FLT3 Internal Tandem Duplication Mutations Research Articles

A heavy metal baseline score predicts outcome in acute myeloid leukemia.

Despite abundant epidemiological data linking metals to leukemia and other cancers, baseline values of toxic and essential metals in patients with leukemia and the clinical impact of these metals remain unknown. Thus, we sought to quantify metal values in untreated patients with acute myeloid leukemia (AML) and controls and determine the impact of metal values on AML patients' survival. Serum samples from patients with untreated AML and controls at Hospices Civils de Lyon were analyzed and compared for trace metals and copper isotopic abundance ratios with inductively coupled plasma mass spectrometry. Survival analysis was performed as a function of metal values, and a multi-metal score was developed for patients with AML. Serum samples were collected from 67 patients with untreated AML and 94 controls. Most patients had intermediate-risk cytogenetics (63.1%) without FLT3 internal tandem duplication mutations (75.6%) or NPM1 mutations (68.1%). Most metal values differed significantly between AML and control groups. Patients with lower magnesium and higher cadmium values had the worst survival rates, with only 36% surviving at 6 months (P = .001). The adverse prognostic effect of this combination was maintained on multivariate analysis. Based on this, we developed a novel metal score, which accounts for multiple relative abnormalities in the values of five toxic and five essential metals. Patients with a higher metal score had significantly worse survival, which was maintained on multivariate analysis (P = .03). This baseline metal scoring system was also prognostic when we applied it to a separate population of front-line AML patients.

AXL/Mertk Inhibitor ONO-7475 Potently Synergizes with BCL2 Inhibitor ABT-199, Overcomes ABT-199 Resistance Mechanisms, and Kills FLT3 ITD AML Cells

AXL/Mertk Inhibitor ONO-7475 Potently Synergizes with BCL2 Inhibitor ABT-199, Overcomes ABT-199 Resistance Mechanisms, and Kills FLT3 ITD AML Cells

SM09419, a Novel, Small-Molecule CDC-like Kinase (CLK) Inhibitor, Demonstrates Strong Inhibition of the Wnt Signaling Pathway and Antitumor Effects in FMS-like Tyrosine Kinase 3 (FLT3)-Mutant Acute Myeloid Leukemia Models

Acute myeloid leukemia (AML) with the FLT3 internal tandem duplication (FLT3-ITD) mutation accounts for ~25% of all AMLs, carries a poor prognosis, and is prone to relapse despite targeted therapy. FLT3 mutations are associated with aberrant activation of the Wnt signaling pathway, which itself is implicated in AML initiation/progression and is required for the self-renewal and survival of leukemic stem cells. CLKs regulate the activity of serine/arginine-rich splicing factors (SRSFs) that modulate spliceosome assembly, mRNA splicing, and gene expression. SM09419 is a novel, oral, small-molecule pan-CLK inhibitor that potently inhibits the Wnt pathway. These studies examined the antitumor activity of SM09419 as a single agent and in combination with targeted and standard therapies in preclinical models of FLT3-ITD AML. In MV-4-11 and MOLM-13 AML cells carrying the FLT3-ITD mutation, SM09419 dose-dependently inhibited SRSF6 phosphorylation and potently suppressed expression of Wnt pathway-related genes (CCND1, MYC, TCF7, DVL2). The effect on cell proliferation was tested in 8 AML cell lines with varying mutation profiles as well as 26 different leukapheresis-derived primary human AML cells. Proliferation was strongly impaired by SM09419 across all tested cell lines (average EC50=0.2 + 0.048 µM]); MV-4-11 and MOLM-13 cells had EC50 of 0.049 and 0.144 µM, respectively. SM09419 also potently inhibited proliferation in all primary AML cells (average EC50=0.048 + 0.0097 µM) regardless of FLT3 mutation status, cytogenetics, or AML diagnosis (de novo or relapsed/refractory). SM09419 also induced apoptosis in MV-4-11 and MOLM-13 cells, increasing caspase 3/7 activation and PARP cleavage while reducing survivin and MCL-1 expression relative to vehicle. In vivo antitumor effects and tolerability of oral SM09419 (QD) alone or combined with either midostaurin (FLT3 inhibitor) or venetoclax (BCL2 inhibitor) and/or azacitidine were assessed in FLT3-ITD xenograft models (n=5-6/group). In MOLM-13 xenografts, SM09419 (12.5 and 25 mg/kg) induced strong tumor growth inhibition (TGI) vs. vehicle at Day 14 (TGI 52% [p<0.05] and 74% [p<0.001], respectively). Midostaurin (50 mg/kg) induced significant TGI vs. vehicle (50%, p<0.05), which was increased when administered in combination with 12.5 mg/kg SM09419 (81%, p<0.001). In MV-4-11 xenografts, single-agent SM09419 (6.25, 12.5, and 25 mg/kg) induced significant TGI vs. vehicle (56% [p<0.05], 94%, and 95% [p<0.001], respectively) at Day 26 with tumor regression in all mice dosed at 12.5 mg/kg and 25 mg/kg. In a subsequent experiment, midostaurin (50 mg/kg) alone and combined with 6.25 mg/kg SM09419 for 23 days induced tumor regression in MV-4-11 xenografts (100% TGI vs. vehicle, p<0.0001). After treatment discontinuation, tumor regression was maintained in all mice (6/6) treated with the combination for 26 days, whereas tumor regrowth was immediately observed in midostaurin-treated mice. In another MV-4-11 xenograft study, the combination of 6.25mg/kg SM09419 with azacitidine (0.8 mg/kg QD) and/or venetoclax (25 mg/kg QD) induced significant TGI (95-98% vs. vehicle, p<0.001) with tumor regression at Day 26. Azacitidine + venetoclax induced 79% TGI (p<0.001), but no tumor regression was observed. The triple combination induced tumor regression in all mice and complete regressions in 4/6 mice (67%); it had a greater effect on slowing tumor regrowth after treatment discontinuation vs. a single agent or doublet. SM09419 alone or in combination was well tolerated in these xenograft models based on body weight measurements. In summary, SM09419 potently inhibited SRSF6 phosphorylation and Wnt signaling pathway activity and induced apoptosis in FLT3-ITD cell lines. It also inhibited proliferation in cell lines and primary AML cells regardless of FLT3 status. The strong in vivo antitumor effects observed as combination treatment suggest that SM09419 combined with standard therapies may provide a clinical benefit by slowing or preventing relapse in AML with a marker of poor prognosis such as FLT3-ITD. A Phase 1 study assessing safety, tolerability, and pharmacokinetics of SM09419 in subjects with advanced hematologic malignancies is being initiated. Disclosures Chung: Samumed, LLC: Employment, Equity Ownership. Creger:Samumed, LLC: Employment, Equity Ownership. Sitts:Samumed, LLC: Employment, Equity Ownership. Chiu:Samumed, LLC: Employment, Equity Ownership. Mak:Samumed, LLC: Employment, Equity Ownership. KC:Samumed, LLC: Employment, Equity Ownership. Tam:Samumed, LLC: Employment, Equity Ownership. Bucci:Samumed, LLC: Employment, Equity Ownership. Stewart:Samumed, LLC: Employment, Equity Ownership. Phalen:Samumed, LLC: Employment, Equity Ownership. Cha:Samumed, LLC: Employment, Equity Ownership.

Characterization of Response and Transfusion Independence in Patients with FLT3-Internal Tandem Duplication (FLT3-ITD)-Mutated Relapsed/Refractory Acute Myeloid Leukemia Treated with Quizartinib or Salvage Chemotherapy in the Quantum-R Trial

Characterization of Response and Transfusion Independence in Patients with FLT3-Internal Tandem Duplication (FLT3-ITD)-Mutated Relapsed/Refractory Acute Myeloid Leukemia Treated with Quizartinib or Salvage Chemotherapy in the Quantum-R Trial

Metabolic Reprogramming of Human Mitochondrial NAD(P)+-Dependent-Malic Enzyme 2 in Acute Myeloid Leukemia

Yu-Nan Huang1, Kang-Hsi Wu4, Te-fu Weng4, Su-Ching Liu4, Hui-Chih Hung1*, Ching-Tien Peng4,5* FLT3 internal tandem duplication (FLT3-ITD) mutations in patients with acute myeloid leukemia (AML) are usually associated with other mutations resulting in unfavorable outcome. Tyrosine kinase inhibitors (TKI) have shown promising responses, however, these responses are almost transient in therapy-resistant AML. Here, we show that human mitochondrial NAD(P)+-dependent-malic enzyme 2 (ME2) have significantly increased in CD34+ cell of patients with AML. To determine how ME2 establish metabolic reprogramming of leukemogenesis, we performed a comprehensive analysis of metabolism in CRISPR-mediated ME2 knockout leukemic cells (THP-1 and MV4-11) and purified leukemic blast cells (CD34+) derived from patients with AML. We demonstrate that disrupting ME2 signaling exerts potent activities against proliferation, reduced oxidative metabolism and lactate metabolism. We also show that genetic inhibition of RUNX1/FLT3/ME2 markedly repressed AML cell leukemogenesis. In conclusion, our findings provide a rationale for clinical development of this strategy for treating RUNX1 and FLT3-mutated leukemic patients. Disclosures No relevant conflicts of interest to declare.

Sorafenib in Combination with Standard Chemotherapy for Children with High Allelic Ratio FLT3/ITD+ AML Improves Event-Free Survival and Reduces Relapse Risk: A Report from the Children's Oncology Group Protocol AAML1031

INTRODUCTION Sorafenib, a multi-kinase tyrosine kinase inhibitor (TKI) targets FLT3 internal tandem duplication (FLT3/ITD) mutations and has efficacy in adult FLT3/ITD+ AML. High allelic ratio (HAR) FLT3/ITD mutations (allelic ratio of > 0.4, referred to as FLT3/ITD+) confer poor prognosis in de novo pediatric AML. COG AAML1031 Arm C studied the feasibility and efficacy of adding sorafenib to standard chemotherapy and as a single agent maintenance therapy in pediatric HAR FLT3/ITD+ patients. METHODS The AAML1031 primary randomization (Arm A vs. B) has been previously reported (Aplenc et al. Blood 2016;128:899). Patients with FLT3/ITD+ AML were offered secondary enrollment on Arm C and toxicities were monitored on respective treatment arms. An initial safety phase (Cohort 1, C1; N=12) defined the maximum tolerated dose (MTD) of sorafenib when added to induction II chemotherapy and subsequent courses. Following completion, the study was amended (Cohort 2; C2) to start sorafenib on day 11 of induction I and concomitantly with chemotherapy in subsequent cycles. A year of sorafenib maintenance was also added. Due to concerns for cardiac toxicity on interim analyses, the study was amended (Cohort 3; C3) to initiate sorafenib after chemotherapy completion in all courses. Sorafenib pharmacokinetics (PK) and plasma inhibitory activity (PIA) were measured in a subset of patients. Clinical outcome analysis was limited to cohorts 2/3 given lack of induction I sorafenib exposure in C1. Arm C results were compared to N=34 HAR FLT3/ITD+ AML patients on the control arm of COG AAML0531 (Arm A), a group that received comparable chemotherapy without sorafenib. Complete remission (CR), event-free survival (EFS), overall survival (OS) and relapse risk (RR) of patients with HAR FLT3/ITD AML enrolled on the 2 trials were compared to assess the impact of sorafenib use. Cox proportional hazards were used for multivariable analyses. RESULTS In the dose finding phase (C1), 12 patient were enrolled and the MTD of sorafenib defined as 200 mg/m2; dose limiting toxicities included rash (N=2; 1 grade III, 1 grade II), grade II hand foot syndrome and grade III fever. For the evaluation of efficacy, 80 C2/C3 (C2: N=33, C3: N=47) patients were enrolled. 30/80 (37.5%) received at least 1 maintenance cycle; 20/80 (25%) completed all courses. As outcomes were compared to N=34 HAR FLT3/ITD+ AML patients enrolled on the preceding trial AAML0531 Arm A as a historical comparison, clinical characteristics were described for the 2 groups (Table 1). For AAML1031 C2/C3, rates of induction I CR were 73% vs. 56% for AAML0531 Arm A (p=0.078). Following induction II, CR rates were 91% and 70% respectively (p=0.007). 3 year EFS from study entry was 57.5% for Arm C vs. 34.3% for AAML0531 Arm A; (p=0.007) whereas 3 year OS from study entry was not significantly different (63.9% vs. 54.1%, p=0.375, Figure 1A). RR from CR was reduced with sorafenib treatment (Arm C 18.2% vs. historical 52.5%, p=0.006, Figure 1B). The improvement in EFS and RR observed with sorafenib was retained in those with wild-type nucleophosmin (NPM-) FLT3/ITD+ AML but lacked statistical significance compared to historical in NPM+ FLT3/ITD+ disease (Figure 2A/2B). As 21/34 (62%) patients in the historical control did not undergo HSCT there was concern that the improved outcome observed for Arm C reflected increased use of HSCT in AAML1031. Multivariable analysis that adjusted for HSCT resulted in significantly lower EFS and higher RR for FLT3/ITD+ patients on AAML0531, suggesting that increased use of HSCT on Arm C did not explain the improved outcomes observed (EFS: HR 2.04, CI 1.18 - 3.53; p=0.01, RR: HR 3.09, CI 1.28 - 7.49; p=0.012). While dose modifications were more frequent for FLT3/ITD+ patients on Arm C versus arms A/B of AAML1031, targeted toxicity rates were otherwise no higher (Table 2). PIA assays demonstrated median trough FLT3 inhibition of 92% (n=29, 95% CI 79-94%), 91% (n=84, 95% CI 87-94%) and 81% (n=70, 95% CI 76-90%) for induction I, induction II and intensification I, respectively. CONCLUSION Addition of sorafenib to Arm C of AAML1031 was safe and resulted in potent FLT3 inhibition, particularly early in therapy. Sorafenib improved rates of induction II CR as well as 3 year EFS and reduced RR from CR compared to historical controls. These data support use of sorafenib in pediatric patients with HAR FLT3/ITD+ AML. Disclosures Fisher: Pfizer: Research Funding; Astellas: Other: Data Safety Monitoring Board Chair for an antifungal study; Merck: Research Funding. Levine:Viracor: Patents & Royalties: biomarker patent; Biogen: Other: non-financial support; Ironwood: Consultancy; Bluebird Bio: Consultancy; National Cancer Institute: Research Funding; Novartis: Consultancy; Incyte: Consultancy, Research Funding; Kamada: Research Funding. Loken:Hematologics, Inc: Employment, Equity Ownership. OffLabel Disclosure: sorafenib for high allelic ratio FLT3/ITD+ AML

Mutational Profiling Predicts Clinical Outcomes to Azacytidine and Low Dose Cytarabine Plus Fludarabine (FLUGA) in Elderly Acute Myeloid Leukemia Patients Enrolled in the Pethema Phase III Flugaza Trial

Mutational Profiling Predicts Clinical Outcomes to Azacytidine and Low Dose Cytarabine Plus Fludarabine (FLUGA) in Elderly Acute Myeloid Leukemia Patients Enrolled in the Pethema Phase III Flugaza Trial

FF-10101 Retains Potent Inhibitory Activities Against Resistant Mutations to FLT3 Inhibitors, Newly Identified in Random Mutagenesis Screens

Background: FLT3-internal tandem duplication (FLT3-ITD) mutation is a poor prognostic factor for acute myeloid leukemia (AML). Several second-generation FLT3-targeted tyrosine kinase inhibitors with high selectivity and potency have been developed to date. Recently, gilteritinib was approved for the FLT3 mutation-positive relapsed or refractory AML patients. However, acquired mutations at the F691 residue in FLT3 kinase domain were identified in the patients who had disease progression after the treatment with gilteritinib, as with quizartinib treatment that caused resistant mutations at F691 and D835 residues. Therefore, it is very important to clarify the potencies of each FLT3 inhibitors against acquired FLT3 mutations for clinically selecting an appropriate FLT3 inhibitor according to the mutation type. In this study, we explored the resistant mutations against FLT3 inhibitors by random mutagenesis analysis and evaluated the cross-reactivity of FLT3 inhibitors against each resistant mutation. Methods: For random mutagenesis assay, 32D cells were infected with retroviruses encoding randomly mutagenized human FLT3-ITD. FLT3-ITD dependent 32D cells were established without an addition of IL-3, and then treated with FLT3 inhibitors, gilteritinib, FF-10101 and quizartinib, at concentrations of GI95 and 3 x GI95. Two weeks after treatment, full length FLT3-ITD sequences of viable clones were analyzed. The identified mutated FLT3-ITDs were introduced into 32D cells to confirm the resistance to FLT3 inhibitors. For cell growth assay, 32D transfectants were incubated with 5 FLT3 inhibitors (gilteritinib, FF-10101, quizartinib, crenolanib and midostaurin) for 3 days followed by determination of cell viability. Results: We identified the gilteritinib-resistant mutation (FLT3-ITD+D698N), quizartinib-resitant mutation (FLT3-ITD+N676T), and FF-10101-resistant mutation (FLT3-ITD+C695W) (Table). Inhibitory activity of gilteritinib against FLT3-ITD+D698N-expressing 32D cells (GI50, 27 nM) was decreased by 12-fold as compared with that against original FLT3-ITD-expressing 32D cells (GI50, 2.3nM). FF-10101 (GI50, 0.73 nM), quizartinib (GI50, 0.99 nM) and crenolanib (GI50, 19 nM) retained potency against FLT3-ITD+D698N, while midostaurin (GI50, 47 nM) did not have a potency. In FLT3-ITD+N676T-expressing 32D cells, inhibitory activities of quizartinib and midostaurin were decreased by 11 and 15-fold (GI50, 6.6 nM and 83 nM, respectively), while FF-10101 (GI50, 0.73 nM), gilteritinib (GI50, 6.6 nM) and crenolanib (GI50, 19 nM) retained potency. In FF-10101-resistant mutation (FLT3-ITD+C695W)-expressing 32D cells, the other inhibitors retained growth inhibitory activity. Conclusions: Resistant mutations to gilteritinib and quizartinib were newly identified in FLT3 kinase domain by random mutagenesis analysis. FF-10101 retained potent inhibitory activities against FLT3-ITD+N676T conferring resistance to quizartinib and midostaurin, and FLT3-ITD+D698N resistant to gilteritinib and midostaurin, although FF-10101 was vulnerable to FLT3-ITD+C695W substituted for C695 residue which forms covalent bond with FF-10101. These results indicated that FF-10101 was a promising agent for the treatment of patients with AML with FLT3 inhibitor-resistant mutations newly identified in this study. Disclosures Ishikawa: Bristol-Myers Squibb: Honoraria; Abbvie GK.: Honoraria; Celgene Co., Ltd.: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria. Saito:FUJIFILM Corporation: Employment. Morimoto:FUJIFILM Corporation: Employment. Murao:FUJIFILM Corporation: Employment. Terada:FUJIFILM Corporation: Employment. Yamaura:FUJIFILM Corporation: Employment. Hagiwara:FUJIFILM Coporation: Employment. Kiyoi:Daiichi Sankyo Co., Ltd: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Pfizer Japan Inc.: Honoraria; Bristol-Myers Squibb: Research Funding; Perseus Proteomics Inc.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Astellas Pharma Inc.: Honoraria, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding.

Phase II Study of Midostaurin + Chemotherapy in Pediatric Patients with Untreated, Newly Diagnosed, FLT3-Mutated Acute Myeloid Leukemia (AML)

Background: In pediatric patients with AML, the presence of FLT3-internal tandem duplication (ITD) mutation is indicative of high-risk disease and confers a worse prognosis and a greater chance of relapse. Midostaurin is a multikinase inhibitor with potent activity against both mutated and non-mutated FLT3. In adult patients with FLT3-mutated AML, midostaurin in combination with chemotherapy has demonstrated good safety/tolerability and significantly improved overall survival (OS) and event-free survival (EFS) compared with placebo + chemotherapy. We present here the design of an ongoing phase II study (NCT03591510) evaluating midostaurin combined with standard chemotherapy and as a single-agent post-consolidation therapy in newly diagnosed pediatric patients with FLT3-mutated AML, other than acute promyelocytic leukemia. Study Design and Methods: This open-label, single-arm study is composed of 2 parts. For Part 1, the primary objective is to determine the recommended phase II dose (RP2D) of midostaurin in combination with chemotherapy, as determined by the occurrence of dose-limiting toxicities. For Part 2, in which patients will receive the RP2D of midostaurin, the primary objective is to evaluate the efficacy of midostaurin + chemotherapy as measured by the EFS rate at 24 mo (for regulatory purposes outside the US) or to assess the safety/tolerability of midostaurin + chemotherapy (for regulatory purposes within the US). Secondary objectives include safety/tolerability (outside US) and EFS at 24 mo (within US), other efficacy parameters (including OS, complete remission [CR], CR with incomplete blood count recovery [CRi], cumulative incidence of relapse, and minimal residual disease-negative status), and characterization of the pharmacokinetics of midostaurin. Eligible patients are aged 3 mo to < 18 yr with expected survival of > 12 wk, have a Lansky or Karnofsky performance status of ≥ 60, and have previously untreated de novo AML with FLT3 mutation: ITD and/or mutation in the tyrosine kinase domain, with mutant/wild-type signal ratio cutoff of ≥ 0.05. Exclusion criteria include any concurrent malignancy, Philadelphia chromosome or BCR-ABL1-positive AML, AML associated with Down syndrome, secondary AML, symptomatic leukemic central nervous system involvement, bone marrow failure syndrome, or prior treatment with a FLT3 inhibitor. Patients are to receive 5 treatment blocks: 2 of standard induction (Blocks 1-2) and 3 of consolidation (Blocks 3-5) chemotherapy, each with sequential twice-daily (BID) oral midostaurin. In Part 1 of the study, midostaurin will be given at a starting dose of 30 mg/m2 BID; dose escalation and determination of RP2D will be facilitated by a Bayesian hierarchical logistic regression model guided by escalation with overdose control principle. In Part 2, midostaurin will be administered at the RP2D. Induction therapy will be the local standard chemotherapy regimen with midostaurin starting after 24 hr from identification of FLT3 abnormalities and continuing for 14 d for Block 1, and FLADx (fludarabine + high-dose cytarabine on D1-5, daunorubicin on D2, 4, 6) with sequential midostaurin on D8-21 for Block 2. Patients demonstrating CR or modified CRi will proceed to consolidation with the following chemotherapy regimens, each with sequential midostaurin on D8-21: HAM (high-dose cytarabine on D1-3, mitoxantrone on D3, 4) for Block 3, HA3E (high-dose cytarabine on D1-3, etoposide on D1-5) for Block 4, and HiDAC (high-dose cytarabine on D1-3) for Block 5. Patients must be in continued remission (CR or modified CRi) to receive each block of consolidation therapy. Patients who complete Block 5 and remain in remission will proceed to 12 × 28-d cycles of post-consolidation therapy with single-agent midostaurin. Patients may undergo allogeneic hematopoietic stem cell transplantation at any time at the investigator's discretion. Overall, the aim is to recruit a minimum of 52 patients, including at least 49 at the RP2D dose. This study is currently enrolling patients at 13 centers in 7 countries (as of July 26, 2019), and a total of 37 centers in 14 countries are planned. Disclosures Reinhardt: Novartis: Other: Participation in Advisory Boards; Jazz: Other: Participation in Advisory Boards, Research Funding; CSL Behring: Research Funding; Roche: Research Funding. Zwaan:BMS: Research Funding; Servier: Consultancy; Sanofi: Consultancy; Celgene: Consultancy, Research Funding; Janssen: Consultancy; Pfizer: Research Funding; Novartis: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmaceuticals: Other: Travel support; Roche: Consultancy; Incyte: Consultancy. Hoenekopp:Novartis Pharma AG Basel: Employment, Equity Ownership. Niolat:Novartis Pharma: Employment. Ifrah:Novartis: Employment. Noel-Baron:Novartis: Employment, Equity Ownership. Locatelli:Miltenyi: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BluebirdBio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Midostaurin is a kinase inhibitor approved in combination with chemotherapy for the treatment of FLT3-mutated AML in adults; the study reported here is in pediatric patients with FLT3-mutated AML.

BMT CTN Protocol 1506: A Phase 3 Trial of Gilteritinib As Maintenance Therapy after Allogeneic Hematopoietic Stem Cell Transplantation in Patients with FLT3-ITD+ AML

BMT CTN Protocol 1506: A Phase 3 Trial of Gilteritinib As Maintenance Therapy after Allogeneic Hematopoietic Stem Cell Transplantation in Patients with FLT3-ITD+ AML

Improved Outcomes of All-Trans-Retinoic Acid and Arsenic Trioxide Plus Idarubicin As a Frontline Treatment in Adult Patients with Acute Promyelocytic Leukemia

Objective To analyze the outcomes of all-trans-retinoic acid (ATRA) and IV arsenic trioxide (ATO) plus idarubicin (IDA) as a frontline induction therapy in 118 acute promyelocytic leukemia (APL) patients with high risk (HR) and standard risk (SR) from January 2008 to December 2017. Methods The medical records of 118 APL patients (HR, n=45; SR, n=73) who received the frontline triple combination regimen at Henan Provincial People's Hospital and Tongji Hospital were retrospectively reviewed. Consolidation therapy comprised 6 cycles of ATO and ATRA plus IDA, and IDA was administered in 1~4 cycles of consolidation therapy based on the comparable clinical efficacy compared with 6 cycles and fewer side effects to myocardium without subsequent maintenance therapy. Results Of 118 patients, there were 3 (2.5%) early deaths and 115 (97.5%) hematologic complete remissions (hCR), and 102 (88.7%) of 115 patients achieved molecular complete remissions (mCR) following induction therapy, and all patients were PCR-negative for PML-RARa after the first cycle of consolidation therapy. The 5-year overall survival (OS) and event-free survival (EFS) were 93.0±2.9% and 92.4±3.0%, respectively. Early death, hCR, mCR and toxicities were comparable between the HR and SR groups. The cumulative incidence of relapse (CIR) in the HR group was 4.7% (n=2), and the CIR in the SR group was 0. The OS and EFS of the SR and HR groups were comparable (92.3±4.5 vs. 92.8±4.0%, X2=0.263, P=0.608; 92.3±4.5 vs. 91.1±4.2%, X2=0.917, P=0.338). The total dosage of IDA was approximately 181~258 mg, and no patient experienced cardiotoxicity. OS and EFS were not influenced by fms-related tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation status (P=0.405, 0.528, respectively). Conclusion The triple combination of ATRA and ATO plus IDA as both an induction and consolidation therapy for the HR and SR groups attained excellent outcomes, and this regimen was effective, safe and easy, without maintenance therapy. The triple combination treatment might be a preferred frontline therapy for APL patients, especially for HR patients or patients with the APL FLT3-ITD mutation. Disclosures No relevant conflicts of interest to declare.

Clinical Outcomes and Characteristics of Patients (pts) with FLT3-Internal Tandem Duplication (FLT3-ITD)-Mutated Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) Undergoing Hematopoietic Stem Cell Transplant (HSCT) after Quizartinib (Q) or Salvage Chemotherapy (SC) in the Quantum-R Trial

Clinical Outcomes and Characteristics of Patients (pts) with FLT3-Internal Tandem Duplication (FLT3-ITD)-Mutated Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) Undergoing Hematopoietic Stem Cell Transplant (HSCT) after Quizartinib (Q) or Salvage Chemotherapy (SC) in the Quantum-R Trial

Prognosis and outcome of patients with acute myeloid leukemia based on FLT3-ITD mutation with or without additional abnormal cytogenetics.

The FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) gene mutation is present in ~20% of patients with de novo acute myeloid leukemia (AML). Patients with an FLT3-ITD mutation have a poor prognosis. However, the prognostic function of FLT3-ITD combined with other cytogenetic abnormalities are not clear. In the present study, a retrospective analysis of 103 newly diagnosed patients with AML was performed. The results revealed that the overall survival (OS) and recurrence-free survival (RFS) times were significantly longer in patients with an FLT3-ITD mutation combined with other favorable risk genes, compared with in those patients with a single FLT3-ITD mutation (P=0.0361 and P=0.0426). Sorafenib combined with chemotherapy significantly improved the overall response rate (ORR) when compared with mono-chemotherapy (P=0.039), but no significant differences were observed in the OS and RFS. In conclusion, favorable-risk cytogenetics may improve the clinical outcomes of patients with FLT3-ITD-mutated AML, but adverse-risk cytogenetics may not further worsen the prognosis. Sorafenib combined with chemotherapy may increase the ORR but would not result in a longer OS and RFS.

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.

Prognostic Significance of bcr-1 and bcr-3 Isoforms of PML-RARA and FLT3-ITD in Patients With Acute Promyelocytic Leukemia

BackgroundAcute promyelocytic leukemia (APL) has a characteristic peculiar morphologic and genetic features as well as a more favorable outcome. We studied the differential effect of bcr-1 and bcr-3 isoforms of the promyelocytic leukemia/retinoic acid receptor alpha (PML-RARA) transcript together with FLT3 internal tandem duplication (FLT3-ITD) mutation status on the outcome of newly diagnosed de novo APL patients. Patients and MethodsThis cohort study included all patients diagnosed with APL at outpatient medical and pediatric oncology clinics of the National Cancer Institute, Cairo University, Cairo, Egypt, from May 2012 to January 2018. ResultsThe study included 118 patients with APL, 71 adults (60.2%) and 47 children (39.8%). Median (range) age was 25 (1.5-70) years. Children had significantly higher total leukocyte count (≥10 × 109/L), disseminated intravascular coagulation (DIC), and thrombocytopenia (< 40 × 109/L) than adults (P = .04, .03, and .04, respectively), while the latter group had significantly higher hemorrhage than children (P = .04). FLT3-ITD mutation was detected in the whole group, children, and adults in 23.7%, 30.6%, and 24.6%, respectively. FLT3-ITD mutation was significantly associated with leukocytosis in the whole group (P = .039). bcr-3 was significantly associated with FLT3-ITD mutation in the whole APL cohort and in adults (P = .011, P = .022). All children (both bcr-1 and bcr-3) and all adult patients with bcr-3 experienced CR, while 22 (78.5%) of 28 patients with bcr-1 experienced CR (P = .04). APL patients with DIC and hemorrhage had significantly lower overall survival (P = .002 and < .001, respectively). Overall survival for APL in children was significantly better than in adults (P = .005). Multivariate analysis indicated that age was an independent prognostic variable affecting survival (hazard ratio = 2.6; 95% confidence interval, 1.3-5.3; P = .007) (adults had hazard ratio of death 2.6 times higher than children). DIC and FLT3-ITD were independent prognostic variables affecting survival in children with APL (hazard ratio = 12.3; 95% confidence interval, 1.46-104.61; P = .021; and hazard ratio = 5.2; 95% confidence interval, 1.01-26.95; P = .048, respectively). ConclusionAge is an independent prognostic factor for APL. bcr-3 is significantly associated with FLT3-ITD in adults with APL. DIC and FLT3-ITD are adverse prognostic factors in children with APL. Despite children being at higher risk, outcome is better than in adults.

Mutation analysis of 77 patients with normal-karyotype myelodysplastic syndrome

To carry out mutation analysis for patients with myelodysplastic syndromes (MDS) and a normal karyotype. Targeted capture and next-generation sequencing (NGS) was carried out using a customized 49-gene panel. FLT3 internal tandem duplication (FLT3-ITD), CALR, NPM1 and CEBPA mutations were detected by PCR and Sanger sequencing. Sixty-two patients (80.5%) were found to harbor at least one mutation. Each patient has carried 2.21 mutations in average. Coexistence of ≥ 3 mutations was common (43.7%). The most commonly mutated genes were RUNX1 (23.4%, 18/77), ASXL1 (18.2%, 14/77), NPM1 (15.6%, 12/77), U2AF1 (15.6%, 12/77), DNMT3A (11.7%, 9/77). Patients with SF3B1 mutations were significantly older than those with ASXL1 mutations (P=0.023). Mutations of the DNMT3A gene were significantly associated with the blood platelet level compared with BCOR mutations (P=0.02). No significant difference was found in the number and rate of mutations between those under or above 60-year-old. Among 67 patients with clinical follow-up, 20 (29.8%) has transformed to acute myeloid leukemia, and the time of transformation has ranged from 1 to 44 months, with a average of 5.3 months. RUNX1, U2AF1 and FLT3 mutations are associated with leukemic transformation. Coexistence of ≥ 3 mutations are frequent among patients with normal-karyotype MDS. Certain mutations are associated with age and leukemic transformation.

Efficacy and Safety of Single-Agent Quizartinib, a Potent and Selective FLT3 Inhibitor (FLT3i), in Patients (pts) With FLT3-Internal Tandem Duplication (FLT3-ITD) – Mutated Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) Enrolled in the Global, Phase 3, Randomized Controlled QuANTUM-R Trial

FLT3-ITD mutations occur in ≈25% of pts with AML and are associated with poor outcomes; pts with R/R FLT3-ITD AML have worse prognosis. Quizartinib (Q) is a once-daily, oral, highly potent and selective FLT3i.

Gilteritinib for the treatment of relapsed and/or refractory FLT3-mutated acute myeloid leukemia

ABSTRACTIntroduction: The receptor tyrosine kinase FLT3 is the most commonly mutated gene in acute myeloid leukemia (AML). FLT3-internal tandem duplication mutations are associated with an increased risk of relapse, and a number of small molecule inhibitors of FLT3 have been developed. The highly potent and selective FLT3 kinase inhibitor gilteritinib is the first tyrosine kinase inhibitor approved as monotherapy for the treatment of relapsed and/or refractory FLT3-mutated AML.Areas covered: We review the biology and prognostic significance of FLT3 mutations in AML and discuss the pharmacology, clinical efficacy, and toxicity profile of gilteritinib. We also summarize important differences among the various FLT3 inhibitors that are currently approved or under development and highlight areas of ongoing research.Expert opinion: Gilteritinib has been shown to improve survival compared to salvage chemotherapy in relapsed and/or refractory FLT3-mutated AML. Gilteritinib is orally available with a favorable toxicity profile and as such is quickly becoming the standard of care for this patient population. Ongoing clinical trials are evaluating gilteritinib in combination with frontline chemotherapy, in combination with other agents such as venetoclax and azacitidine for patients who are ineligible for standard induction therapy, and as a maintenance agent.

Association between increased mutation rates in DNMT3A and FLT3-ITD and poor prognosis of patients with acute myeloid leukemia.

A total of 133 patients with acute myeloid leukemia (AML) were enrolled in the current study and were subdivided into 4 groups: 34 harboring DNA methyltransferase 3 α (DNMT3A) + fms related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations, 37 harboring only FLT3-ITD mutation, 32 harboring only DNMT3A mutation and 30 harboring no mutations in DNMT3A and FLT3-ITD (control). Patients in all groups were administered daunorubicin and cytarabine chemotherapy regimens. The rates of complete remission (CR), 1-year relapse (RR) and 3-year overall survival (OS) were compared. Patients in the DNMT3A + FLT3-ITD mutation group exhibited higher proportions of peripheral white blood cells (WBCs) and myeloid progenitor cells compared with those in DNMT3A mutation only, FLT3-ITD mutation only and control groups (P<0.05). The rates of CD15+ and HLA-DR+ in the DNMT3A + FLT3-ITD mutation and DNMT3A mutation only groups were significantly higher than those in the FLT3-ITD mutation only and control groups (P<0.05); in addition, the rate of CD38+ in the DNMT3A + FLT3-ITD mutation and FLT3-ITD mutation only groups was significantly higher compared with that in the DNMT3A mutation only and control groups (P<0.05). The overall chemotherapy effectiveness rate, CR, 1-year RR and the 3-year OS rates of patients in the DNMT3A + FLT3-ITD mutation group were significantly worse compared with FLT3-ITD mutation only, DNMT3A mutation only and control groups (P<0.05). The results of this study indicated that increased mutation rates in DNMT3A and FLT3-ITD may be associated with increased WBC and myeloid progenitor cell counts, an inferior chemotherapy efficacy and prognosis, a lower CR rate, and higher 1-year RR and mortality rate.

Abstract 342: S63845, a novel BH3 mimetic Mcl-1 inhibitor synergizes with midostaurin to induce potent apoptosis in acute myeloid leukemia cells carrying FLT3-ITD mutations

Abstract Myeloid cell leukemia 1 (Mcl-1) is one of the key anti-apoptotic Bcl-2 family proteins that binds and neutralizes pro-apoptotic BIM, BAX and BAK at the mitochondrial outer membrane, preventing cytochrome c release and caspase activation. Selective upregulation of Mcl-1 functionally contributes to resistance of acute myeloid leukemias (AML) with FMS-like tyrosine kinase-3-internal tandem duplications (FLT3-ITD) to chemotherapy (Kasper S. et al. 2012, Blood Cancer J. 2:e60, doi:10.1038/bcj.2012.5). Here we show that a novel Mcl-1 inhibitor S63845 (Kotschy A. et al. 2016, Nature 538, 477-482), has synergistic proapoptotic activity in combination with FLT3-ITD kinase inhibitor midostaurin in pre-clinical models of AML. Our studies demonstrate that S63845 has potent single agent activity in AML cell lines and primary AML samples harboring FLT3-ITD with IC50 values in low nanomolar range. Co-targeting of Mcl-1 and FLT3-ITD with S63845 and midostaurin, respectively, significantly increased apoptosis in FLT3-ITD cells with caspase-3 activation and PARP cleavage occurring rapidly within 6 hours of treatment. Consistent with markedly reduced cell growth and viability, analysis of drug combinations efficacy using Bliss independence model revealed strong synergistic interactions between S63845 and midostaurin in FLT3-ITD cell lines and primary AML samples. Midostaurin caused de-phosphorylation of FLT3-ITD and its downstream targets such as STAT5, AKT and MAPK. This was accompanied by significant downregulation of MAPK-mediated phosphorylation of Mcl-1 at Thr163 required for Mcl-1 stability. Consequently, midostaurin reduced Mcl-1 protein levels, with no major changes in antiapoptotic Bcl-2 or Bcl-XL. Importantly, midostaurin increased expression of pro-apoptotic Bim, which could in turn bind and negate residual Mcl-1 pro-survival activity. Elevated Bim was sustained upon S63845 co-treatment, suggesting that Bim plays functional role in midostaurin/S63845-mediated lethality. Dynamic BH3 profiling showed that midostaurin primed FLT3-ITD cells to Mcl-1 and Bcl-2 inhibitors and facilitated general apoptosis priming in response to Bim peptide. Importantly, given that Mcl-1 is a major contributing factor to resistance of AML to Bcl-2 selective BH3-mimetic venetoclax, S63845/midostaurin treatment induced cell death in venetoclax-resistant FLT3-ITD mutants. In summary, S63845/midostaurin is highly synergistic in FLT3-ITD mutated AML cells including those resistant to venetoclax. In vivo experiments of tolerability and efficacy are ongoing and will be reported. Citation Format: Anna Skwarska, Qi Zhang, Shelley M. Herbrich, Natalia Baran, Ensar Halilovic, Peter Ruvolo, Vivian Ruvolo, Erick Morris, Andrew Wei, Donia Moujalled, Michael Andreff, Marina Konopleva. S63845, a novel BH3 mimetic Mcl-1 inhibitor synergizes with midostaurin to induce potent apoptosis in acute myeloid leukemia cells carrying FLT3-ITD mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 342.