FLT3 Tyrosine Kinase Domain Mutations Research Articles

A study of the synthetic route and resistance of gilteritinib

Gilteritinib is used to treat acute myeloid leukemia (AML) carrying FMS-like tyrosine kinase 3 (FLT3) mutations. As it is a new drug, there are still many aspects that need improvement. This article will be written according to the following three points: Analysis of its mechanism of action with the receptor; Analysis of the three main synthetic routes of gilteritinib and reasonable suggestions are given; Causes and solutions to the emergence of drug resistance. The following 4 points have been found that:(1) FLT3-internal tandem duplication (ITD) mutation and FLT3-tyrosine kinase domain (TKD) mutation, leukemic cells' ability to proliferate, differentiate, and survive aberrant changes due to both kinds of mutations. (2) Gilteritinib combines with FLT3's ATP pocket, and the compound shaped from FLT3 to gilteritinib was synthesized by the juxtamembrane (JM) domain. (3) Synthesis route 3 in this paper is most suitable for industrial mass production. (4) Resistance to gilteritinib is divided into primary resistance and secondary resistance, research in the future should focus on finding new therapeutic targets.

V-FAST Master Trial: Subgroup Analysis of Outcomes with CPX-351 Plus Midostaurin in Adults with Newly Diagnosed Acute Myeloid Leukemia By FLT3 Mutation Type

CPX-351 (United States: Vyxeos®; European Union/United Kingdom: Vyxeos® liposomal), a dual-drug liposomal encapsulation of daunorubicin and cytarabine in a synergistic 1:5 molar ratio, is approved for newly diagnosed, therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes in patients aged ≥1 year in the United States and in adults in the European Union/United Kingdom. In a phase 3 study in older adults with newly diagnosed, high-risk/secondary AML, CPX-351 significantly improved overall survival and remission rates versus conventional 7+3 chemotherapy, with a comparable safety profile. Preclinical data suggest CPX-351 may have synergistic activity with targeted agents, including the FLT3 inhibitor midostaurin (MID). V-FAST (Vyxeos - First Phase Assessment with Targeted Agents) is an open-label, multicenter, multi-arm, nonrandomized, phase 1b master trial (NCT04075747) to evaluate the safety and preliminary efficacy of CPX-351 combined with targeted agents (MID, venetoclax, enasidenib). Eligible adults in the CPX-351 + MID cohort were aged 18 to 75 years, had newly diagnosed AML (de novo AML or secondary AML) with a FLT3 mutation, were fit for intensive chemotherapy, and had an ECOG performance status of 0 to 2. The dose-exploration phase (3+3 design) determined a recommended phase 2 dose of CPX-351 100 units/m2 (daunorubicin 44 mg/m2 + cytarabine 100 mg/m2) on Days 1, 3, and 5 + MID 50 mg twice daily on Days 8 to 21. There were no dose-limiting toxicities, and additional patients were enrolled in the expansion phase at this dose. Herein, we report outcomes for a subgroup analysis based on the presence of a FLT3 internal tandem duplication (ITD) and/or tyrosine kinase domain (TKD) mutation within the cohort of adults primarily with de novo AML who were treated with CPX-351 + MID in the V-FAST trial. A total of 23 patients had sufficient data available to be included in the general safety and efficacy analyses in this ongoing trial at the time of the data cutoff on March 22, 2022. Of the patients included in this analysis, 18 patients had a FLT3 ITD mutation and 6 patients had a FLT3 TKD mutation; 1 patient had both FLT3 mutation types and was included in both subgroups. Patient baseline characteristics are shown in the Table. A diagnosis of de novo AML was reported for 78% of patients with FLT3 ITD mutation and all (100%) patients with a FLT3 TKD mutation. Treatment-emergent adverse events (TEAE) occurring in >50% of patients with a FLT3 ITD or TKD mutation, respectively, included febrile neutropenia (78%; 83%), nausea (67%; 67%), alanine aminotransferase increased (61%; 33%), leukopenia (61%; 50%), and thrombocytopenia (56%; 67%). All patients experienced a ≥3 grade TEAE, primarily hematologic events and infections. Serious TEAEs were reported for 6 (33%) patients with a FLT3 ITD mutation and 2 (33%) patients with a FLT3 TKD mutation; no serious TEAE was reported more than once. Four (22%) patients with a FLT3 ITD mutation and 2 (33%) patients with a FLT3 TKD mutation died during the study; there were no deaths on or before study Day 60. Complete remission (CR) was achieved by 14/17 (82%) patients with a FLT3 ITD mutation and by 5/6 (83%) patients with a FLT3 TKD mutation (Figure). Among patients in CR and with a measurable residual disease (MRD) assessment by multicolor flow cytometry, MRD negativity was achieved by 5/10 (50%) patients with a FLT3 ITD mutation and 1/3 (33%) patients with a FLT3 TKD mutation, all after the first induction cycle. Median (interquartile range) recovery times to an absolute neutrophil count ≥500/μL and platelet count ≥50,000/μL, respectively, were 35 (34, 43) and 34 (28, 36) days for patients in CR with a FLT3 ITD mutation and 30 (29, 35) and 30 (29, 40) days for those in CR with a FLT3 TKD mutation. A total of 10/18 (56%) patients with a FLT3 ITD mutation and 2/6 (33%) patients with a FLT3 TKD mutation have proceeded to hematopoietic cell transplantation to date. In conclusion, preliminary results from the V-FAST trial suggest the combination of CPX-351 + MID is feasible with a manageable safety profile and promising remission rates in adults with newly diagnosed, FLT3-mutated AML. Although conclusions are limited by the small numbers of patients in each subgroup, results are generally consistent for patients with FLT3 ITD and TKD mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Inhibitor of FLT3 and Its Drug-Resistant Mutants with Superior Activity to Gilteritinib in Molm-13 Preclinical Acute Myeloid Leukemia Xenograft Model

Acute Myeloid Leukemia (AML) is a complex heterogenous disease characterized by the aberrant growth of undifferentiated myeloid progenitor cells typically driven by a combination of epigenetic and signaling mutations. In particular, fms-like tyrosine kinase 3 (Flt3) internal tandem duplication (FLT3-ITD) is associated with poor relapse-free and overall survival. Several FLT3 inhibitors have been developed such as the first generation multikinase inhibitor Midostaurin which is approved for newly diagnosed FLT3 mutant AML in combination with induction chemotherapy. Second generation inhibitors such as gilteritinib are more selective and potent and are also approved for relapsed/refractory FLT3 mutant AML. Although initial response has been positive with this class of agents, prolonged remission is rarely achieved, and diverse mechanisms of resistance often arise with FLT3 inhibitors representing an ongoing challenge. Common mechanisms of resistance to FLT3 inhibitors includes the acquisition of tyrosine kinase domain (TKD) secondary mutations such as the gatekeeper F691L TKD mutation, or the emergence of additional mutations in alternative signaling pathways such as RAS/MAPK. Thus, it is important to continue developing novel inhibitors capable of circumventing these resistance mechanisms. Herein we describe 2082-0047, a novel tyrosine kinase inhibitor with sub-nanomolar potency against FLT3 mutant AML, including TKD mutations. Compound 2082-0047 is a highly selective pan-FLT3 kinase inhibitor, as in both scanMAX and KdELECT KINOMEscan™ Profiling assays (Eurofins DiscoverX) it has shown 400-1000 fold selectivity. In our initial screening, 2082-0047 was only effective in FLT3-ITD mutant cells, however the KINOMEscan™ Profiling data described above demonstrates that 2082-0047 is potent against FLT3, FLT3 ITD, D835H, D835V, D835Y, D835V, K663Q, N841I, and R834Q FLT3 TKD mutations. We confirmed this data in a cellular assay using BaF3 cell lines expressing the TKD mutations D835Y, D835H or F691L alone or with Flt3-ITD. Next, we confirmed using immunoblot analysis of phosphorylated Flt3 that 2082-0047 is a potent inhibitor of FLT3-ITD signaling (superior to gilteritinib) in FLT3-ITD mutant AML cell lines. Using wildtype B6 mice, we conducted pharmacokinetic (PK) studies and found that 2082-0047 had 79 % oral bioavailability, achieved 1.2 μΜ Cmax after 4 hours, and had half-life of 4.7 hours. Next, our absorption, distribution, metabolism and excretion (ADME) studies showed lack of CYP inhibition of major human isoforms. Overall, our PK studies demonstrated overall favorable drug like properties enabling in vivo investigation of therapeutic activity. Therefore, we proceeded with using the MOLM-13 disseminated xenograft model in NCG recipient mice to determine the overall survival of 2082-0047 compared to gilteritinib. We enrolled mice 10 days post engraftment to receive daily oral vehicle, 3, 10, 20 or 30 mg/kg of 2082-0047 or 30 mg/kg of gilteritinib and found that 10 and 20 mg/kg of 2082-0047 were well tolerated and superior to gilteritinib at 30 mg/kg (Figure 1). In summary, we describe a novel and potent inhibitor of FLT3, including both internal tandem duplication and drug-resistant tyrosine kinase domain (TKD) mutants, with promising preclinical activity which is superior to the most potent FDA approved FLT3 inhibitor gilteritinib in an aggressive AML in vivo model. Our non-GLP in vivo safety studies confirmed a broad therapeutic window of 2082-0047. Ongoing Investigational New Drug (IND) enabling studies will include comparing the effect of 2082-0047 to gilteritinib in an immunocompetent syngeneic AML murine model harboring both a Flt3-ITD and F691 TKD gatekeeper mutation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

V-FAST master trial: Preliminary results of treatment with CPX-351 plus midostaurin in adults with newly diagnosed FLT3-mutated acute myeloid leukemia.

7043 Background: CPX-351 (US: Vyxeos; Europe: Vyxeos liposomal), a dual-drug liposomal encapsulation of daunorubicin and cytarabine in a synergistic 1:5 molar ratio, is approved for newly diagnosed, therapy-related AML or AML with myelodysplasia-related changes in patients aged ≥1 year in the US and in adults in Europe. In a phase 3 study in older adults with newly diagnosed, high-risk/secondary AML, CPX-351 significantly improved overall survival and remission rates versus conventional 7+3, with a comparable safety profile. Preclinical data suggest CPX-351 may have synergistic activity with targeted agents, including the FLT3 inhibitor midostaurin (MID). Herein, we report preliminary results for the cohort of adults treated with CPX-351 + MID in the V-FAST (Vyxeos – First Phase Assessment with Targeted Agents) trial. Methods: V-FAST is an open-label, multicenter, multiarm, nonrandomized, phase 1b master trial (NCT04075747) to evaluate the safety and preliminary efficacy of CPX-351 combined with targeted agents (midostaurin, venetoclax, enasidenib). Eligible adults in the CPX-351 + MID cohort were aged 18 to 75 years, had newly diagnosed AML with a FLT3 internal tandem duplication (ITD) or tyrosine kinase domain (TKD) mutation, were fit for intensive chemotherapy, and had an ECOG performance status of 0 to 2. The dose-exploration phase (3+3 design) determined a recommended phase 2 dose of CPX-351 100 units/m2 (daunorubicin 44 mg/m2 + cytarabine 100 mg/m2) on Days 1, 3, and 5 + MID 50 mg BID on Days 8 to 21. There were no dose-limiting toxicities, and additional patients were enrolled in the expansion phase at this dose. Results: A total of 23 patients received CPX-351 + MID and had sufficient data to be included in the analysis (cutoff date: 1/20/2022). Patient baseline characteristics are shown in the Table. Treatment-emergent adverse events (TEAEs) in ≥40% of patients included febrile neutropenia (78%), nausea (65%), increased alanine aminotransferase (57%), leukopenia (57%), thrombocytopenia (57%), headache (43%), and hyponatremia (43%). All patients experienced a grade 3/4 TEAE, primarily hematologic events. Nonhematologic grade 3/4 TEAEs in ≥2 patients included pneumonia (17%), lung infection (13%), and hyperglycemia (9%). There were no grade 5 TEAEs and no deaths on or before Day 60. Complete remission was achieved by 18/22 (82%) evaluable patients after the first induction cycle. Conclusions: Preliminary results from the V-FAST trial suggest CPX-351 + MID is feasible, with a manageable safety profile and promising remission rates in adults with newly diagnosed AML who have a FLT3 mutation. Clinical trial information: NCT04075747. [Table: see text]

Midostaurin in the treatment of a patient with acute myeloid leukaemia with FLT3-TKD mutation and NPM1 mutation

Acute myeloid leukaemia (AML) is aggressive cancer with a diverse clinical course which mainly results from the heterogeneous and complex molecular landscape of the disease. For some genetic alterations, in particular mutations in the FMS-like tyrosine kinase 3 ( FLT3 ) gene, the prognosis of patients with newly diagnosed AML depends on the coexistence of other genetic disorders. A thorough understanding of the interactions between mutations is key to improving risk stratification in newly diagnosed AML and personalizing therapy. The addition of midostaurin, an FLT3 tyrosine kinase inhibitor, to standard chemotherapy improved outcomes in the unfavourable prognostic group of AML patients. This research describes a therapeutic strategy in a patient with newly diagnosed AML with FLT3-tyrosine kinase domain (TKD) mutation and wild-type NPM1 who has received a standard remission induction and consolidation chemotherapy in combination with midostaurin, followed by maintenance therapy with an FLT3 inhibitor and haploidentical allogeneic hematopoietic stem cell transplantation.

FLT3 Gene Mutation in Acute Myeloid Leukemia: Correlation with Hematological, Immunophenotypic, and Cytogenetic Characteristics

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.

Midostaurin Plus Intensive Chemotherapy for Younger and Older Patients with Acute Myeloid Leukemia and FLT3 Internal Tandem Duplications

Midostaurin Plus Intensive Chemotherapy for Younger and Older Patients with Acute Myeloid Leukemia and FLT3 Internal Tandem Duplications

The FLT3 F691L Gatekeeper Mutation Promotes Clinical Resistance to Gilteritinib + Venetoclax (GILT + VEN) in AML

Background: FMS-like tyrosine kinase (FLT3) is one the most frequently mutated genes in AML and is associated with poor prognosis. FLT3 internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations occur in up to 30% and 5-10% of AML, respectively. Several small molecule FLT3 inhibitors (FLT3i) have been developed but their use as single agents is limited due to the development of drug resistance. Our lab developed a two-step model of early and late resistance to FLT3i that recapitulates resistance in AML patients (Traer et al. Cancer Res. 2016; Joshi et al. Cancer Cell 2021). Early resistance, also known as AML persistence, is the stage when residual AML cells are dependent upon the marrow microenvironment for survival and patients are clinically responding. Late resistance to GILT was characterized by expansion of intrinsic mutations, with NRAS mutations being the most frequent mutation, in addition to a few gatekeeper FLT3 mutations. Current therapies are looking at combinations to overcome GILT resistance, including chemotherapy, hypomethylating agents (HMAs), and venetoclax (VEN) +/- HMAs. GILT+VEN, in particular, has shown good initial activity in relapsed/refractory FLT3 AML patients (Daver et al. ASH 2020), however the mechanism of resistance to this combination is unknown.Results: Early resistance cell cultures to GILT+VEN were created by exposing MOLM14 cells to GILT 25nM + VEN 25nM alone or supplemented with microenvironmental ligands FGF2 or FLT3 ligand (FL; N=3/group). Media, drugs, and ligands were replenished twice weekly. After 25 weeks, only the cultures exposed to ligand resumed growth (N=1 for FGF2 and N=3 for FL). Ligands were then removed from these early resistant cultures to induce late resistance. There was an initial drop in cell viability but cells resumed growth after only 3.5 weeks (Fig. 1). In contrast, the time to develop early and late resistance to GILT monotherapy was 8 and 15 weeks, respectively. Immunoblot analysis of GILT + VEN early and late resistant cultures demonstrated restoration of FLT3 signaling, as well as phosphorylation of downstream AKT/MAPK pathways. These results also contrasted to late GILT monotherapy resistant cultures, which had downstream AKT/MAPK activation via outgrowth of NRAS mutations. Since FLT3 appeared to be functionally active, we sequenced FLT3 and found that all early and late GILT + VEN resistance cultures had gatekeeper FLT3 F691L mutations. F691L accounted for only in a minority of resistance cultures to GILT monotherapy. To test if FLT3 signaling was important for resistance, we exposed parental cells to higher concentrations of gilteritinib, which have been shown to partly overcome F691L, as well as the FLT3i FF-10101, which binds FLT3 at a different site and is not affected by the F691L mutation. Both of these approaches restored sensitivity to FLT3i in vitro. As expected, the F691L mutation provided broad resistance to most FLT3i (Fig. 2).To validate this mechanism of resistance in patients, we identified a relapsed FLT3-ITD patient who was treated with GILT monotherapy for 5 months, followed by GILT + HMA for 4 cycles, and then GILT + VEN for resistant proliferative disease. After an initial response to GILT + VEN, the leukemia cells began to increase again in the peripheral blood. A repeat genetic test was ordered and the patient was found to have developed a FLT3 F691L mutation at a high variant allele frequency (Fig. 3).Conclusion: We have developed a robust cell line model of early and late resistance to FLT3i that mimics the timing and expansion of resistance mutations in the clinic. Our model of early and late resistance to GILT combinations can prospectively predict mechanisms of resistance. Although uncommon as a mechanism of resistance to GILT monotherapy, our model and early patient data predicts that F691L mutations are more important for GILT + VEN resistance. [Display omitted] DisclosuresTyner: Seattle Genetics: Research Funding; Astrazeneca: Research Funding; Array: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Petra: Research Funding; Constellation: Research Funding; Genentech: Research Funding; Agios: Research Funding; Schrodinger: Research Funding. Traer: ImmunoGen: Membership on an entity's Board of Directors or advisory committees; Schrodinger: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Servier/Agios: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Distinctive Flow Cytometric and Mutational Profile of Acute Myeloid Leukemia With t(8;16)(p11;p13) Translocation.

Acute myeloid leukemia (AML) with t(8;16)(p11;p13) abnormalities is a rare, aggressive, and diagnostically challenging subtype that results in KAT6A-CREBBP gene fusion. To investigate their immunophenotype and genomic features, we identified 5 cases of AML with t(8;16) through a retrospective review of the databases at Northwestern Memorial Hospital in Chicago, IL, and Washington University Medical Center, in St Louis, MO. In all, 4 of 5 cases were therapy related and 1 was possibly therapy related. The leukemic blasts showed distinctive features, including bright CD45 expression and remarkably high side scatter that overlapped with maturing myeloid elements, making the blasts difficult to identify on initial examination. They were positive for CD13, CD33, and CD64 and negative for CD34 and CD117. Next-generation sequencing profiling of 4 cases revealed pathogenic ASXL1 (2 cases), FLT3-tyrosine kinase domain (TKD) mutations (2 cases), and other pathogenic mutations. In 3 patients, t(8;16) was the sole cytogenetic abnormality; additional aberrations were found in 2 patients. Single nucleotide polymorphism microarray revealed 1 case with 7q deletion as a secondary clone. Our data highlight the distinctive immunophenotypic profile of AML with t(8;16), which, along with its unique morphology, often presents a diagnostic challenge. We showed that mutations of either ASXL1 or FLT3-TKD are seen in most cases of this leukemia.

Abstract 1050: Efficacy of AMG 176 in combination with gilteritinib in preclinical models of acute myeloid leukemia

Abstract Acute Myeloid Leukemia (AML) is the most common and aggressive acute leukemia in adults, with a 25% 5-year survival rate. FLT3 is the most frequently mutated gene in AML. About 25% of AML patients harbor FLT3 internal tandem duplication (ITD) mutations, and about 8 % of patients harbor FLT3 tyrosine kinase domain (TKD) mutations. Both FLT3-ITD and FLT3-TKD mutations constitutively activate the protein, which causes poor survival. Gilteritinib is a highly potent and selective oral FLT3 inhibitor recently approved by the FDA. Although gilteritinib showed strong single agent activity in AML patients with FLT3 mutations, the development of gilteritinib resistance limits the long-term efficacy of this treatment, indicating that combination therapy may be advantageous for AML patients with FLT3 mutations. FLT3 mutations are known to be anti-apoptotic. Myeloid cell leukemia-1 (MCL-1), an anti-apoptotic protein, expressed in a large percentage of the AML patient population, plays a critical role in AML cell survival and drug resistance. AMG 176 is a potent, selective and orally bioavailable MCL-1 inhibitor, which induces rapid commitment to apoptosis in AML. Here we demonstrated that AMG 176 and gilteritinib combination treatment synergistically targeted FLT3-ITD mutated AML. A strong synergistic effect (Combination index <0.2) was observed in two FLT3-ITD mutated AML cell lines treated with the AMG 176 plus gilteritinib combination. Increased Bcl-2-like protein 11 (BIM) expression and decreased BIM/MCL-1 interaction was observed under the combination treatment, coupled with a significant increase in apoptosis. In the MV4-11 AML xenograft model, mice treated with two cycles of AMG 176 (2 days on 5 days off) at 30 mg/kg or with 3 mg/kg gilteritinib daily showed 76% or 74% tumor growth inhibition respectively. A statistically significant increase in efficacy was observed in the combination treatment group, with 92.8% tumor regression, including 50% of mice being tumor free. No significant difference in body weight was found between groups. Our results show that AMG 176 plus gilteritinib treatment was effective and well tolerated in mouse models of AML, which supports further evaluation in a clinical setting. Citation Format: Xiaoyue Chen, Sean Caenepeel, Brian Belmontes, Patricia L. McElroy, Karen Rex, Tao Osgood, Paul Hughes. Efficacy of AMG 176 in combination with gilteritinib in preclinical models of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1050.

Mutation Patterns Identify Adult Patients with De Novo Acute Myeloid Leukemia (AML) Aged 60 Years or Older Who Respond Favorably to Standard Chemotherapy: an Analysis of Alliance Studies

Abstract Background: Despite changes in treatment regimens, the patient's age (and the corresponding treatment intensity) remains one of the most important prognostic factors in AML. Thus far, only 5-15% of AML patients aged ≥60 years (y) are cured with chemotherapy. Identification of patients who are more (or less) likely to respond to standard cytarabine/daunorubicin-based chemotherapy might help to enable early risk stratification toward alternative treatment regimens. Aims: To provide a comprehensive molecular profile of a large cohort of adults with de novo acute myeloid leukemia (AML) aged ≥60 y, and to determine if specific molecular and/or cytogenetic features could identify patients who are more likely to respond to standard chemotherapy. Methods: We used a next-generation sequencing panel of 80 cancer- and/or leukemia-associated genes to profile molecularly 423 patients aged ≥60 y with de novo AML and available cytogenetic data similarly treated on Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology. Results: Overall, we detected 1377 mutations, with a median of 3 mutations per patient (range: 0-8). The outcome of the whole patient cohort was poor, with complete remission (CR) rate of 55% and disease-free (DFS) and overall survival (OS) 3y-rates of 14%. In multivariable analyses (MVA), mutated NPM1 was the only factor positively associated with achieving CR (P Summary: We identified combinations of mutations whose presence is associated with favorable outcome in older AML patients treated with standard chemotherapy. Patients with intermediate and adverse molecular features should be considered for alternative treatment regimens. Download : Download high-res image (186KB) Download : Download full-size image Figure 1 . Disclosures Stone: Otsuka: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Ono: Membership on an entity's Board of Directors or advisory committees; Orsenix: Membership on an entity's Board of Directors or advisory committees; Cornerstone: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Argenix: Other: DSMB; Arog: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Actinium: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Fujifilm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Jazz: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Seattle genetics: Membership on an entity's Board of Directors or advisory committees; Sumitomo: Membership on an entity's Board of Directors or advisory committees. Kolitz: Gilead, Magellan, Novartis, Pharmacyclics, and Seattle Genetics: Consultancy; Gilead, Magellan, and Novartis: Honoraria; Celgene, Jazz: Equity Ownership; Boehringer Ingelheim, Cantex, Erytech, and Millennium: Research Funding; Gilead, Novartis, and Seattle Genetics: Other: Travel Support. Powell: Rafael Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Byrd: Janssen: Research Funding; Genentech: Research Funding; Acerta Pharma: Research Funding; Pharmacyclics: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S.

TP-0903, a Novel Axl Inhibitor with Activity in Drug Resistant FLT3-ITD+ AML through a Mechanism That Includes FLT3 Inhibition

Abnormal expression of receptor tyrosine kinase Axl has been associated with poor prognosis in different cancers including acute myeloid leukemia (AML) and has been linked to drug resistance against chemotherapy as well as targeted therapies including FLT3 inhibitors (Park et al. Leukemia 2015). With its role in tumorigenesis, Axl has been actively pursued as a potential therapeutic target with small molecule inhibitors. TP-0903, a type I inhibitor, was designed in silico on an Axl scaffold that does not clash with the Axl gatekeeper residue, Leu620 (Mollard et al. ACS Med Chem Lett 2011). Since Axl inhibitors are known to also interact with FLT3 (Myers et al. J Med Chem 2016), we evaluated the hypothesis that TP-0903 demonstrates preclinical activity against FLT3 -ITD+AML through a mechanism that potentially involves both Axl and FLT3 inhibition.TP-0903 was evaluated against Axl and FLT3 wildtype (WT), ITD and tyrosine kinase domain (TKD) mutations in binding and kinase assays. Inhibition of cell viability was evaluated in Ba/F3 cells transfected with GFP vector control (VC), FLT3 -ITD, or TKD mutations, FLT3 -ITD+ AML cell lines (MOLM13, MOLM13-Res harboring a ITD/D835Y mutation, and MV4-11), and primary diagnostic FLT3 -ITD+ blast samples. Inhibition of FLT3 signaling was determined by Western blot analysis. In vivo activity was evaluated in systemic MOLM13 and MOLM13-Res cell xenograft models in NOD scid gamma (NSG) mice; 1x106 luciferase expressing cells were injected intravenously to female NSG mice, and TP-0903 or vehicle was administered by oral gavage for 3 weeks, starting on day 7-10 post tail vein injection. TP-0903 40-60 mg/kg was administered once daily (qd), and 80 mg/kg was administered on Monday, Wednesday, and Friday (M/W/F). Progression of leukemia was monitored using bioluminescence imaging and Kaplan-Meir analysis was performed for animal survival.TP-0903 had potent binding affinities to Axl, FLT3 WT and all FLT3 mutants evaluated, expressed as Kd: Axl (8.2 nM), FLT3 WT (0.93 nM), ITD (5.6 nM), D835H (1.9 nM), D835V (2.1 nM), D835Y (1.4 nM), ITD/D835Y (0.79 nM), and ITD/F691L (1.9 nM). In a kinase assay, TP-0903 inhibited FLT3 ITD and D835Y with IC50 values of 3.9 nM and 0.12 nM, respectively. TP-0903 inhibited the viability of Ba/F3 transfected with VC (mean IC50 of 3 independent experiments, 47 nM), and activity was more potent against all cells expressing different FLT3 mutants: FLT3 -ITD (15 nM), D835H (16 nM), D835Y (22 nM), ITD/D835H (16 nM), ITD/D835Y (20 nM), and ITD/F691L (16 nM). Western blot analysis of lysates from TP-0903-treated Ba/F3 cells expressing ITD, D835Y, ITD/D835Y, or ITD/F691L mutants showed inhibition of phospho-FLT3 and phospho-STAT5 at 10-100 nM. In FLT3 -ITD+ AML cell lines, TP-0903 demonstrated potent activity with mean IC50 values of 21 nM, 16 nM, and 17 nM in MOLM13, MOLM13-Res, and MV4-11 cells, respectively; the IC50 in primary blast samples ranged from 38-80 nM. In vivo, TP-0903 delayed the outgrowth of MOLM13 cells and significantly prolonged survival in a dose/schedule dependent manner compared to vehicle-treated mice with the following median survival: vehicle (16 days), TP-0903 40 mg/kg qd (26 days, P=0.0039), TP-0903 60 mg/kg qd (32 days, P=0.002), and 80 mg/kg M/W/F (24 days, P=0.0015). In an ongoing MOLM13-Res xenograft model, TP-0903 60 mg/kg qd has suppressed the outgrowth of leukemic cells up to 11 days of treatment compared to vehicle-treated mice (P=0.0002).TP-0903, a novel Axl inhibitor with FLT3 inhibition activity, shows promising in vitro and in vivo activity in de novo and drug resistant FLT3 -ITD+ AML. Given that TP-0903 was initially designed to bind to the gatekeeper leucine residue in Axl, TP-0903 may overcome resistance to the clinically relevant FLT3 gatekeeper F691 substitution mutation to leucine, which most FLT3 inhibitors are vulnerable to. Further in vitro studies to evaluate mechanism of action and in vivo studies for dose optimization and efficacy evaluations in additional models of FLT3 -ITD+ AML are ongoing. DisclosuresWhatcott:Tolero Pharmaceuticals, Inc.: Employment. Warner:Tolero Pharmaceuticals, Inc.: Employment. Blum:Boerhinger Ingelheim: Research Funding; Pfizer: Consultancy; Astellas: Consultancy.

Molecular Profiling in Extramedullary Acute Myeloid Leukemia Reveals Distinct Subgroups Defined By Mutations in KIT and RUNX1

Background: Between 2 to 23% of patients with acute myeloid leukemia manifest extramedullary involvement including myeloid sarcoma. While the genomic landscape of AML and the respective clinical and outcome correlations are well characterized, our understanding of the molecular underpinnings of extramedullary AML remains limited. Various cytogenetic abnormalities have been described in association with extramedullary AML, including core binding factor (CBF) translocations t(8;21) and inv(16). Recent studies have also applied targeted gene sequencing approaches (21 to 39 genes) in small patient cohorts (6-18) to generate limited mutational profiles of extramedullary AML [Li et al. Leukemia 2015; Pastoret et al. Leukemia & Lymphoma 2016; Kashofer et al. Leukemia & Lymphoma]. Here we utilize a comprehensive next generation sequencing (NGS) panel interrogating 585 genes implicated in hematological malignancies in 17 patients and provide the most comprehensive mutational profile of extramedullary AML to date.Methods: 17 pathologically confirmed cases of extramedullary AML were identified. Genomic DNA extraction was performed from formalin-fixed, paraffin-embedded (FFPE) tumor samples. Isolated DNA was sent for sequencing using the HemePACT panel, a custom hybridization capture-based NGS assay encompassing 585 genes related to hematologic malignancies. All HemePACT data were compared to an unmatched normal sample. Data were passed to standard pipelines used to detect candidate substitutions and indels (Caveman and pindel respectively). Candidate variants were manually reviewed to eliminate single nucleotide polymorphisms and artifacts and to find a subset of high-confidence putative oncogenic variants. Cytogenetic data was obtained from standard clinical karyotyping and fluorescence in-situ hybridization assays from bone marrow aspirates from patients with bone marrow AML involvement.Results: Demographic data for sequenced extramedullary AML patients is provided in Table 1. We identified a total of 80 mutations. The median number of mutations /sample is 4 (range:1-11). The most frequently mutated genes are KIT found in 7/17 patients (41%) and RUNX1 found in 6/17 (35%) patients. Intriguingly, KIT and RUNX1 mutations are mutually exclusive(p<0.05), and 13/17 (76.5%) sequenced extramedullary AML samples have mutations in either KIT or RUNX1 . (Figure 1) DNMT3A mutations are found in 3/17 (17%) patients. FLT3-tyrosine kinase domain (TKD) mutations are found in 4/17 (23%) of patients. DNMT3A and FLT3-TKD mutations co-segregate with RUNX1 (2/3 and 2/4 respectively) but not with KIT mutations. As only 2/17 patients underwent cytogenetic evaluation from extramedullary AML, mutational data from extramedullary AML was largely integrated with cytogenetic data from bone marrow aspirates. 7/17 (41.1%) of sequenced patients had CBF translocations (6/7 with inv(16)/t(16;16), and 1/7 with t(8;21) present in bone marrow cytogenetic evaluation by karyotype or FISH. Out of 7 patients with CBF translocations, 6/7 (85.7%) have KIT mutations present in extramedullary AML. 1 patient with inv(16) found by karyotyping from myeloid sarcoma did not have a KIT mutation in extramedullary AML. No patients with RUNX1 mutations in extramedullary AML (0/6) have CBF translocations. Kaplan-Meier analysis from this small cohort did not show a statistically significant difference in overall survival between patients with KIT and RUNX1 mutations (p=0.57).Overall, our data suggest the potential for distinct and specific mutational profiles associated with AML extramedullary involvement.Conclusions: Our study provides the most comprehensive mutational profiling of extramedullary AML to date. Combined molecular and cytogenetic profiling demonstrate that extramedullary AML is characterized by a distinct repertoire of acquired mutations enriched for mutations in KIT and RUNX1. Importantly our data identify two distinct and non-overlapping molecular subgroups. One subgroup is defined by CBF alterations and KIT mutations, while the second most prominent subtype is defined by mutations in RUNX1. Recognition of these subtypes and their co-mutations may form the basis for genotype-clinical correlations, as well as experimental models to explore the biology of extramedullary AML. Additional extramedullary AML patient samples are being sequenced and will be presented. [Display omitted] DisclosuresGoldberg:Genentech: Research Funding; Pfizer: Research Funding; ADC Therapeutics: Research Funding. Levine:Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership; Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership.

81030 Utilizing a synergistic drug combination to target relapsed/refractory FLT3 mutant AML

ABSTRACT IMPACT: This work will help to understand a novel therapeutic approach to a common type of acute myeloid leukemia. OBJECTIVES/GOALS: FMS-like tyrosine kinase 3 (or FLT3) mutations occur in ˜30% of acute myeloid leukemia (AML) cases. FLT3 tyrosine kinase domain (TKD) mutations are particularly important in relapsed/refractory FLT3 mutant AML, which portends poor prognosis. This study describes a therapeutic approach to overcoming resistance conferred by FLT3-TKD mutations. METHODS/STUDY POPULATION: To understand the efficacy of a novel type 1 FLT3 inhibitor (NCGC1481), as a monotherapy and combination therapy, several assays were utilized to interrogate functionality of these therapies. Cell lines and patient samples containing aspartate 835 to tyrosine mutations (D835Y, the most common TKD alteration) and phenylalanine 691 to leucine (F691L) were utilized to examine the effects of NCGC1481 with and without other targeted therapies like MEK inhibitors. Specifically, assays measuring viability, cell death using flow cytometry, in vitro clonogenicity, cellular signaling, and xenograft survival were examined in these FLT3-TKD AML models. Synergy was also measured using well described methods, which also allowed for appropriate dose finding for drug combination experiments. RESULTS/ANTICIPATED RESULTS: Our novel type 1 FLT3 inhibitor (NCGC1481) was particularly effective in the most common FLT3 TKD mutant, D835Y. NCGC1481 reduced viability and cell signaling, while also inducing cell death and prolonging xenograft survival in the FLT3-D835Y model system. In contrast, clinically approved FLT3 inhibitors were less effective at suppressing AML cells expressing FLT3-D835Y. In the case of FLT3-F691L, most of the FLT3 inhibitors tested, including NCGC1481, suppressed canonical FLT3 signaling, but did not significantly reduce viability or leukemic clonogenicity. However, when NCGC1481 was combined with other targeted agents like MEK inhibitors, at synergistic doses, eradication of the FLT3-F691L AML clone was substantially increased. DISCUSSION/SIGNIFICANCE OF FINDINGS: In AML, response to FLT3 inhibitor therapy is often short-lived, with resistance sometimes occurring via FLT3-TKD mutations. Given the dismal prognosis of relapsed FLT3 mutant AML, novel therapies are necessary. This study describes efficacy of a novel FLT3 inhibitor, along with its synergistic activity when combined with other targeted agents.

Midostaurin in patients with acute myeloid leukemia and FLT3-TKD mutations: a subanalysis from the RATIFY trial

AbstractThe results from the RATIFY trial (ClinicalTrials.gov: NCT00651261; CALGB 10603) showed that midostaurin combined with standard chemotherapy significantly improved outcomes in patients with FMS-like tyrosine kinase 3 (FLT3)–mutated acute myeloid leukemia (AML), compared with placebo. In this post hoc subgroup analysis from the trial, we evaluated the impact of midostaurin in 163 patients with FLT3-tyrosine kinase domain (TKD) mutations. At a median follow-up of 60.7 months (95% CI, 55.0-70.8), the 5-year event-free survival (EFS) rate was significantly higher in patients treated with midostaurin than in those treated with placebo (45.2% vs 30.1%; P = .044). A trend toward improved disease-free survival was also observed with midostaurin (67.3% vs 53.4%; P = .089), whereas overall survival (OS) was similar in the 2 groups. Patients with AML and NPM1mut/FLT3-TKDmut or core binding factor (CBF)–rearranged/FLT3-TKDmut genotypes had significantly prolonged OS with or without censoring at hematopoietic cell transplantation (HCT), compared with NPM1WT/CBF-negative AMLs. The multivariable model for OS and EFS adjusted for allogeneic HCT in first complete remission as a time-dependent covariable, revealed NPM1 mutations and CBF rearrangements as significant favorable factors. These data show that NPM1 mutations or CBF rearrangements identify favorable prognostic groups in patients with FLT3-TKD AMLs, independent of other factors, also in the context of midostaurin treatment.

Detection and Quantification of FLT3 Internal Tandem Duplication Mutations Do Not Vary Significantly Between Whole Blood and Blast-Enriched Samples.

Many commonly used FLT3 mutational assay protocols require a tedious blast enrichment step. We investigated whether elimination of this step would still give equivalent results and compared the accuracy of variant allele fraction (VAF) between polymerase chain reaction/capillary electrophoresis (PCR/CE) vs next-generation sequencing (NGS) methods. Total leukocyte vs blast-enriched whole-blood aliquots were tested for FLT3 internal tandem duplication (ITD) and tyrosine kinase domain mutations by PCR/CE. VAF of the ITD mutations was also compared with NGS VAF. Blast-enriched vs total leukocyte specimens showed 100% concordance in the 25 positive specimens. VAF was consistently lower by NGS, with poorer fidelity to PCR/CE VAF as the ITD size increased. Our study supports elimination of the blast enrichment step without compromising results or sensitivity. In addition, since NGS shows a loose correlation with PCR/CE quantitative results, NGS VAF should not be reported for FLT3 ITDs.

Hybridization capture-based next generation sequencing reliably detects FLT3 mutations and classifies FLT3-internal tandem duplication allelic ratio in acute myeloid leukemia: a comparative study to standard fragment analysis

FLT3-internal tandem duplication occurs in 20–30% of acute myeloid leukemia and confers an adverse prognosis with its allelic ratio being a key risk stratifier. The US Food and Drug Administration recently approved FLT3 inhibitors midostaurin and gilteritinib in FLT3 mutation-positive acute myeloid leukemia. Historically, FLT3 was tested by fragment analysis, which has become the standard method endorsed by international guidelines. However, next generation sequencing is increasingly used at acute myeloid leukemia diagnosis given its ability to simultaneously evaluate multiple clinically informative markers. As FLT3-internal tandem duplication detection was known to be challenging by next generation sequencing and the results carry profound prognostic and therapeutic implications, it is important to thoroughly examine its performance in FLT3-internal tandem duplication detection and allelic ratio classification. In a comparative study with fragment analysis, we retrospectively reviewed our experience using a custom-designed, hybridization capture-based, targeted next generation sequencing panel. Among 7902 cases, FLT3-internal tandem duplication was detected in 335 with variable sizes (3–231 bp) and insertion sites. Fragment analysis was also performed in 402 cases, demonstrating 100% concordance in FLT3-internal tandem duplication detection. In 136 dual-tested, positive cases, 128/136 (94%) exhibited concordant high/low allelic ratio classifications. The remaining 6% showed borderline low allelic ratio by next generation sequencing. The two methods were concordant in FLT3-tyrosine kinase domain mutation detection at the hotspot D835/I836 targeted by fragment analysis. Furthermore, seven mutations which may benefit from FLT3 inhibitor therapy were detected by next generation sequencing, in regions not covered by fragment analysis. Our study demonstrates that using a hybridization capture-based chemistry and optimized bioinformatics pipeline, next generation sequencing can reliably detect FLT3-internal tandem duplication and classify its allelic ratio for acute myeloid leukemia risk stratification. Next generation sequencing also exhibits superior comprehensiveness in FLT3 mutation detection and may further improve personalized, targeted therapy in acute myeloid leukemia.

Significance of FLT3-tyrosine kinase domain mutation as a prognostic factor for acute myeloid leukemia.

The prognostic significance of FLT3-tyrosine kinase domain (TKD) mutations remains unknown. To investigate the prognostic impact of FLT3-TKD, 676 de novo acute myeloid leukemia (AML), we retrospectively analyzed cases and conducted a review of the literature. Of the 676 de novo AML cases, 34 (5.0%) were FLT3-TKD-positive; both FLT3-TKD and FLT3-ITD were noted in only two cases (0.3%). Although no significant differences in relapse-free survival (RFS) were noted, FLT3-TKD-positive cases showed better prognoses than FLT3-ITD-positive cases (FLT3-TKD versus FLT3-ITD, p = 0.152). For overall survival (OS), although FLT3-TKD-positive cases showed prognoses similar to those for FLT3-WT cases, their prognoses were significantly better than those of FLT3-ITD-positive cases (FLT3-TKD versus FLT3-ITD, p = 0.032). Moreover, the 5-year OS for FLT3-TKD-positive cases was 46.1%, indicating that this as an intermediate prognosis group. Although no reports from Asia have indicated a frequency of FLT3-TKD-positive cases > 10%, several reports from Europe and the United States have indicated frequencies > 10%. This suggests the possibility that FLT3-TKD-positive cases are less common in Asia than in Europe and the United States. We anticipate that in the future, the appearance of targeting agents, such as FLT3 inhibitors, will improve the prognosis of FLT3-TKD-positive AML relative to that of FLT3-WT AML.

Activity of TP‐0903 in FLT3 inhibitor resistant AML models

BackgroundThe development of tyrosine kinase inhibitors (TKIs) for the treatment of acute myeloid leukemia (AML) with FLT3 internal tandem duplication mutations (FLT3‐ITD+) has been challenging due to intrinsic and acquired drug resistance. Important resistance mechanisms that have been described include: 1) the emergence of secondary FLT3 tyrosine kinase domain (TKD) mutations; 2) compensatory signaling pathways (intrinsic to the leukemia cell or due to tumor‐microenvironment interactions), such as Gas6/Axl upregulation; and 3) the co‐occurrence of somatic mutations with FLT3‐ITD, such as IDH1/2, NRAS and MLL‐PTD. TP‐0903, is a new TKI in development as an Axl inhibitor. Previously, we demonstrated that TP‐0903 is a potent inhibitor of FLT3‐ITD and drug resistant FLT3 TKD mutations in preclinical in vitro and in vivo models (Jeon JY et al. ASH 2017). Here, we present the activity of TP‐0903 in additional TKI‐resistant models of FLT3‐ITD+ AML in comparison to other FLT3 TKIs.MethodsFLT3‐ITD+ MOLM13 cells were seeded in direct co‐culture with or without HS5‐GFP bone marrow stromal cells (MSCs) for 24h followed by 72h treatment with TP‐0903 or 5 other clinical candidate FLT3 TKIs; cell viability of co‐cultured MOLM13 cells was measured via MTT assay. Soluble Gas6 and Axl were measured in co‐cultured media over 24–72h by ELISA. Ex vivo activity of TKIs on the viability of murine primary leukemia cells with FLT3‐ITD+/−/IDH2‐R140Q+/− or FLT3‐ITD+/−/MLL‐PTD co‐occurring mutations was assessed after 72h TKI treatment using a CellTiter Glo assay.ResultsThe effect of TP‐0903 and other FLT3 TKIs on the inhibition of viability of MOLM13 cells cultured with or without MSCs is shown in Table 1. While co‐culture induced a minimal 1.7‐fold shift in the TP‐0903 IC50 value, the other FLT3 TKIs were more resistance in co‐culture, with gilteritinib and sorafenib showing the greatest change in IC50 values (4.9‐ and 5.0‐fold). Soluble Axl and Gas6 levels increased over 24–72h in co‐cultured media with the highest concentrations achieved of 1455 ± 322 pg/mL and 5098 ± 974 pg/mL, respectively. In primary FLT3‐ITD+/−/IDH2‐R140Q+/− leukemia cells, TP‐0903 was 8‐ to 10‐fold more potent than gilteritinib and crenolanib, whereas midostaurin, quizartinib and sorafenib did not show activity in these cells (Table 1). TP‐0903 was also 5‐fold more potent than gilteritinib in primary FLT3‐ITD+/−/MLL‐PTD leukemia cells. Ongoing studies are evaluating the effect of soluble Gas6 on TKI sensitivity in AML cells, as well as the in vivo activity of TP‐0903 in FLT3‐ITD+/−/IDH2‐R140Q+/− or FLT3‐ITD+/−/MLL‐PTD spontaneous mouse models.ConclusionsTP‐0903 retains activity in preclinical models of intrinsic/acquired FLT3 TKI resistance with therapeutic potential to overcome drug resistant FLT3‐ITD+ AML.Support or Funding InformationThis work is supported by Eli Lilly fellowship and NIH grant 5R01CA138744‐09This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

48. Identification of a novel CEBPA homozygous duplication by NGS and SNP microarray

The prognostic significance of CEBPA variants in acute myeloid leukemia (AML) is less favorable for individuals with a single CEBPA variant compared to individuals with biallelic CEBPA variants at the N- and C-termini of the protein. Few AML cases with homozygous CEBPA variants have been reported, and a better understanding of such cases could aid determination of prognostic impact of these variants. Here, we report a patient with newly diagnosed AML that was negative by outside testing for both the FLT3 internal tandem duplication (FLT3-ITD) and tyrosine kinase domain mutations. Cytogenetic testing showed a bone marrow karyotype of 46,XY[20] and FISH results were within normal limits for AML-related abnormalities. Next generation sequencing analysis revealed an NRAS pathogenic variant (LRG_92t1:c.38G>T, p.(Gly13Val)), three GATA2 variants of clinical significance/possible clinical significance (LRG_295t2:c.1085G>A, p.(Arg362Gln)(;)1018-14_1036dup, p.(?)(;)989G>C, p.(Arg330Pro)), and a C-terminal in-frame duplication of 30 nucleotides in the CEBPA gene (LRG_456t1:c. c.950_979dup, p.(Leu317_Lys326dup)). Wild-type sequence was not detected in the region surrounding the CEBPA variant and a SNP chromosomal microarray revealed loss of heterozygosity spanning 19q13.11-19q13.43, which includes the CEBPA gene (arr[GRCh37] 19q13.11q13.43(33,365,429-59,097,752) hmz). Together, these NGS and microarray results are consistent with a homozygous CEBPA duplication. Although homozygous CEBPA variants are rare in AML, they have a similar gene expression signature to AML with biallelic CEBPA variants. While it remains to be determined if the prognosis for AML patients with these variants is similar to AML patients with biallelic CEBPA variants, these results may aid prognostic studies in the future.