FLT3-positive Acute Myeloid Leukemia Research Articles

Fms-like tyrosine kinase 3 positive acute myeloid leukemia.

Fms-like tyrosine kinase 3 (FLT3) mutations are common in newly diagnosed patients with acute myeloid leukemia (AML). They are associated with a high risk of relapse. The identification of FLT3 mutations has important implications for the management of AML. FLT3 inhibitors have shown improved outcomes in FLT3-positive AML when used as a single agent in the salvage setting. However, the combination of inhibitors and chemotherapy in the first-line setting is the real game changer in FLT3mutant AML. The introduction of these drugs has improved the prognosis of FLT3-mutant AML, but the development of resistance is common. There are still many unanswered questions about FLT3-mutant AML. This article will analyze recent advances for FLT3-mutant AML, focusing on front-line therapy and post-transplant maintenance. Novel drug combinations and strategies against FLT3 mutated AML are currently under investigation and will be the focus of future studies. The development of more selective and potent FLT3 inhibitors may further improve outcomes for patients with FLT3-positive AML. Monitoring minimal residual disease and overcoming resistance are key issues for the future.

Abstract CT025: NMS-03592088, a novel, potent FLT3, KIT and CSF1R inhibitor with activity in FLT3 positive acute myeloid leukemia patients with prior FLT3 inhibitor experience

Abstract Background: FLT3 mutations occur in approximately 30% of AML patients and are associated with aggressive disease. Despite the approval of midostaurin and gilteritinib, the prognosis for FLT3+ patients with relapsed or refractory disease is poor. NMS-088 is a novel, potent FLT3, KIT and CSF1R inhibitor with superior preclinical activity compared with approved FLT3 inhibitors in different FLT3-driven models. In addition, NMS-088 is active on FLT3 resistance mutation F691L. Dose escalation results from a Phase I/II study to establish safety, dose selection and preliminary clinical activity for NMS-088 in patients with R/R AML and CMML are described. Methods: In the Phase I 3+3 escalations, NMS-088 is administered daily for 21 of 28 days (schedule A) or continuously (schedule B). Patients must have R/R AML or CMML unsuitable for standard therapy. The primary objective is the MTD or MAD as assessed by DLTs. Secondary endpoints include safety, PK and ELN response. Results: as of January 26, 44 R/R AML or CMML patients were treated at doses from 20 to 360 mg/day in A or from 120 to 250 mg/day in B. Median age was 64 yrs, 41 pts had AML and 3 pts had CMML, median number of prior lines was 2 (range 1 to 10). FLT3 mutations were present in 24 out of 41 AML pts (20 FLT3-ITD, 2 FLT3 D835 and 2 FLT3-ITD and D835). The majority of pts with FLT3+ AML had received prior FLT3 inhibitors (86.4%). NMS-088 showed manageable safety with no MTD characterized. One pt had DLT (abnormal posture, decreased activity, dyspnea G3 and eyelid ptosis G1) at 360 mg in A (at day 21) and one pt had DLT (eyelid ptosis G3) at 180 mg in B (at day 29), both suggestive for myasthenic syndrome. Three additional pts experienced possible myasthenic syndrome at doses ≥ 180 mg. Overall the most frequent treatment emergent related adverse events (≥10%) were nausea (any grade 20.5%), vomiting (13.6%), asthenia (11.4%). Discontinuations due to related AEs were as follows: 2 DLT pts per protocol, 2 pts due to nausea (G1; day 161 at 270 mg) and myasthenia gravis (G3; day 39 at 300 mg in a pt with baseline AChR antibodies). There was a dose-dependent trend for response. A total of 5 out of 12 evaluable pts with FLT3+ AML treated at dose ≥ 300 mg achieved a response with 2 CRi, 1 CRi/MLFS and 2 MLFS. Remarkably, all these pts had received prior midostaurin and 2 pts received both midostaurin and gilteritinib. Two pts with response withdrew from treatment to receive HSCT (DoR 1.0+ mos each). For other responding pts DoR was 1.3, 2.8 and 7.9 mos. Conclusions: NMS-088 showed clinical efficacy in pts with FLT3+ R/R AML, including pts who have failed prior FLT3 inhibitors. Together with the manageable safety observed, these results warrant further development of this drug including potential as a novel valuable therapeutic option for pts who have exhausted available treatments. The trial is currently opened for enrollment (NTC03922100). Citation Format: Antonio Curti, Alessandro Rambaldi, Chiara Cattaneo, Roberto Cairoli, Matteo Della Porta, Patrizia Chiusolo, Federico Lussana, Marta Ubezio, Valentina Mancini, Isabel Cano, Carmen Besliu, Christian Hove Claussen, Rosalinda Gatto, Patrizia Crivori, Elena Colajori, Alessio Somaschini, Cristina Davite, Antonella Isacchi, Elena Ardini, Lisa Mahnke, Pau Montesinos. NMS-03592088, a novel, potent FLT3, KIT and CSF1R inhibitor with activity in FLT3 positive acute myeloid leukemia patients with prior FLT3 inhibitor experience [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT025.

Synthesis and Structural Optimization of 2,7,9-Trisubstituted purin-8-ones as FLT3-ITD Inhibitors.

Therapy of FLT3-positive acute myeloid leukemia still remains complicated, despite the availability of newly approved kinase inhibitors. Various strategies to avoid the reduced efficacy of therapy have been explored, including the development of dual targeting compounds, which inhibit FLT3 and another kinase necessary for the survival and proliferation of AML cells. We have designed new 2,7,9-trisubstituted 8-oxopurines as FLT3 inhibitors and report here the structure-activity relationship studies. We demonstrated that substituents at positions 7 and 9 modulate activity between CDK4 and FLT3 kinase, and the isopropyl group at position 7 substantially increased the selectivity toward FLT3 kinase, which led to the discovery of compound 15a (9-cyclopentyl-7-isopropyl-2-((4-(piperazin-1-yl)phenyl)amino)-7,9-dihydro-8H-purin-8-one). Cellular analyses in MV4-11 cells revealed inhibition of autophosphorylation of FLT3 kinase in nanomolar doses, including the suppression of downstream STAT5 and ERK1/2 phosphorylation. We also describe mechanistic studies in cell lines and activity in a mouse xenograft model in vivo.

AML-190 Targeted Therapy for R/R FLT3-Positive Acute Myeloid Leukemia is as Effective as Salvage Intensive Chemotherapy but Less Toxic and With Better Long-Term Results

Patients with FLT3-mutated acute myeloid leukemia (AML) have a poor prognosis. The choice of the most optimal therapy is an actual problem. To compare the efficacy, safety, and survival rates of salvage intensive chemotherapy (IC) and targeted therapy in patients with FLT3-mutated relapsed/refractory (R/R) AML. A retrospective study was conducted, which included patients with FLT3-mutated R/R AML. Patients received IC (FLAG±Ida) or gilteritinib (as monotherapy or in combination with venetoclax, azacitidine). All patients received inpatient treatment. The study included 24 patients (12 women, 12 men) with a median age of 42 years (18-83 years); 54.2% (13/24) had relapsed and 45.8% (11/24) had refractory AML. Of the 13 relapsed patients, 8 (61.5%) had early relapse. Thirteen patients received IC and 11 patients received gilteritinib (5 as monotherapy, 2 in combination with venetoclax, 4 in combination with venetoclax and azacitidine). Some patients received salvage IC therapy; others received gilteritinib therapy. Overall response rates (CR, CRMRD-; CRi, MFLS) did not differ significantly between the two groups (63% in the gilteritinib group and 60% in the IC group). The median time to remission was shorter in the IC group than in the gilteritinib group (0.8 vs. 1.5 months, respectively). There was no early mortality in the gilteritinib group; in the IC group, it was 23% (3/11). Serious adverse events were more common in the IC group than in the gilteritinib group (61.5% vs. 18.1%, respectively, P=0.032). In the gilteritinib group, more patients underwent allogeneic bone marrow transplantation (BMT) (77.7% vs. 58.3%). The overall survival and relapse-free survival were longer in the gilteritinib group than in the IC group (20.4 vs. 15.3 months; 7.9 vs. 3.37 months, respectively). Gilteritinib therapy is comparable in efficacy to salvage IC in patients with FLT3-mutated R/R AML but has been shown to be safer and have better survival rates. Gilteritinib is an optimal bridge therapy to allogeneic BMT.

Abstract 726: Effects of diet composition and timing on treatment for FLT3 positive acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) with a mutation in the fms-like tyrosine kinase receptor 3 gene (FLT3) confers poor survival for pediatric patients, indicating a significant need to improve treatment efficacy. A significant percentage of pediatric patients consume diets high in fat and sugar content. These macronutrients modulate oxidative stress yet their impact on AML treatment is unexplored. Studies have implicated a role for BMAL1 rhythmicity, a circadian clock protein, in leukemogenesis. However, BMAL1 has not been studied in FLT3 mutant AML. Given that both nutrition and circadian rhythmicity can be modulated by behavior, we hypothesized that modulating redox and BMAL1 status through diet composition and timing would enhance treatment efficacy in an orthotopic mouse model. In all in vivo experiments, mice were injected via tail vein with 100,000 MOLM13 luciferase labelled cells, a FLT3 AML human cell line. After engraftment was confirmed 3 days later, mice were put on diet modification regimens. In three subsequent experiments, distinct diet interventions were delivered: low fat (30%)/low sucrose (10%), low sucrose alone (10%), or an isocaloric time feeding regimen (TRF) restricted to mice’s active phase (7 PM to 7 AM). Standard chow, high sucrose diet (45%) and ad libitum feeding were controls, respectively. In each experiment, doxorubicin (2 mg/kg) or PBS was injected twice weekly via tail vein. Tumor burden was monitored via bioluminescent imaging. Reactive oxygen species (ROS) levels were assessed with flow cytometry, and changes in circadian, redox, and apoptotic protein expression were measured by western blot and mass cytometry. On day 17, in the presence of doxorubicin, mice on a low fat/low sucrose diet showed significantly lower tumor burden compared to control. Doxorubicin induced oxidative stress was reduced in the mice fed the modified diet as demonstrated by a decrease in ROS levels in peripheral blood mononuclear cells. Even in the absence of doxorubicin treatment, the modified diet group showed significantly lower tumor burden on day 17. For mice on a low sucrose diet, changes in redox and circadian protein expression were observed on day 17. Of note, total BMAL1 protein showed a two-fold increase in spleen from the low sucrose group. In the timed feeding experiments, we observed TRF mice had significantly reduced leukemic burden on day 14. Importantly, doxorubicin efficacy was not compromised by TRF. In addition, a significant increase in total BMAL1 protein expression was observed in liver from these TRF mice on day 14. These results point to a novel finding: diet composition and timing slows progression of FLT3 AML growth in vivo and are accompanied by BMAL1 modulation. In future studies, we aim to further define the role of circadian rhythmicity and diet composition in AML progression and identify methods to complement treatment to positively impact survival outcomes for patients diagnosed with AML. Citation Format: Megan Rodriguez, Mary Figueroa, Brianna Murphy, Tiewei Cheng, Baharan Fekry, Kendra Allton, Michelle Barton, Marina Konopleva, Kristin Mahan, Joya Chandra. Effects of diet composition and timing on treatment for FLT3 positive acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 726.

High Incidence of Invasive Fungal Diseases in Patients with FLT3-Mutated AML Treated with Midostaurin: Results of a Multicenter Observational SEIFEM Study.

The potential drug-drug interactions of midostaurin may impact the choice of antifungal (AF) prophylaxis in FLT3-positive acute myeloid leukemia (AML) patients. To evaluate the incidence of invasive fungal diseases (IFD) during the treatment of FLT3-mutated AML patients and to correlate it to the different AF prophylaxis strategies, we planned a multicenter observational study involving 15 SEIFEM centers. One hundred fourteen patients treated with chemotherapy + midostaurin as induction/reinduction, consolidation or both were enrolled. During induction, the incidence of probable/proven and possible IFD was 10.5% and 9.7%, respectively; no statistically significant difference was observed according to the different AF strategy adopted. The median duration of neutropenia was similar in patients with or without IFD. Proven/probable and possible IFD incidence was 2.4% and 1.8%, respectively, during consolidation. Age was the only risk factor for IFD (OR, 95% CI, 1.10 [1.03–1.19]) and complete remission achievement after first induction the only one for survival (OR, 95% CI, 5.12 [1.93–13.60]). The rate of midostaurin discontinuation was similar across different AF strategies. The IFD attributable mortality during induction was 8.3%. In conclusion, the 20.2% overall incidence of IFD occurring in FLT3-mutated AML during induction with chemotherapy + midostaurin, regardless of AF strategy type, was noteworthy, and merits further study, particularly in elderly patients.

Therapeutic Management of Patients with FLT3 + Acute Myeloid Leukemia: Case Reports and Focus on Gilteritinib Monotherapy

Acute myeloid leukemia is a malignant disorder of the bone marrow, characterized by differentiation, clonal expansion, and uncontrolled proliferation of malignant myeloid progenitor cells and by several molecular and genetic abnormalities. A mutation of FMS-like tyrosine kinase 3 gene can be observed in about one-third of cases of acute myeloid leukemia. Two FLT3 inhibitors are actually approved for FLT3 mutated acute myeloid leukemia: midostaurin, a multikinase first generation inhibitor with lower affinity for FLT3 binding, and gilteritinib fumarate, a potent second-generation inhibitor of both FLT3-ITD and TKD. Gilteritinib is a new effective and well-tolerated drug for patients with relapsing or refractory FLT3-positive acute myeloid leukemia. Thanks to its efficacy, low toxicity, its good manageability (oral formulation), this drug is suitable for all the patients, including elderly frail patient with concomitant therapies or pre-existing or underlying diseases, and can be used also in the outpatient setting, reducing risks and costs related to the hospitalization. We report and discuss seven cases of different patients with FLT3 positive acute myeloid leukemia successfully managed with gilteritinib in the real clinical practice.

Expression of a recombinant FLT3 ligand and its emtansine conjugate as a therapeutic candidate against acute myeloid leukemia cells with FLT3 expression

BackgroundMost patients with acute myeloid leukemia (AML) remain uncurable and require novel therapeutic methods. Gain-of-function FMS-like tyrosine kinase 3 (FLT3) mutations are present in 30–40% of AML patients and serve as an attractive therapeutic target. In addition, FLT3 is aberrantly expressed on blasts in > 90% of patients with AML, making the FLT3 ligand-based drug conjugate a promising therapeutic strategy for the treatment of patients with AML. Here, E. coli was used as a host to express recombinant human FLT3 ligand (rhFL), which was used as a specific vehicle to deliver cytotoxic drugs to FLT3 + AML cells.MethodsRecombinant hFL was expressed and purified from induced recombinant BL21 (DE3) E. coli. Purified rhFL and emtansine (DM1) were conjugated by an N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) linker. We evaluated the potency of the conjugation product FL-DM1 against FLT3-expressing AML cells by examining viability, apoptosis and the cell cycle. The activation of proteins related to the activation of FLT3 signaling and apoptosis pathways was detected by immunoblotting. The selectivity of FL-DM1 was assessed in our unique HCD-57 cell line, which was transformed with the FLT3 internal tandem duplication mutant (FLT3-ITD).ResultsSoluble rhFL was successfully expressed in the periplasm of recombinant E. coli. The purified rhFL was bioactive in stimulating FLT3 signaling in AML cells, and the drug conjugate FL-DM1 showed activity in cell signaling and internalization. FL-DM1 was effective in inhibiting the survival of FLT3-expressing THP-1 and MV-4-11 AML cells, with half maximal inhibitory concentration (IC50) of 12.9 nM and 1.1 nM. Additionally, FL-DM1 induced caspase-3-dependent apoptosis and arrested the cell cycle at the G2/M phase. Moreover, FL-DM1 selectively targeted HCD-57 cells transformed by FLT3-ITD but not parental HCD-57 cells without FLT3 expression. FL-DM1 can also induce obvious apoptosis in primary FLT3-positive AML cells ex vivo.ConclusionsOur data demonstrated that soluble rhFL can be produced in a bioactive form in the periplasm of recombinant E. coli. FL can be used as a specific vehicle to deliver DM1 into FLT3-expressing AML cells. FL-DM1 exhibited cytotoxicity in FLT3-expressing AML cell lines and primary AML cells. FL-DM1 may have potential clinical applications in treating patients with FLT3-positive AML.

Safety profile and impact on survival of tyrosine kinase inhibitors versus conventional therapy in relapse or refractory FLT3 positive acute myeloid leukemia patients

Relapsed or refractory (R/R) acute myeloid leukemia (AML) has a poor prognosis, and new therapies are a major clinical need. When mutated, FLT3 drives neoplastic cell proliferation. New drugs (i.e., tyrosine kinase inhibitors, TKIs) showed effectiveness in FLT3-AML and promise to change disease history and outcome. We evaluated the benefit conferred by TKIs in terms of survival, burden of complications and surrogate endpoint of quality of life in a retrospective cohort of 49 FLT3 positive, R/R AML patients. Patients who received TKIs were compared to those treated with conventional chemotherapy. Treatment with TKIs conferred a better OS and wea associated with a lower burden and severity of adverse events. Importantly, patients who received TKIs showed reduced time of hospitalization. In conclusion, treatment with TKI in R/R FLT3-AML was related to a better survival, less and milder AEs, and shorter hospitalization.

ERRATUM: A unique case of durable complete remission after salvage with azacitidine and DLI for high risk flt-3 positive acute myeloid leukemia, following relapse 18 months post allogeneic stem cell transplant.

Due to an error, Dr. Nikolousis’s last name was mistakenly spelled in this article (published in 2019 in Hematology Reports DOI: 10.4081/hr.2019.7800) [...]

Interim Analysis of Hematologic Malignancies (HM)-Screen-Japan 01: A Mutation Profiling Multicenter Study of Patients with AML

Interim Analysis of Hematologic Malignancies (HM)-Screen-Japan 01: A Mutation Profiling Multicenter Study of Patients with AML

FLT3 Ligand-DM1 Conjugate Selectively Targets Acute Myeloid Leukemia Cells with FLT3 Expression

Acute myeloid leukemia (AML) is a malignant hematopoietic neoplasm featured by impaired differentiation and uncontrolled proliferation of myeloid progenitors. Gain-of-function mutations of FMS-like tyrosine kinase 3 (FLT3) present in 30-40% of patients with AML. In addition, more than 90% of AML blasts aberrantly express FLT3, making FLT3 an attractive therapeutic target for AML. Currently, several small molecule tyrosine kinase inhibitors (TKIs) targeting FLT3 have been approved in the treatment of AML, but they need to be used in combination with chemotherapy because of their limited potency to eliminate leukemic cells as single agents, largely due to the development of secondary inhibitor-resistant FLT3 mutations. Therefore, novel therapeutic strategies targeting FLT3 are needed. In the present study, we developed a FLT3 ligand-emtansine drug conjugate (FL-DM1) that targeted FLT3-positive AML cells with high potency and selectivity. We expressed recombinant human FLT3 ligand (rhFL) in the periplasm of recombinant E. Coli. The protein was purified by a two-step purification system containing Ni-NTA and Phenyl Sepharose. Our purified rhFL was bioactive to stimulate phosphorylation of FLT3 and proliferation of THP-1 cells. Next, we conjugated purified rhFL and emtansine (DM1) with SPDP linker. Reducing and non-reducing SDS-PAGE revealed that rhFL and DM1 were successfully conjugated, evidenced by a band with higher molecular weight of the conjugation product. Previous studies show that DM1 is a drug preferentially targeting proliferating cells by depolymerizing microtubules through binding at the vinca binding site of tubulin. We found that FL-DM1 reserved the physiological function of FLT3 ligand to stimulate proliferation of AML cells by inducing phosphorylation of FLT3 and the downstream signaling protein AKT in immunoblot, potentially enhancing the potency of FL-DM1 to inhibit FLT3-positive AML cells. Furthermore, flow cytometry showed that the surface expression of FLT3 significantly decreased on cells treated by FL-DM1 within two hours, which indicated the internalization of FL-DM1/FLT3 complex on these FLT3-positive AML cells, providing a mechanism of FL-DM1 entering target cells. In cell viability assay, we found that FL-DM1 effectively inhibited FLT3-positive AML cells THP-1 and MV-4-11 with IC50 around 10-30 nM. Also, FL-DM1 induced apoptosis and cell cycle arrest at G2/M phase in these cells detected by flow cytometry. In our previous studies, we generated FLT3-ITD transformed HCD-57 cells. HCD-57 cells are FLT3-negative erythroleukemia cells that depend on erythropoietin for survival. When infected with recombinant retroviruses carrying FLT3-ITD, they acquired ability to proliferate in the absence of EPO. We found that FL-DM1 inhibited HCD-57 cells transformed by FLT3-ITD, but not parental HCD-57 cells without FLT3 expression, indicating the selectivity of FL-DM1 to target FLT3-positive AML cells. In conclusion, our data demonstrated that FL-based drug conjugate can serve as an effective drug to target FLT3-expressing AML cells. Further studies will focus on in-vivo evaluation of FL-DM1 in animal models, the production of uncleavable SMCC linked FL-DM1 with improved in-vivo pharmacokinetic properties, and screening of FL muteins-DM1 conjugates with desired pharmacological properties. Disclosures No relevant conflicts of interest to declare.

Nonclinical Safety Assessment of AMG 553, an Investigational Chimeric Antigen Receptor T-Cell Therapy for the Treatment of Acute Myeloid Leukemia.

Feline McDonough Sarcoma-like tyrosine kinase 3 (FLT3), a tyrosine-protein kinase involved in hematopoiesis, is detectable on the cell surface of approximately 80% of leukemia isolates from adult patients with acute myeloid leukemia (AML). AMG 553 is an investigational chimeric antigen receptor (CAR) T-cell immunotherapy for the treatment of AML. FLT3 expression analysis and in vitro and in vivo studies were leveraged to evaluate the nonclinical safety of AMG 553. Cynomolgus monkeys administered autologous anti-FLT3 CAR T cells demonstrated no evidence of CAR T-cell-mediated toxicity, expansion, or persistence, likely due to restricted cell surface FLT3 protein expression in healthy animals. This highlights the limited value of such in vivo studies for safety assessment of the CAR T-cell modality when directed against a target with restricted expression. To complement these studies and directly evaluate the potential toxicities of eliciting T-cell-mediated cytotoxicity against cells with surface expression of FLT3 protein in vivo, data from cynomolgus monkey toxicology studies with 2 bispecific T-cell engager molecules targeting FLT3 were leveraged; findings were consistent with the targeted killing of bone marrow cells expressing cell surface FLT3. Potential AMG 553-induced cytotoxicity was assessed against a wide range of normal human primary cells and cell lines; cytotoxicity was observed against FLT3-positive AML cell lines and a percentage of primary bone marrow CD34+ cells. In conclusion, the nonclinical safety data suggest that AMG 553 can target FLT3 protein on AML cells, whereas only affecting a percentage of normal hematopoietic stem and progenitor cells, supporting clinical development.

Acute myeloid leukemia transformed to a targetable disease.

Acute myeloid leukemia (AML) is a heterogeneous neoplasm characterized by the monoclonal proliferation of immature progenitors. It is the most common acute leukemia in adults and its incidence increases with age. The standard traditional treatment in fit patients was the '3 + 7' regimen and cytarabine consolidation followed or not with allogeneic stem cell transplantation. Recently, several targeted therapies such as gemtuzumab ozogamicin targeting the CD33+ AML, midostaurin, gilteritinib and crenolanib inhibiting FLT3-positive AML and ivosidenib and enasidenib blocking IDH-mutated AML have been approved. These new drugs led to the change of the landscape of the treatment of AML and transforming this disease to a targetable one. We aimed in this paper to review the implications of each new target, the mechanisms of action of these new drugs and we discuss all the studies leading to the approval of these new drugs in their indications according to each target.

Azacytidine in Newly Diagnosed FLT3-ITD-Positive Acute Myeloid Leukemia Presenting with Pneumonia: A Case Series.

Acute myeloid leukemia (AML) is an aggressive disease that predisposes the patients to infections. FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) positive AML is a type of high-risk AML. Pneumonia is a common complication in patients of AML both due to the disease itself and as a result of induction chemotherapy. Treating AML patients who present with pneumonia is a challenge as induction chemotherapy further increases the severity and mortality of pneumonia as it causes myelosuppression. We report four patients with newly diagnosed FLT3-ITD-positive AML who had pneumonia at presentation. All four cases required induction chemotherapy with 7+3 which could not be given due to their poor general condition, secondary to pneumonia. Therefore, they were given low-intensity therapy, in the form of azacytidine, to prevent further progression of AML while they were recovering from pneumonia and became well enough to tolerate intensive induction chemotherapy. This treatment strategy of using a bridge before intensive chemotherapy was successful in our patients and 3 out 4 achieved documented remission. In our opinion, patients with newly diagnosed FLT3 positive AML with pneumonia can be given low-intensity chemotherapy such as azacytidine until the remission of pneumonia for better patient outcomes.

PB1724 FLT3 MUTATED AML: THE ROLE OF CLADRIBINE AND MIDOSTAURIN ADDED TO THE STANDARD INTENSIVE CHEMOTHERAPY ‐ SINGLE CENTRE EXPERIENCE FROM UMC LJUBLJANA, SLOVENIA

Background:The FLT3 ITD mutation in a patient with acute myeloid leukemia (AML) is associated with a poor outcome. Midostaurin is the first multikinase inhibitor approved for the treatment of adults with newly diagnosed FLT3 positive AML. The drug has been available in Slovenia from July 2017 and used in all eligible FLT3 patients. Furthermore, following encouraging results shown by the Polish Adult Leukemia Group, we have been adding cladribine to standard combination DA3+7 induction (namely DAC) for all AML patients aged 60 years or less since 2014.Aims:To compare the outcome of FLT3 positive AML patients treated with DA3+7 or DAC with further addition of midostaurin.Methods:We retrospectivelly analyzed all consecutive FLT3 positive AML patients, aged 60 years or less, treated with intensive chemotherapy between years 2012‐2018. Bone marrow (or in some cases peripheral blood) RNA was used for FLT3 mutation detection. FLT3 ITD mutation was detected by reverse transcription PCR (RT‐PCR) and gel electrophoresis analysis. FLT3 D836 mutation detection was carried out by RT‐PCR and Restriction Fragment Length Polymorphism technique using restriction enzyme EcoRV. From 01/2012 – 04/2014 DA3 + 7 was used, while from 05/2014 – 12/2018 DAC (addition of 5 mg/m2 of cladribine in a 3‐hour infusion on days 1‐5 to DA3 + 7) was used for all AML patients aged 60 years or less eligible for intensive chemotherapy. Remission was assessed by bone marrow aspiration in the aplastic phase and before the first consolidation therapy. Consolidation treatment was intermediate or high dose cytosar. Midostaurin was added according to indication no later than in the first consolidation phase (2x50 mg from day 8‐21). All patients with FLT3 ITD mutation and available donor were transplanted. Patients who had not achieved remission after induction chemotherapy proceeded with rescue chemotherapy and allogeneic transplantation. Patients who were not transplanted continued maintenance treatment with midostaurin 2x50 mg continuously for one year.Results:There were 39 FLT3 positive AML patients aged 60 years or less who were treated with intensive chemotherapy in the period 2012‐2018: 16 with DA3 + 7 and 23 with DAC. There was no difference between the baseline characteristics between the groups (Table 1). Remission after induction was achieved in 9/16 patients in the DA group (1 patient was treated with midostaurin during induction) and 19/23 in the DAC group (5 patients received midostaurin during the induction), p = 0,072. Survival was significantly better in the DAC group in which overall 12/23 patients were treated with midostaurin compared to DA3 + 7 where only 1 patient was treated with midostaurin; p = 0,028. The proportion of patients with allogeneic transplant was similar (7/16 in the DA and 11/23 in the DAC group; p = 0,802). When comparing patients with or without midostaurin treatment the survival was significantly better in patients treated with midostaurin (p = 0,005), however, the median observation time was only 11 months. The addition of midostaurin to DAC did not increase toxicity.Summary/Conclusion:Addition of cladribine and midostaurin to standard DA3 + 7 improved survival in FLT3 positive AML patients. No new safety signals were noted in patients receiving DAC + midostaurin. Further follow‐up is required to evaluate influence of midostaurin in combination with DAC induction chemotherapy on progression free survival and overall survival.image

High-throughput Identification of FLT3 Wild-type and Mutant Kinase Substrate Preferences and Application to Design of Sensitive In Vitro Kinase Assay Substrates

Acute myeloid leukemia (AML) is an aggressive disease that is characterized by abnormal increase of immature myeloblasts in blood and bone marrow. The FLT3 receptor tyrosine kinase plays an integral role in hematopoiesis, and one third of AML diagnoses exhibit gain-of-function mutations in FLT3, with the juxtamembrane domain internal tandem duplication (ITD) and the kinase domain D835Y variants observed most frequently. Few FLT3 substrates or phosphorylation sites are known, which limits insight into FLT3's substrate preferences and makes assay design particularly challenging. We applied in vitro phosphorylation of a cell lysate digest (adaptation of the Kinase Assay Linked with Phosphoproteomics (KALIP) technique and similar methods) for high-throughput identification of substrates for three FLT3 variants (wild-type, ITD mutant, and D835Y mutant). Incorporation of identified substrate sequences as input into the KINATEST-ID substrate preference analysis and assay development pipeline facilitated the design of several peptide substrates that are phosphorylated efficiently by all three FLT3 kinase variants. These substrates could be used in assays to identify new FLT3 inhibitors that overcome resistant mutations to improve FLT3-positive AML treatment.

A unique case of durable complete remission after salvage with azacitidine and DLI for high risk flt-3 positive acute myeloid leukemia, following relapse 18 months post allogeneic stem cell transplant.

A unique case of primary refractory FLT3-itd mutated acute myeloid leukemia in an elderly patient, who achieved completed morphological remission, and FLT3-itd negativity, following 9 cycles of azacitadine in combination with escalating doses of donor lymphocyte infusions following relapse 18 months post reduced intensity HLAA mismatch Campath conditioning allogeneic stem cell transplant.

Midostaurin in Combination with Idarubicin and Cytarabine (3+7) Induction for FLT3 Positive AML - Very High Complete Response Rates and Transition to Allogeneic Transplantation

Background:The CALGB 10603 RATIFY trial demonstrated that midostaurin, a multi-targeted small molecule FLT3 inhibitor, when given in combination with standard 3+7 induction consisting of daunorubicin 60mg/m2 for 3 doses and cytarabine 200mg/m2/day for 7 days significantly prolonged overall survival (OS) compared to placebo plus standard induction in FLT3 positive acute myeloid leukemia (AML) patients. The safety and efficacy of the other anthracycline commonly utilized in standard AML induction, idarubicin, has not been evaluated to date.Methods:A single-center retrospective review from May 2017 to July 2018 was performed. Patients were included if they had a diagnosis of FLT3 positive AML and received induction chemotherapy with idarubicin. The primary outcome was incidence of adverse effects attributed to midostaurin. Grading of adverse effects was in accordance with the Common Terminology and Criteria of Adverse Effects (CTCAE) version 5. Additional outcomes included OS, relapse-free survival (RFS), similar as in the Ratify trial, as well as detection of FLT3 on subsequent bone marrow biopsies.Results:Ten patients were included. All patients received induction therapy with idarubicin 12mg/m2 for 3 doses and cytarabine 100mg/m2/day for 7 days. Median age was 53 years (range: 33 to 66) and 6/10 were male. Eight of 10 patients exhibited internal tandem duplication (ITD) on diagnosis; two had FLT3 tyrosine kinase domain (TKD) D835. Eight patients had diploid cytogenetics; the other two patients had core-binding factor AML. Midostaurin was initiated on day 8 of induction in all but 2 patients, who started on day 11 and 12, respectively.Nine of ten patients completed all 28 planned doses of midostaurin. All patients received antifungal prophylaxis with micafungin throughout the course of midostaurin. The median time from day 1 of induction to neutrophil (>500/µl) and platelet (>100,000/µl) recovery was 23 days and 26 days, respectively. Granulocyte colony stimulating factor (G-CSF) was initiated for all patients after day 14 bone marrow biopsy, as per institutional procedure. Four of 10 patients experienced an adverse event attributed to midostaurin. Maculopapular rash was observed in 3 patients a median of 5 days after midostaurin initiation: 2 of 3 patients had a grade 2 rash and continued therapy with topical steroids; one patient had a grade 3 rash and discontinued midostaurin after 17 of 28 planned doses. A grade 1 drug fever was attributed to midostaurin in a fourth patient. Fevers persisted despite neutrophil recovery and subsequently dissipated after completion of the final midostaurin dose.Persistent FLT3 mutation was detected in 4/9 (1 not reported) day 14 bone marrow biopsies but was negative in 9/10 pts on day 28. The lone positive FLT3 result on day 28 occurred in a patient with refractory disease (>5% blasts) necessitating salvage therapy. Notably, this patient only completed 17 of 28 planned doses. All other patients exhibited a complete response (CR) on day 28.The median follow-up time was 243 days (range: 57 to 394). Nine patients are alive at the time of reporting. Six patients proceeded to allogeneic transplantation -one death was attributed to transplant-related complications, occurring in the same patient needing salvage and reduced duration midostaurin. Two patients relapsed a median of 184 days after start of induction -both FLT3-ITD positive and neither having undergone allogeneic transplant prior to relapse.Conclusions:Midostaurin in combination with idarubicin-based induction 3+7 therapy in this first case series appears to be safe. While the incidence of rash was higher (30%) than reported in RATIFY, this only resulted in discontinuation of therapy in one patient. Although patient numbers are limited, 90% achieved a FLT3 negative CR after completion of induction therapy and six patients proceeded to allogeneic transplantation. A confounding variable includes the routine use of G-CSF, which likely contributed to the shorter duration from induction to neutrophil recovery observed compared to RATIFY (23 days vs 26 days). The influence of G-CSF use on outcome is uncertain, however represents an interesting observation. A randomized, prospective trial comparing midostaurin in combination with idarubicin versus daunorubicin at both 60mg/m2 and 90mg/m2 is warranted to establish the optimal anthracycline induction therapy for FLT3 mutated AML patients. DisclosuresAl-Homsi:Celyad: Membership on an entity's Board of Directors or advisory committees. Diefenbach:Denovo: Research Funding; Merck: Consultancy, Research Funding; Acerta: Research Funding; Incyte: Research Funding; Trillium: Research Funding; Millenium/Takeda: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Genentech: Consultancy; Seattle Genetics: Consultancy, Research Funding.

PHP281 - THE METHODOLOGICAL DIFFERENCES OF EUROPEAN JOINT ASSESSMENT AND GERMAN HTA: AN EMPIRICAL APPROACH

With the legislatory endorsement of European Joint Assessments for pharmaceuticals (JAs) the HTA landscape in Europe is approaching fundamental structural change. Since 2016, only two pharmaceuticals (both in oncology) have undergone both European JA and national assessment in Germany. This is the first time that the methodology applied in the different assessments is compared based on concrete examples. Differences between the JA and the corresponding German assessment report were analyzed for Midostaurin in the indication of FLT3-positive acute myeloid leukemia (JA published November 2017) and Alectinib in the indication of ALK-positive advanced non-small cell lung cancer (JA published January 2018). The comparison features five key items previously identified: body of evidence, comparators, endpoints, evidence synthesis, and subgroups. While in Germany only RCT data with a specific comparator was accepted in each case, non-RCTs and multiple comparators were included in the JA leading to a larger body of evidence (1 study vs. 3 studies in each example). Indirect comparisons were considered only in the JAs (e.g. Alectinib vs. Ceritinib). Furthermore, some endpoints not regarded as relevant in Germany, such as progression- and disease-free survival, were presented within the JAs. The methodology used to evaluate the certainty of results differed significantly. However, there were also similarities between JA and the German assessment: both approaches follow the principles of evidence-based medicine; central endpoints were overall survival, health-related quality of life, and safety; subgroups investigated were consistent. The examples reviewed confirm that the outcome of European JAs and their German equivalent can be expected to differ for methodological reasons. In general, JAs offer a broader spectrum of possibilities in terms of suitable evidence and analytical methods. Nevertheless, a high degree of consensus was observed in the final evaluations suggesting a strong potential for future harmonization of the different procedures.