor prognosis. In an analysis of over 1.5 million HSAFM-imaged amplicons from cell line and clinical samples containing FLT3-ITDs, dPCR-HSAFM returned the expected variant length and variant allele frequency, down to 5% variant samples. As a high-throughput method with single-molecule resolution, dPCR-HSAFM thus represents an advance in HSAFM analysis and a powerful tool for the diagnosis of length polymorphisms.

Detection of FLT3 gene mutations in acute myeloid leukemia is now recognized as an unfavorable factor that affects the disease course, emerging the risk of relapses and overall survival shortening and disease-free survival of patients. The aim of the study was to determine the frequency of mutations of the gene FLT3 and to assess their impact on clinical indicators, overall survival and disease-free survival in patients with acute myeloid leukemia. We compared complete blood count parameters, karyotype, duration of overall survival and disease-free survival in 199 patients with acute myeloid leukemia depending on the presence or absence of mutations of the FLT3 gene. Significant differences across these groups were discovered only in WBC and blasts between the group of patients with acute myeloid leukemia (FLT3+) and without mutations in the FLT3 gene (FLT3-). The differences between two groups were also identified in patients chromosomal aberrations. Significant differences (p=0,00024) in the duration of overall survival between groups of patients with acute myeloid leukemia with mutations of FLT3-ITD+, FLT3-TKD+ and FLT3- were demonstrated. Median overall survival was: 1 6 months for patients with mutation FLT3-ITD+ and 17 months for FLT3-TKD+ patients and not achieved for FLT3- patients. The use of modern molecular genetic methods of research in acute myeloid leukemia allows to improve the diagnosis of the disease, as well as to carry out risk stratification and individualize therapy. The use of targeted therapy for FLT3-positive patients who are not candidates for hematopoietic stem cell transplantation will increase the effectiveness of the treatment and improve the performance of overall survival and disease-free survival.

The molecular consequences of coding mutations can often be predicted simply from their effect on a gene’s sequence. Noncoding mutations require more work. In this issue of Blood, Yang and colleagues(1) use 3D genomics to make an important contribution to the list of functional noncoding mutations in cancer. They show that microdeletions at 13q12.2 in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) eliminate the boundary of a topologically associated domain (TAD) at the FLT3 locus, which results in higher expression of FLT3, an important driver gene in acute leukemias.

Data on outcome in older (≥70 years) patients with acute promyelocytic leukemia after treatment with arsenic trioxide (ATO) compared with standard chemotherapy (CTX) is scarce. We evaluated 433 patients (median age, 73.4 years) treated either with ATO+ all-trans retinoic acid (ATO/ATRA; n = 26), CTX/ATRA + ATO during consolidation (CTX/ATRA/ATO; n = 148), or with CTX/ATRA (n = 259). Median follow-up for overall survival (OS) was 4.8 years. Complete remissions (CR) were achieved in 92% with ATO/ATRA and 82% with CTX/ATRA; induction death rates were 8% and 18%, respectively. For analysis of postremission outcomes we combined the ATO/ATRA and CTX/ATRA/ATO groups (ATO/ATRA ± CTX). Cumulative incidence of relapse (CIR) was significantly lower after ATO/ATRA ± CTX compared with CTX/ATRA (P P = 0.20). High (>10 × 109/l) white blood cell (WBC) counts at diagnosis were associated with higher CIR (P P = 0.48). ATO, when added to ATRA or CTX/ATRA is feasible and effective in elderly patients for remission induction and consolidation, particularly in patients with high WBC at diagnosis.

The association of FLT3 mutations with white blood cell (WBC) counts at diagnosis and early death was studied in patients with acute promyelocytic leukemia (APL). Publications indexed in databases of biomedical literature were analyzed. Potential publication bias was evaluated by analyzing the standard error in funnel plots using the estimated relative risk (RR). Mixed-effect models were used to obtain the consolidated RR. All analyses were conducted using the R statistical software package. We used 24 publications in the final meta-analysis. Of 1005 males and 1376 females included in these 24 publications, 645 had FLT3-ITD (internal tandem duplication) mutations. Information on FLT3-D835 mutations was available in 10 publications for 175 patients. Concurrent occurrence of the two mutations was rare. WBC count at diagnosis was ≥10 × 109/L in 351 patients. For patients with the FLT3-ITD mutation, RR was 0.59 for overall survival (OS) and 1.62 for death during induction. For those with FLT3-D835 mutations, the RR was 0.50 for OS and 1.77 for death during induction. RR for WBC count ≥10 × 109/L was 3.29 and 1.48 for patients with FLT3-ITD and FLT3-D835, respectively. APL patients with FLT3-ITD or FLT3-D835 are more likely to present with elevated WBC counts and poorer prognosis than those without these mutations.

To the editor: Whether, and for which patients with acute myeloid leukemia, the dose of daunorubicin in induction should be 90 mg/m2 continues to be discussed. There seems little doubt that 90 mg/m2 is superior to a 45 mg/m2 dose.[1][1][⇓][2]-[3][3] In this context, the reports from the Eastern

Acute myeloid leukemia (AML) is a cancerous disease affecting the myeloid line of the bone marrow cells. FLT3, also known as CD135, is a proto-oncogene, which, if mutated, leads to different types of cancer. The protein it encodes presents tyrosine-kinase activity, and its intratandem mutation, FLT3-ITD, leads to uncontrolled proliferation of myeloblasts and worse outcomes in AML patients. There are currently several pharmacological agents that can inhibit the effect of either the proteins with tyrosine-kinase activity or the mutated FLT3 gene. We present the case of a 68-year-old patient, smoker, with a history of arterial hypertension, chronic obstructive pulmonary disease, presenting with headache unresponsive to antalgics, dyspnea after physical exertion, and epistaxis, with onset 2 months prior to his presentation. The patient was diagnosed with AML with positive FTL3 mutation for which conventional induction therapy was initiated. Within the next days, the patient presented several complications related to the disease itself or caused by the treatment, which eventually led to his death.

Whether or not FLT3 mutations are present and expressed within a leukemic hematopoietic stem cell has engendered some controversy. New evidence has now been presented on this issue that could change the way we manage the disease in the future.

Objective: The most frequent acquired molecular abnormalities and important prognostic indicators in patients with Acute Myeloid Leukaemia (AML) are fms-like tyrosine kinase-3 gene (FLT3) and nucleophosmin-1 (NPM1) mutations. Our study aims to develop a cost effective and comprehensive in-house conventional PCR method for detection of FLT3-ITD, FLT3-D835 and NPM1 mutations and to evaluate the frequency of these mutations in patients with cytogenetically normal (CN) AML in our population. Methods: A total of 199 samples from AML patients (95 women, 104 men) were included in the study. Mutation analyses were performed using polymerase chain reaction (PCR) and gene sequencing. Result: Sixty-eight patients were positive for the mutations. FLT3-ITD mutations were detected in 32 patients (16.1%), followed by FLT3-D835 in 5 (2.5%) and NPM1 in 54 (27.1%). Double mutations of NPM1 and FLT3-ITD were detected in 23 cases (11.6%). Assays validation were performed using Sanger sequencing and showed 100% concordance with in house method. Conclusion: The optimized in-house PCR assays for the detection of FLT3-ITD, FLT3-D835 and NPM1 mutations in AML patients were robust, less labour intensive and cost effective. These assays can be used as diagnostic tools for mutation detection in AML patients since identification of these mutations are important for prognostication and optimization of patient care.

TO THE EDITOR: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy, and T-cell ALL (T-ALL) accounts for approximately 15% of cases of childhood ALL.[1][1],[2][2] Early T-cell precursor ALL (ETP-ALL) is a recently recognized subtype of T-ALL.[3][3],[4][4] Hallmarks of T-ALL

FLT3-internal tandem duplication (FLT3-ITD) mutations are common in acute promyelocytic leukemia (APL) and render the disease more difficult to manage or cure. In this issue of Blood, Esnault et al begin to unravel how the mutation-activated FLT3 receptor impedes the effects of all-trans retinoic acid (ATRA) and how arsenic counters this.1

When the FLT3 gene is mutated, it originates a modified receptor with structural changes, which give survival advantage and malignant hematopoietic cell proliferation. Thus, the presence of mutations in this gene is considered an unfavorable prognostic factor. A total of 85 consecutive samples of newly diagnosed untreated patients with AL were included in the study after they provided their informed consent. FLT3 gene mutations were detected by PCR. For the pediatric group, a positive correlation was observed between WBC count and the presence of FLT3-ITD in patients with AML and ALL. Furthermore, children with AML who had the FLT3-ITD mutation showed a tendency to express CD34 in blast cells. In the adult group, the AML patients with FLT3-ITD who expressed CD34 in blast cells had a tendency to worse progression. The present data indicate no association between the prognostic factors evaluated and FLT3 gene mutations in adult with AL. Yet, the presence of FLT3-ITD mutation was significantly related with WBC count in the pediatric group. These findings demonstrate that FLT3 gene mutations can be considered as independent poor prognostic factors.

In contrast to FLT3 ITD mutations, in-frame deletions in the FLT3 gene have rarely been described in adult acute leukemia. We report two cases of AML with uncommon in-frame mutations in the juxtamembrane domain of the FLT3 gene: a 3-bp (c.1770_1774delCTACGinsGT; p.F590_V592delinsLF) deletion/insertion and a 12-bp (c.1780_1791delTTCAGAGAATAT; p.F594_Y597del) deletion. We verified by sequencing that the reading frame of the FLT3 gene was preserved and by cDNA analysis that the mRNA of the mutant allele was expressed in both cases. Given the recent development of FLT3 inhibitors, our findings may be of therapeutic value for AML patients harboring similar FLT3 mutations.

FLT3 internal tandem duplication (FLT3ITD) are common mutations in acute myeloid leukemia (AML) associated with poor patient prognosis. Although new generation FLT3 tyrosine kinase inhibitors (TKI) have shown promising results, the outcome of FLT3ITD AML patients remains poor and demands the identification of novel, specific and validated therapeutic targets for this highly aggressive AML subtype. Utilizing an unbiased genome-wide CRISPR/Cas9 screen, we identify GLS, the first enzyme in glutamine metabolism, as synthetically lethal with FLT3-TKI treatment. Using complementary metabolomic and gene-expression analysis, we demonstrate that glutamine metabolism, through its ability to support both mitochondrial function and cellular redox metabolism, becomes a metabolic dependency of FLT3ITD AML, specifically unmasked by FLT3-TKI treatment. We extend these findings to AML subtypes driven by other tyrosine kinase (TK) activating mutations, and validate the role of GLS as a clinically actionable therapeutic target in both primary AML and in vivo models. Our work highlights the role of metabolic adaptations as a resistance mechanism to several TKI, and suggests glutaminolysis as a therapeutically targetable vulnerability when combined with specific TKI in FLT3ITD and other TK activating mutation driven leukemias.

SEL24/MEN1703 is a first-in-class, orally available, dual PIM/FLT3 kinase inhibitor currently investigated in patients with Acute Myeloid Leukemia (AML) in the first-in-human study CLI24-001 (NCT03008187). PIM and FLT3 kinases are considered to play an important role in AML and are inhibited by SEL24/MEN1703. Moreover, there is evidence that inhibition of PIM kinases might contribute to overcoming acquired resistance induced by approved FLT3 inhibitors1. In AML, different signal transducers in the FLT3 pathway are substrates of kinases. Therefore, their phosphorylation levels might be modulated by kinase inhibitors and may be exploited as a potential pharmacodynamic biomarker in clinical development. In particular, phosphorylation of S6, 4E-BP1, and STAT5 is regulated by both FLT3 and PIM1/2. Thus, the objective of this investigation was to identify the most promising pharmacodynamic biomarker/s for implementation in the clinical trials of SEL24/MEN1703. Initially, we assessed the in vitro cytotoxic effect of SEL24/MEN1703 in a panel of 26 AML cell lines harboring different genetic mutations, to identify suitable cell lines for subsequent experiments. In the selected panel of AML cell lines, SEL24/MEN1703 resulted in the inhibition of phosphorylation of S6, 4-EBP1 and STAT5 as measured by immunoblotting. Notably, the reduction in phosphorylated S6 (pS6) in response to SEL24/MEN1703 was particularly evident. Since SEL24/MEN1703 displays a broad cytotoxic activity in AML cell lines, we clustered sensitive and resistant cell lines considering 0.5 μM as the IC50 cut-off value. Then, we investigated the relationship between SEL24/MEN1703 cytotoxic activity in AML cell lines and the inhibition of the above mentioned phosphorylated proteins in a 24-hour cytotoxic assay, showing a correlation between IC50 and the reduction of pS6 (Pearson correlation coefficient: -0.6905, R2= 0.477). To further confirm the in vitro data, SEL24/MEN1703 ability to modulate phosphoproteins was assessed also in xenograft mice bearing MOLM-16 cell line. The phosphorylation status of S6, 4E-BP1 and STAT5 was analyzed by immunoblot in tumor tissues from mice treated at 25 mg/kg of SEL24/MEN1703 at baseline and at 4, 8, and 16 hours after treatment. Results showed that also in vivo, SEL24/MEN1703 administration resulted in a decrease of pS6, with maximum reduction in this parameter observed 4 hours after the administration of the investigational compound. Based on these results, pS6 was identified as the pharmacodynamic biomarker to be implemented in the CLI24-001 clinical trial. Among different available methods, flow cytometry was selected as the preferred platform to analyze patient samples, because of its ability to provide quantitative assessment of cellular events and pharmacodynamic evaluation in a selected, relevant cell subpopulation, such as the AML blast cells. The assessment of pS6 in the clinical trial is planned both at baseline and at cycle 1 day 14 for whole blood and bone marrow. In addition, pS6 levels will be measured in whole blood at additional time points during treatment cycles. We have implemented the measurement of pS6 in the CLI24-001 trial, and pS6 levels as well as their relationship with the main pharmacokinetic parameters in patients treated with SEL24/MEN703 at 100 and 125 mg will be presented. 1Green A.S. et al., Pim kinases modulate resistance to FLT3 tyrosine kinase inhibitors in FLT3-ITD acute myeloid leukemia, Sci Adv, 2015 Disclosures Tomirotti: Menarini Ricerche S.p.A.: Employment. Merlino:Menarini Ricerche S.p.A.: Employment. Fiascarelli:Menarini Ricerche S.p.A.: Employment. Baldini:Menarini Ricerche S.p.A.: Employment. Tagliavini:Menarini Ricerche S.p.A: Employment. Borella:Menarini Ricerche S.p.A.: Employment. Mazan:Selvita S.A.: Employment. Brzózka:Selvita S.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Bressan:Menarini Ricerche S.p.A.: Employment. Pellacani:Menarini Ricerche S.p.A.: Employment; Amgen: Equity Ownership. Salerno:Menarini Ricerche S.p.A.: Employment. Binaschi:Menarini Ricerche S.p.A.: Employment. Bellarosa:Menarini Ricerche S.p.A.: Employment.

Background: FLT3-mutated (mu) AML pts have poor outcomes. IDH1mutated (IDH1m) and IDH2m co-occur with FLT3 mutations in 15-27% and 8-30% of AML, respectively (Boddu P, et al. Leukemia 2017; 31:2526-2529). FLT3-ITD is a strong risk factor for relapse and since multiple FLT3 inhibitors (FLT3i's) are available, it is important to further determine how the efficacy of these targeted is impacted by concurrent mutation/s. Methods: We reviewed FLT3-mu AML pts with concurrent IDHm's between Jan 2011-Dec 2018 who had received at least one first or second generation FLT3-inhibitor (FLT3i)-based therapy in frontline and/or relapsed/refractory (R/R) setting. We are not discussing IDHi based therapies in double mutant pts this abstract. Mutation testing was performed using a NGS-based analysis for the detection of somatic mutations in the coding sequences of 28 or 81 genes on DNA extracted from BMA, as previously described (33). We analyzed the characteristics of these pts, responses to therapy, and outcomes. Results: Eighty two FLT3, IDH 'double mutated' AML pts were identified. One pt had FLT3-TKD mu and IDH1m, and 2 pts had FLT3-TKD mu and IDH2m and were excluded. We focus here on the 79 pts with concurrent FLT3-ITD mu and IDH1m or IDH2m. 30 pts had concurrent FLT3/IDH1m: 19 (63%) in frontline and 11 (37%) in the R/R setting. 49 had concurrent FLT3/IDH2m: 31 (63%) in frontline and 18 (37%) in the R/R setting. The median follow-up was 8.3 months [1.2 - 115] and 10.5 months [0.3 - 57.8] months for pts with concurrent FLT3/IDH1m and FLT3/IDH2m, respectively. The median number of prior therapies was 2 [1 - 8] in both R/R settings. The most frequent mutations were IR132H (77%) and IR140Q (98%) in pts with FLT3/IDH1m and FLT3/IDH2m, respectively. Patient characteristics are shown in Table 1. As expected, the incidence of adverse cytogenetics in FLT3/IDH mutated pts was low (7/79; 9%). The most common co-occurring mutations in FLT3/IDH1 pts were NPM1 (66%) and DNMT3A (43%). Similarly in FLT3/IDH2: NPM1 (53%) and DNMT3A (31%). FLT3i's were given as a single agent or in combination with cytotoxic chemotherapy (CCT) or low intensity therapy (LIT) (hypomethylating agents and low-dose cytarabine) in the frontline or R/R setting (Table 2). CR/CRi rate was highest with CCR with FLT3i, with 83% and 78% of frontline and R/R FLT3/IDH pts achieving CR/CRi with this approach. Among pts who could not receive CCT based therapies, LIT with FLT3i showed encouraging activity with CR/CRi rates of 47% and 40% in frontline and R/R FLT3/IDH pts, respectively. LIT alone was not very effective with CR/CRi rates of 0 and 33% in frontline and R/R FLT3/IDH pts, although numbers are small. Similarly, single agent FLT3 showed modest activity with CR/CRi in 16% of the R/R FLT3/IDH pts. As expected FLT3/IDH2 pts had improved overall survival (OS) and event free survival (EFS) compared with FLT3/IDH1 pts. The median OS was 11.2 and 2.88 in frontline and R/R FLT3/IDH1 pts, respectively. The median OS was 20.6 and 10.3 in frontline and R/R FLT3/IDH2 pts, respectively. The median EFS was 4.98 and 4.27 in frontline FLT3/IDH1 pts who received CCT+FLT3i and LIT+FLT3i, respectively (p=0.10). The median EFS was 14.2 and 8.2 in frontline FLT3/IDH2 pts who received CCT+FLT3i and LIT+FLT3i, respectively (p=0.09). Conclusion:FLT3/IDH2 pts have better EFS and OS than FLT3/IDH1 pts in frontline and R/R setting with FLT3i in combination with CCT or LIT. CCT in combination with FLT3i appears to be optimal therapy for FLT3/IDH co-mutated pts with CR/CRi rates of 83% in frontline, and 78% in R/R setting. In pts unable to tolerate CCT a combination of LIT with FLT3i is a reasonable approach yielding CR/CRi rates of 40-45% in frontline and R/R FLT3/IDH pts. LIT alone and FLT3-inhibitor alone demonstrated lower activity with CR/CRi rates of 14-16% in double mutant pts. Disclosures Kadia: Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Bioline RX: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding. Konopleva:Astra Zeneca: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Genentech: Honoraria, Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Eli Lilly: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Calithera: Research Funding; Agios: Research Funding. Ravandi:Macrogenix: Consultancy, Research Funding; Xencor: Consultancy, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Menarini Ricerche: Research Funding; Cyclacel LTD: Research Funding; Selvita: Research Funding. Cortes:Novartis: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding; BiolineRx: Consultancy; Forma Therapeutics: Consultancy, Honoraria, Research Funding; Immunogen: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Biopath Holdings: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Astellas Pharma: Consultancy, Honoraria, Research Funding; Merus: Consultancy, Honoraria, Research Funding; Sun Pharma: Research Funding. Jabbour:Pfizer: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Cyclacel LTD: Research Funding. Garcia-Manero:Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding; Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding. Short:Takeda Oncology: Consultancy, Research Funding; AstraZeneca: Consultancy; Amgen: Honoraria. Sasaki:Pfizer: Consultancy; Otsuka: Honoraria. Pemmaraju:novartis: Consultancy, Research Funding; plexxikon: Research Funding; Daiichi-Sankyo: Research Funding; abbvie: Consultancy, Honoraria, Research Funding; sagerstrong: Research Funding; affymetrix: Research Funding; celgene: Consultancy, Honoraria; cellectis: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; samus: Research Funding; incyte: Consultancy, Research Funding; mustangbio: Consultancy, Research Funding. Borthakur:Argenx: Membership on an entity's Board of Directors or advisory committees; FTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Tetralogic Pharmaceuticals: Research Funding; Janssen: Research Funding; NKarta: Consultancy; Bayer Healthcare AG: Research Funding; Novartis: Research Funding; Incyte: Research Funding; Agensys: Research Funding; Merck: Research Funding; GSK: Research Funding; Arvinas: Research Funding; Xbiotech USA: Research Funding; Cyclacel: Research Funding; BioTheryX: Membership on an entity's Board of Directors or advisory committees; Strategia Therapeutics: Research Funding; BMS: Research Funding; AstraZeneca: Research Funding; PTC Therapeutics: Consultancy; Cantargia AB: Research Funding; BioLine Rx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai: Research Funding; Oncoceutics: Research Funding; Eli Lilly and Co.: Research Funding; Oncoceutics, Inc.: Research Funding; Polaris: Research Funding; AbbVie: Research Funding. Kantarjian:Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Amgen: Honoraria, Research Funding; BMS: Research Funding; Cyclacel: Research Funding; Takeda: Honoraria; Immunogen: Research Funding; Ariad: Research Funding; Novartis: Research Funding; Agios: Honoraria, Research Funding; Jazz Pharma: Research Funding; Astex: Research Funding; Pfizer: Honoraria, Research Funding. DiNardo:celgene: Consultancy, Honoraria; jazz: Honoraria; syros: Honoraria; daiichi sankyo: Honoraria; agios: Consultancy, Honoraria; medimmune: Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees; abbvie: Consultancy, Honoraria. Daver:Celgene: Consultancy; Glycomimetics: Research Funding; NOHLA: Research Funding; BMS: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Immunogen: Consultancy, Research Funding; Otsuka: Consultancy; Karyopharm: Consultancy, Research Funding; Jazz: Consultancy; Forty-Seven: Consultancy; Novartis: Consultancy, Research Funding; Agios: Consultancy; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Servier: Research Funding; Hanmi Pharm Co., Ltd.: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Astellas: Consultancy.

BACKGROUND: FLT3 alterations in Acute Myeloid Leukemia (AML) occur as either point mutations in the tyrosine kinase domain (TKD) or internal tandem duplications (ITD), both of which result in constitutive activation of FLT3; however, only FLT3-ITD has prognostic value. Per NCCN guidelines, when NPM and FLT3-ITD alterations co-occur, an allele frequency ≥ 0.5 for FLT3-ITD (FLT3-ITDhigh) confers an intermediate prognosis, while allele frequencies < 0.5 (FLT3-ITDlow) confers a favorable prognosis. For allele frequency to be ≥ 0.5, loss of heterozygosity (LOH) or copy number gains at the FLT3 locus are required. In this study, we examined a large cohort of FLT3 mutated AML samples to study LOH at the FLT3 locus. DESIGN: During routine clinical care, 2129 AML samples were evaluated by comprehensive genomic profiling (CGP) for 406 genes via DNAseq for all classes of genomic alterations and 265 genes via RNAseq for rearrangements, using a hybrid-capture next generation sequencing assay (FoundationOne®Heme). Of these samples, 1379 met analytic specifications required for LOH analysis. LOH analysis was performed by first generating copy number models from exon and SNP log ratio and minor allele fractions (maf). LOH was then determined using the modeled copy number, maf, and tumor purity. Samples with low aneuploidy were reviewed manually. RESULTS: The median age of the overall cohort was 60 y (range LOH analysis was performed for 236 AML samples with a FLT3 alteration and 224 randomly selected wild-type FLT3 AML samples as controls. Samples with a FLT3 alteration had a significantly higher rate of LOH compared to the control samples (34/236 (14.4%) versus 2/224 (0.9%), p = 9.0x10-9). Samples with a FLT3-ITD were under significantly greater LOH than samples with a point mutation (33/171 (19.3%) versus 4/115 (3.5%), p = 2.8x10-5). Samples with only a point mutation rarely had LOH (1/94, 1.0%), not significantly different than FLT3 wild-type controls. Copy number analysis revealed that FLT3-altered samples had a rate of FLT3 copy number gains not significantly different than control samples (10/236 (4.2%) versus 7/224 (3.1%)). The allele frequency was significantly higher for FLT3-ITD alterations compared to FLT3-TKD alterations (median 0.11 versus 0.24, p < 0.001), and FLT3-ITD alterations under LOH had a higher allele frequency than those not under LOH (median 0.35 versus 0.24, p< 0.01). Only 1/115 FLT3-TKD alterations had an allele frequency > 0.5 versus 7/171 of the FLT3-ITD alterations. Five (of 7) FLT3-ITDhigh samples were under LOH and the remaining 2 samples had FLT3 copy number gains. FLT3-ITD mutated AML has co-occurring genomic alterations of clinical significance; however, with this sample size there were no statistically significant differences in the frequencies between samples with and without LOH at the FLT3 locus (NPM1: 64% vs 43%, DNMT3A: 48% vs 37%, WT1: 36% vs 25%, RUNX1: 12% vs 17%, NRAS: 3% vs 16%). CONCLUSIONS: In AML, the FLT3 locus has increased LOH when a FLT3-ITD is present, compared to FLT3 wild-type controls and samples with FLT3-TKD alterations. Copy number alterations in FLT3 are not different in FLT3-altered vs FLT3 wild type AML. Allele frequencies were higher for FLT3-ITDs compared to FLT3-TKDs and were highest for FLT3-ITDs under LOH. An emerging negative prognostic indicator in AML is FLT3-ITDhigh. Determination of FLT3-ITDhigh status requires high tumor purity, copy number gains, and/or LOH. The requirement of high tumor purity makes FLT3-ITDhigh status dependent on factors other than tumor biology, such as tumor sampling. In our dataset, 33 (19%) FLT3-ITD samples were under LOH but only 5 (4%) were FLT3-ITDhigh. LOH of FLT3-ITDs has also been associated with worse prognosis in AML and further studies are warranted to determine if allele frequency or absence of wild-type FLT3 drives these prognostic correlations. FLT3-ITD LOH is more common than FLT3-ITDhigh and could provide more accurate, sample independent risk stratification for patients with FLT3-ITD+ AML. Disclosures Severson: F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Sokol:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Madison:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Duncan:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Hemmerich:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Edgerly:Foundation Medicine, Inc: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Huang:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Britt:Foundation Medicine, Inc: Employment. Vergilio:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Elvin:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Reddy:Foundation Medicine, Inc: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Sathyan:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Alexander:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Ross:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Ali:Foundation Medicine, Inc: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Ramkissoon:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment.

Background: Aberrant expression levels of several miRs have been reported to independently associate with outcome of patients (pts) with CN-AML. In these reports, miR expression was profiled using microarray assays, which interrogate a selected subset of miRs. The advent of next-generation sequencing (NGS) has allowed unbiased measurement of miR expression, but, to our knowledge, NGS has not been used to identify miRs associated with prognosis of AML pts. Here, we analyze small RNA sequencing (smRNA-seq) data from a large cohort of younger adults with CN-AML, for whom outcome data were available, with the goal to identify new prognostic miRs. Methods: We performed smRNA-seq in 281 younger adults (aged 18-59 y) with de novo CN-AML. Cytogenetic analyses were performed in Cancer and Leukemia Group B (CALGB)/Alliance institutional laboratories using standard banding techniques; mutational analyses were done centrally using a targeted DNA sequencing platform. All pts were treated on frontline CALGB/Alliance protocols. Results: We first evaluated which miRs associated with overall survival (OS) in univariable analysis; we detected 9 such miRs. We then used a machine-learning approach, namely random forests, to identify miRs whose concomitant expression could generate an effective outcome predictor in CN-AML. To account for the effect of co-existing prognostic gene mutations, we included the European LeukemiaNet (ELN) risk group status in the random forest analyses. A total of 8 prognostic miRs were identified, 4 of which were also found to be prognostic in univariable analysis (underlined below), thus bringing the total number of unique prognostic miRs to 13: miR-511, miR-1193, miR-155, miR-4517, miR-3681, miR-2355, miR-628, miR-1266, miR-6715a, miR-1180, miR-6715b, miR-132, miR-146b. We used partitioning around medoids to divide our pts into clusters, based on the combined expression levels of the 13 prognostic miRs. Two such clusters were identified: cluster 1 comprised 173 pts and cluster 2 contained 108 pts. Regarding pretreatment and molecular features, pts in cluster 1 had lower percent of bone marrow blasts (P=.04), and had more frequently biallelic CEBPA mutations (P Conclusion: Unbiased profiling of miRs using smRNA-seq has identified a novel set of 13 miRs with prognostic significance in CN-AML. MiR expression-based cluster status independently associates with clinical outcome of CN-AML pts. Our preliminary in vitro experiments have shown that miR-511, whose association with prognosis was the strongest among the newly identified prognostic miRs, is functionally relevant in AML. Disclosures Uy: Astellas: Consultancy; Pfizer: Consultancy; Curis: Consultancy; GlycoMimetics: Consultancy. Powell:Rafael Pharmaceuticals: Consultancy, Research Funding; Novartis: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Research Funding; Janssen: Research Funding. Kolitz:Astellas: Research Funding; Boeringer-Ingelheim: Research Funding; Roche: Research Funding. Byrd:Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Genentech: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; BeiGene: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; BeiGene: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau.

Aging-associated immune remodeling (AAIR) leads to an impaired ability to respond to vaccination and combat infections, and is due to many factors acting in concert. Several studies have linked the T-cell decline that occurs with age to thymic involution. However, there is novel and mounting evidence that also aged lymphoid-primed multipotent progenitors (LMPPs) are immune system intrinsic players in AAIR. However, very little is known on molecular and cellular mechanisms by which aging LMPPs could drive this AAIR phenomenon. Deciphering the underlying mechanisms is of crucial importance for developing new therapies to attenuate AAIR. Here, we present new data demonstrating the dysregulated pathways associated with aged LMPPs and the cellular changes in early thymic differentiation events in driving AAIR. To assess the T-lineage potential of aged LMPPs, we performed single cells ex-vivo OP9D assays using LMPPs (Lin-cKit+Sca1+CD34-Flt3hi) from aged (18-20 month-old) and young (8-10 week-old) C57BL/6 animals as controls. The frequencies of T-cell lineage potential in aged LMPPs and young LMPPs at the single cell level were very similar. This result was also validated in vivo by transplantation assays where 5000 aged or young LMPPs were injected into sub-lethally irradiated young recipients followed by T-cell lineage (CD4+ & CD8+) analysis in the peripheral blood (PB) at 4 weeks post transplantation (Mean; Y:12,75 vs A:16:23 % of total, p=0.46). However, aged LMPPs were associated with a dramatic disadvantage of PB T-cells at 4 weeks post injections (Mean; Y:9 vs A:2.3 % of total, p We also performed RNA-Seq analyses on LMPPs from young and aged mice. Unsupervised hierarchical clustering of differentially expressed genes between young and aged LMPPs highlighted a clear dysregulation of only a few pathways that are involved in T-cell development such as Notch signaling. We next correlated our RNA-Seq data with other immunological signatures in attempt to look for more T-cell specific key factors that are differentially expressed between young and aged LMPPs. Importantly, the results show that the data from our RNA-Seq correlated with more than 400 immunological signatures among which 25 were most highly correlated. Interestingly, this correlation has allowed us to curate a list of the top 30 differentially expressed genes between young and aged LMPPs including T-cell specific transcription factors such as Satb1 and Foxo1. Altogether, our findings reveal that the T-cell immune decline that occurs with age is already imprinted in LMPPs within the bone marrow and translates into a dysregulation of signaling pathways that are directly related to T-cell development. Targeting these pathways could open up new perspectives in attenuating AAIR. Disclosures No relevant conflicts of interest to declare.

Myeloid malignancies can evolve from prior hematologic disorders, most commonly AML evolving from MDS. AA and PNH are not malignant conditions but they can evolve to myeloid malignancies with a lower frequency compared to MDS and other myeloid and myeloproliferative diseases. PNH can evolve to myeloid disorders with an incidence rate of 4-10%. Here we aim to revisit the subject of malignant evolution of PNH to myeloid disorders by analyzing the molecular background of PNH using modern NGS technologies. Clinical characteristics, demographics and mutational profiles of patients were collected at The Cleveland Clinic Foundation. In total a cohort of 243 patients evaluated for hemolytic PNH (n=83), AA (39) and AA/PNH (n=121) followed for a period of 22 years was evaluated. Inclusion criteria were: complete flow cytometric panels of PNH cells, patients without antecedent AA (pPNH, 45), patients evolving from AA with PNH clone >20% (sPNH, 38), karyotype (at diagnosis of PNH and transformation), clinical parameters including time to malignant progression, and molecular characterization resulting from NGS performed using various library preparation systems (TruSeq, TruSight, Nextera), comparison of molecular mutations with control cohorts (AA, 160; MDS, 835). The incidence rate of myeloid disorders in our PNH cohort was 3% (7/243). Among hemolytic PNH patients, 7 patients progressed to AML (n=1), MDS (n=5) or myelofibrosis (n=1). Median age was 48 yr (range, 24-80); M/F, 5/2. Median PNH clone size was 71% (range, 29-99). Three progressors were in the pPNH and 4 in the sPNH group. Time to malignant diagnosis was A total of 45 somatic mutations equally distributed in the 2 groups (pPNH, 22; sPNH, 23) and with similar VAFAVG (pPNH vs. sPNH 38% vs. 33%) were found. PNH showed a higher proportion of individuals with mutations compared to AA/PNH+AA (42 vs. 22% of cases with ≥1 mutation; P=.002) with a median VAF percentage significantly higher in PNH vs. AA/PNH+AA (40 vs. 19%; P55), 17% were equivocal (possibly hits biclonal with a VAFSUM between 45-55), while 13% were more likely a result of clonal chimerism (hits present in different clones; VAFSUM Clonal evolution of hemolytic PNH to MDS/AML is rare, but still occurs and it is accompanied by mutations in typical myeloid genes (BCOR, NPM1, TET2, U2AF1) which in permissive circumstances are capable to change the cell's fate favoring clonal evolution. Our results suggest that most PNH cases can carry additional mutations in the same clone and these mutations can be secondary hits, with PIGA mutations being the founder lesions. However even when mutations in myeloid genes are dominant, the phenotype of the patients is inferred by PIGA. This observation supports the nature of PNH as a monogenic disease with clinical manifestations resulting by PIGA mutations rather than by myeloid genes. Disclosures Sekeres: Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

BACKGROUND: After many decades of stagnation, several promising novel therapies have recently been approved for the management of patients (pts) with Acute Myeloid Leukemia (AML). Many of these target pts in specific clinical and molecular subsets such as midostaurin/gilteritinib for FLT3-mutated AML, liposomal daunorubicin-cytarabine for secondary AML, and fractionated gemtuzumab ozogamicin (GO) for good risk AML. To address the dismal outlook for elderly AML patients, we now have venetoclax or glasdegib combinations. Single agent FLT3 inhibitors (resulting in ~4 m improvement in overall survival (OS)) and IDH inhibitors (response rate 40%, doubled OS in responders) have shown to be superior to traditional approaches in the relapsed setting. Although these agents are exciting, a majority of pts with AML present with intermediate and high-risk disease and only a small subset will have actionable mutations. In order to determine the impact of these newer therapies on outcome for our pts, we investigated AML outcomes for pts diagnosed and treated since 2017 and compared these with survival for similar pts treated in the prior two years (y). METHODS: We performed a retrospective chart review to identify newly diagnosed pts treated with chemotherapy over a 2y period (2015-17) prior to the first FDA approval of Midostaurin in April, 2017 (old AML era or group 1) and the 2y since approval (2017-19; new AML era or group 2). We reviewed charts of 138 AML pts meeting these criteria at our institution: 79 in group 1 and 59 in group 2. Demographics, disease-specific variables, as well as outcomes of interest (overall survival (OS), overall response rate (CR/CRi)) were collected on an IRB-approved protocol. Responses were defined according to the 2003 International Working Group (IWG) criteria. Demographics, baseline characteristics, and treatment responses were analyzed using descriptive statistics. Overall survival was estimated utilizing Kaplan-Meier (KM) survival analysis. RESULTS: Clinical characteristics were comparable in both groups. Median age was 65y with slight differences in gender distribution (Table 1). ELN risk categories (Döhner, Blood. 2017) across the groups were similarly distributed; a majority of pts had intermediate and adverse risk characteristics. As expected, more pts received newer therapies in group 2. 6 pts (7.6%) in group 1 received new drugs (as part of clinical trials), while 31 (52%) received newer therapies in group 2. Newer therapies were mostly GO (28%), midostaurin (11%) and venetoclax (11%) with lesser percentages of the more specific targeted therapies. Median follow-up was 10m for group 1 and 8m for group 2. Median OS was 13m for group 1 and 20m for group 2, but the difference was not statistically significant (p=0.29) [Figure 1]. OS was significantly better in the subgroup of older AML pts (age ≥ 60y); median OS was 7m vs. 11m in groups 1 and 2 respectively (p=0.01).Rates of CR from induction therapy were comparable in both groups (68% in group 1 versus 65% in group 2). A larger number of pts (especially older pts) in group 2 went on to allogeneic bone marrow transplant (allo-HCT). This was perhaps due to increased recognition of performance status over age in selection of pts for allo-HCT and increased utilization of reduced intensity and non-myeloablative conditioning regimens. CONCLUSIONS: Our results indicate a positive impact on survival for pts diagnosed in the era of novel therapies in a real world setting, specifically for pts presenting at older ages. A larger cohort analysis to further evaluate these findings is currently underway. The expansion of the therapeutic armamentarium as well as expanded transplant options is encouraging and offers new hope for pts diagnosed with AML Disclosures Griffiths: Onconova Therapeutics: Other: PI on a clinical trial; Partner Therapeutics: Consultancy; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Appelis Pharmaceuticals: Other: PI on a clinical trial; Onconova Therapeutics: Other: PI on a clinical trial; Appelis Pharmaceuticals: Other: PI on a clinical trial; Boston Scientific: Consultancy; Genentech, Inc.: Research Funding; Genentech, Inc.: Research Funding; New Link Genetics: Consultancy; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Celgene, Inc: Consultancy, Research Funding; Celgene, Inc: Consultancy, Research Funding; Abbvie, Inc.: Consultancy, PI on a clinical trial; Abbvie, Inc.: Consultancy; New Link Genetics: Consultancy; Persimmune: Consultancy; Persimmune: Consultancy; Boston Scientific: Consultancy; Novartis Inc.: Consultancy; Partner Therapeutics: Consultancy; Novartis Inc.: Consultancy. Wang:Kite: Other: Advisory role; Jazz: Other: Advisory role; Amgen: Other: Advisory role; Agios: Other: Advisory role; Abbvie: Other: Advisory role; Daiichi: Other: Advisory role; Stemline: Other: Advisory role, Speakers Bureau; Pfizer: Other: Advisory role, Speakers Bureau; celyad: Other: Advisory role; Astellas: Other: Advisory role, Speakers Bureau. Thota:Incyte, Inc.: Speakers Bureau.

Targeted inhibitors to oncogenic kinases demonstrate encouraging clinical responses early in the treatment course, however, most patients will relapse due to target-dependent mechanisms that mitigate enzyme-inhibitor binding or through target-independent mechanisms, such as alternate activation of survival and proliferation pathways, known as adaptive resistance. One example involves the FMS-like receptor tyrosine kinase (FLT3). Activating mutations of FLT3 result in its autophosphorylation and initiation of intracellular signaling pathways, which induce abnormal survival and proliferation of leukemic cells.One of the most common mutations in acute myeloid leukemia (AML) involves the internal tandem duplication (ITD) of FLT3, which occurs in ~25% of all cases of newly diagnosed AML and confers a particularly poor prognosis. FLT3 inhibitors (FLT3i) evaluated in clinical studies as monotherapy and combination therapies have shown good initial response rates; however, patients eventually relapse with FLT3i-resistant disease. The absence of durable remission in patients treated with potent and selective FLT3i highlights the need to identify resistance mechanisms and develop additional treatment strategies. Several mechanisms contribute to resistance to selective FLT3i, including mutations in the tyrosine kinase domain of FLT3 (20-50%) or activation of parallel signaling mechanisms that bypass FLT3 signaling, referred to as adaptive resistance (30-50%). Here we describe mechanisms of adaptive resistance in FLT3-mutant AML by examining in-cell kinase and gene regulatory network responses after oncogenic signaling blockade by FLT3 inhibitors (FLT3i). Through this integrative approach, we identified activation of innate immune stress response pathways after treatment of FLT3-mutant AML cells with FLT3i. Utilizing genetic approaches, we demonstrated that innate immune pathway activation via IRAK1 and IRAK4 contributes to adaptive resistance in FLT3-mutant AML cells. The immediate nature of IRAK1/4 activation in adaptively resistant FLT3-ITD AML cells requires concomitant inhibition of these targets to avoid compensatory signaling and cell survival. Achieving optimal multi-drug combination regimens that yield extended overlapping exposure while avoiding unwanted toxicities is challenging. Therefore, we desired a small molecule inhibitor that simultaneously targeted the FLT3 and IRAK1/4 kinases to eradicate adaptively resistant FLT3-ITD AML. To overcome this adaptive resistance mechanism, we developed and optimized a novel small molecule that simultaneously inhibits FLT3 and IRAK1/4 kinases. The FLT3-IRAK1/4 inhibitor exhibited potent binding affinity for IRAK1 (KD= 2.9 nM), IRAK4 (KD= 0.3 nM), and FLT3 (KD= 0.3 nM), as well as acceptable pharmacokinetic properties in mice. Moreover, a high-resolution crystal structure demonstrates that the FLT3-IRAK1/4 inhibitor binds as a type I inhibitor (ATP-competitive binding to the active state). The FLT3-IRAK1/4 inhibitor eliminated adaptively resistant FLT3-mutant AML cell lines and patient-derived samples in vitro and in vivo, and displayed superior efficacy as compared to current targeted FLT3 therapies. Our study demonstrates that therapies that simultaneously inhibit FLT3 signaling and compensatory IRAK1/4 activation have the potential to improve the therapeutic efficacy in patients with FLT3-mutant AML. In conclusion, these findings reveal that inflammatory stress response pathways contribute to adaptive resistance in FLT3-mutant AML and suggests that this mechanism may extend to other malignant cells undergoing a stress-induced response to therapy. Disclosures Hoyt: Kurome Therapeutics: Consultancy. Berman:Astellas: Membership on an entity's Board of Directors or advisory committees, Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Amgen: Honoraria; Lilly: Honoraria; Gilead: Consultancy; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Roche: Consultancy, Research Funding; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees. Rosenbaum:Kurome Therapeutics: Consultancy, Employment. Perentesis:Kurome Therapeutics: Consultancy. Starczynowski:Kurome Therapeutics: Consultancy.

Objectives: Constitutive activation of the fms-like tyrosine kinase 3 (FLT3) receptor by internal tandem duplication (ITD) of the juxtamembrane region has been described in patients with acute myeloid leukemia. FLT3/ITDs are present in about 20-30% of all acute myeloid leukemia cases. It has been shown that the mutation is correlated with worse prognosis. However, none of the previous studies investigated which FAB subtype is associated with higher percentage of FLT3/ITD, thus the reason for undertaking the current study.Methods: The prevalence and the potential prognostic impact of FLT3 mutations in 39 acute myeloid leukemia patients were analyzed by genomic polymerase chain reaction. Twelve samples with FLT3/ITDs and 27 acute myeloid leukemia samples without the mutations were compared with respect to clinical prognosis and FAB subtype. Results were correlated with cytogenetic data and the clinical response.Results: FLT3/ITD mutations were found in 31% of patients. FLT3/ITD was associated with similar clinical characteristics and was more prevalent in patients with normal karyotype (83%). Interestingly, half of the FLT3/ITD aberrations were found in patients with FAB M1 (50%), and fewer were found in patients with FAB M2 (8%), M4 (8%), and M5 (8%). Although less frequent in patients with cytogenetic aberrations, FLT3/ITDs were found in 17% of patients with t(15;17). Although the study was powered to 80%, patients with FLT3/ITD mutation did not show shorter complete remission duration or a higher relapse rate.Conclusion: The data confirm that FLT3/ITD mutations represent a common alteration in adult acute myeloid leukemia, mainly with normal karyotype (83%) and de novo acute myeloid leukemia (75%), as compared to secondary acute myeloid leukemia (25%) (p

Objective To compare the clinical and laboratory characteristics, therapeutic efficacy and prognosis of acute myelogenous leukemia (AML) with or without FLT3-ITD gene mutation. Methods The clinical data of 93 AML patients, aged from 10 to 66 years, were retrospectively analyzed. Among them, 21 patients were FLT3-ITD gene (FLT3-ITD+AML group) positive, and 72 patients were negative for FLT3-ITD gene (FLT3-ITD－AML group). The patients' chromosomes were examined, and mutation of FLT3-ITD and NPM1 gene and the expressions of HOX11, ETO, EVI and NPM1 gene were analyzed, along with the 2-year event-free survival and 2-year overall survival were learned. Results At primary diagnosis, the proportion of patients with WBC>100×109/L, and bleeding rate was significantly higher in FLT3-ITD+ AML group than that in FLT3-ITD－AML group (P=0.04; P=0.01), but no statistical difference was found in age, immunophenotype and chromosomal abnormality (P>0.05). The complete remission (CR) rate was 28.57% in FLT3-ITD+ AML group, and it was obviously lower than that in FLT3-ITD－AML group (55.56%, P=0.013). The 2-year event-free survival was 29.2% in FLT3-ITD+ AML group, and it was significantly lower than that in FLT3-ITD－AML group (37.7%; P=0.04). Meanwhile, 33.3% of the patients in FLT3-ITD－AML group were also positive for NPM1 gene expression, and all of them did not get complete remission. However, only 1.39% of the patients expressed NPM1 gene in FLT3-ITD－AML group, but 50% of them got complete remission (P=0.022). Conclusion Patients with FLT3-ITD+AML were easily associated with high WBC counts, bleeding, lower CR rate, poor event-free survival, and poor prognosis.

In this issue of Blood , [Ellegast et al][1] demonstrate that MISTRG mice allow high-level, reproducible engraftment of favorable prognosis acute myeloid leukemia (AML), a group that has been recalcitrant to attempts to develop valuable preclinical patient-derived xenograft (PDX) models.[1][2] Over

FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations resulting in constitutive kinase activity are common in acute myeloid leukemia (AML) and carry a poor prognosis. Several agents targeting FLT3 have been developed, but their limited clinical activity suggests that the inhibition of other factors contributing to the malignant phenotype is required. We examined gene expression data sets as well as primary specimens and found that the expression of GLI2, a major effector of the Hedgehog (Hh) signaling pathway, was increased in FLT3-ITD compared to wild-type FLT3 AML. To examine the functional role of the Hh pathway, we studied mice in which Flt3-ITD expression results in an indolent myeloproliferative state and found that constitutive Hh signaling accelerated the development of AML by enhancing signal transducer and activator of transcription 5 (STAT5) signaling and the proliferation of bone marrow myeloid progenitors. Furthermore, combined FLT3 and Hh pathway inhibition limited leukemic growth in vitro and in vivo, and this approach may serve as a therapeutic strategy for FLT3-ITD AML.

Objective: To identify the well-known common translocations and FLT3 mutations in childhood acute myelogenousleukemia (AML) patients in Turkey. Material and Methods: The study included 50 newly diagnosed patients in which t(15;17), t(8;21), and inv(16)chromosomal translocations were identified using real-time PCR and FLT3 gene mutations were identified via direct PCR amplification PCR-RE analysis. Results: In all, t(15;17) chromosomal aberrations were observed in 4 patients (8.0%), t(8;21) chromosomal aberrationswere observed in 12 patients (24.0%), inv(16) chromosomal aberrations were observed in 3 patients (6.0%), and FLT3-ITD mutations were observed in 2 patients (4.0%); FLT3-D835 point mutation heterozygosity was observed in only 1patient (2.0%) patient. Conclusion: Despite of the known literature, a patient with FLT3-ITD and FLT3-D835 double mutation shows a bettersurvival and this might be due to the complementation effect of the t(15;17) translocation. The reportedmutation ratein this article (4%) of FLT3 gene seems to be one of the first results for Turkish population.

Coexpression of NUP98/NSD1 and FLT3/ITD in AML is associated with very low complete remission rates and poor survival.It is the interaction between NUP98/NSD1 and FLT3/ITD that determines poor outcome in NUP98/NSD1-associated AML.

Introduction: The phase III RATIFY study (Stone et al, NEJM 2017) demonstrated that the addition of the FLT3 inhibitor midostaurin to intensive induction and consolidation courses improves outcome in younger FLT3-positive AML patients. The toxicity and efficacy profile of adding midostaurin to chemotherapy in patients not originally included in the RATIFY study is unknown. We sought to characterize midostaurin use in a 'real-world' setting. Methods: Patients (>18 years) with FLT3-positive AML (ITD/TKD) were eligible to receive midostaurin through the Novartis extended access program that was launched in Israel in April 2016. In order to control for toxicity and efficacy outcomes in the midostaurin-treated patients, a historical control cohort was created that included patients with FLT3-positive AML from 2 participating centers that were not treated with midostaurin (all patients diagnosed after January 2015). Data were extracted from electronic patient records and were collected from several medical centers in Israel. Base-line characteristics, disease- and patient- specific parameters as well as relapse-rates and overall survival were analyzed and compared between the midostaurin treated and untreated cohorts. We used Cox regression to analyze predictors for survival. This study was approved by the Institutional Review Board. Results: Thirty-five patients were included in the analysis. The median age of the patients was 62 years (range 27-78); 40% and 20% of patients were over the age of 65 and 70 years, respectively. FLT3-ITD mutations were detected in 32 patients (91%) and 3 patients had TKD mutations (9%). Eight patients (23%) had secondary leukemia, 83% had normal karyotype and 57% were NPM1-mutated. No differences were noted between the midostaurin-treated group (n=21) and the historical control cohort (n=14) in terms of age, gender, leukemia ontogeny, cytogenetics, presenting blood counts, extramedullary involvement, performance status and comorbidity scales. More patients in the midostaurin group were found to be NPM1-mutated (66 vs. 44%, p=0.03). Furthermore, no differences were noted between the groups in terms of daunorubicin dose for induction (45, 60 and 90 mg/m2/dayin 20, 30 and 50% of patients, respectively), number of consolidations (median number of cycles - 2), cytarabine dose or allogeneic transplantation rate (45 and 36% in the midostaurin and control group, respectively). The full 14 day midostaurin course was given in most patients during induction (73%). In 5 patients midostaurin was initiated only at the post-induction courses due to technical delays in drug supply. Only 4 patients experienced dose reductions or interruptions during therapy: 3 during induction (septic shock, drug interaction and QT prolongation) and 1 during consolidation (new onset atrial fibrillation). Toxicity was comparable between the cohorts. Febrile neutropenia during induction was noted in 95 and 93% of patients in the midostaurin and control groups, respectively. Time to neutrophil and platelet recovery were also comparable (25 vs. 24 days and 24 vs. 20 days in the midostaurin and control groups respectively). Other toxicities were uncommon and not significantly different between the groups. CR/CRi rates were 85% and 58% in the midostaurin and control cohorts, respectively (p=0.17). The median follow-up time for surviving patients in the midostaurin and control cohorts were 426 and 517 days, respectively (p=0.55). During follow-up, 7 deaths occurred in the midostaurin group and 9 in the control arm. Median survival was not reached for the midostaurin treated group and was 281 days for the control group (Figure 1, p=0.42). Nine and 6 patients in remission relapsed in the midostaurin and control group, respectively, translating into a relapse-rate of 53% and 86%, respectively (p=0.19). No difference in early death rate was noted between the groups. The only factor that significantly affected overall survival in the COX-regression analysis was white blood cell count at diagnosis (p=0.03). Conclusions: In the off-trial setting, midostaurin is administered across all age groups and various FLT3-positive subtypes. In this 'real-life' setting, midostaurin is well tolerated and does not significantly add to the toxicity of chemotherapy. Longer follow-up and more patients are needed to assess the efficacy of adding midostaurin to chemotherapy in this group of patients. Figure 1. Figure 1. Disclosures Ofran: Novartis: Other: Served on a Novartis advisory board.