FLT3-mutated Acute Myeloid Leukemia Patients Research Articles

Cladribine Combined with Homoharringtonine and Cytarabine Achieves a High Remission Rate in Adult Patients with De Novo Acute Myeloid Leukemia, Especially for Adverse-Risk Group: A Prospective, Single Center, Single-Arm, Phase 2 Study

Background: For adult de novo acute myeloid leukemia (AML), the standard 3+7 induction regimen has approximately 60% complete remission (CR) rate. Recently, BCL-2 inhibitor plus intensive chemotherapies as novel induction regimens have shown an above 90% composite complete remission (CRc) rate, however still have limited response effect in an adverse risk group, especially for patients with specific cytogenetic abnormalities such as KMT2A rearrangements, FLT3, RAS, and TP53 mutations. Multiple studies have shown that homoharringtonine (HHT) can target leukemia diseases that have switched their anti-apoptotic dependence from BCL-2 to MCL-1. We recently demonstrated that cladribine (CdA) could eradicate BCL-2 inhibitor-resistant monocytic leukemia stem cells driven by RAS mutants and KMT2A rearrangements. (Pei S, et al. Cancer Discovery. 2023) Together, these clinical and basic findings suggest a rationale for combining CdA with HHT to treat AML patients. In this study, we report the efficacy and safety of a newly designed “CHA” regimen composed of CdA, HHT, and cytarabine (AraC) in adult patients with de novo AML. Methods: This is a prospective, single-center, single-arm phase 2 trial. Newly diagnosed AML patients aged 18-59 years were enrolled. The diagnosis, risk classification, and response evaluation were determined according to ELN 2022 criteria. All enrolled patients received the CHA regimen: CdA (5mg/m2, days 1-3), HHT (2mg/m2, days 1-5), and AraC (100mg/ m2, days 1-7) administered intravenously. The response was evaluated on day 28 via bone marrow routine and minimal residual disease (MRD) measurement by flow cytometry. The primary endpoint was CRc (CR plus CRi). Secondary endpoints were MRD, overall survival (OS), disease-free survival (DFS), event-free survival (EFS), and adverse events. Bone marrow (BM) samples on day 1 and 7 and peripheral blood (PB) samples on days 1-7 were collected for laboratory flow analysis to monitor the kinetics of leukemia reduction. We also treated primary AML specimens with single, dual, and triple combinations of CdA, HHT, and Ara-C to evaluate their anti-leukemic activity in vitro. Statistical analyses were carried out using SPSS 24.0. Survival curves were prepared using the Kaplan-Meier method. A two-tailed P value <0.05 was considered statistically significant. This trial is registered on ClinicalTrials.gov (NCT05906914). Results: A total of 33 patients were enrolled from August 1, 2021, to April 15, 2023. The median age was 49 years. 33% (11/33) of patients were at favorable risk, 42% (14/33) were at intermediate risk, and 24% (8/24) were at adverse risk. After one course of CHA induction therapy, the CRc rate was 87% (27 of 31 patients), and the CR rate was 84% (26 of 31 patients). The CR rates in the favorable, intermediate, and adverse-risk groups were 82% (9 of 11 patients), 75% (9 of 12 patients), and 100% (8 of 8 patients), respectively. Notably, six out of six patients with KMT2A rearrangement and ten out of ten patients with RAS mutations achieved MRD-negative CR. The overall MRD negativity was 89% (24 of 27 patients with CRc). In 31 evaluable patients, four patients did not reach CR; three of them held FLT3 mutations. 30-days treatment-related mortality (TRM) was 6% (2 of 33 patients), and both died of sepsis. The median neutrophil recovery time (from the end of chemotherapy to the neutrophil counts ≥0.5×109/L) and platelets recovery time (from the end of chemotherapy to platelets counts ≥20×109/L) were both 11 days. With a median follow-up time of 13 months (IQR: 6-17 months), the one-year OS was 86%, 1-year DFS was 89%, and the 1-year EFS was 76%. Flow cytometry analysis of serially collected PB and BM samples from nine randomly selected patients revealed that an average of 91.0% and 94.7% of leukemia burden was cleared in the PB and BM by day 7, respectively, demonstrating a rapid and effective response induced by CHA. Finally, our in vitro assay demonstrated a synergistic effect of CdA, HHT, and Ara-C on primary AML specimens including three patients with adverse-risk. Conclusion: We propose the CHA regimen as a novel, effective, and safe chemotherapy regimen for newly diagnosed adult AML patients with remarkable potency in treating adverse-risk group patients, especially those with KMT2A rearrangements and RAS mutations that are otherwise resistant to BCL-2 inhibitor-based therapies, but a limited effect on FLT3mutated AML patients.

Real-World Data for FLT3-Mutated Acute Myeloid Leukemia Patients Treated in Resource-Constrained Settings

Background: Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is a well-established prognostic marker in acute myeloid leukemia (AML), whereas the impact of mutations in the Tirosine-Kinase Domain (TKD) is controversial. The association of TK inhibitors (TKIs) with intensive chemotherapy improved the overall survival (OS) of FLT3-mutated AML patients in randomized clinical trials and is recommended by the European LeukemiaNet panel of experts. Nevertheless, TKIs are not available for most patients treated in Low- and Middle-Income Countries (LMIC) and there is a scarcity of data concerning the outcome of treatment in these resource-constrained settings nowadays. Methods: This is a real-world study encompassing newly diagnosed adults with FLT3-mutated AML intensively treated at public hospitals in Brazil between January 2015 and December 2020, who were registered in the International Consortium on Acute Leukemias (ICAL) ICAML2015 Study. No patient received TKIs as part of the treatment. FLT3 mutation was tested in diagnostic samples by fragment analysis method. We used the Adapted Genetic Risk (AGR) score reported by Silveira et al. (2020), which is an adaptation of the European Leukemia Net (ELN) 2017 classification. The primary endpoints were event-free survival (EFS) and OS, calculated by Kaplan-Meier method. Relapse rate was calculated through competing risk analysis. Results: Overall, 350 pts (median age: 51y; 52.8% females) were included. FLT3-ITD and -TKD mutations were detected in 17.5% and 7.2% of the patients, respectively. Ninety patients (35.1%) were classified in the favorable risk group, 110 (42.9%) in the intermediate, and 56 (21.9%) in the adverse risk group, according to the AGR. The most frequent co-mutation was in the NPM1 gene. The remaining features are summarized in Table 1. Seventy-eight patients underwent allogeneic stem-cell transplantation (HSCT) in first complete remission (22.2%), of whom 27 had FLT3-ITD (i.e. 43.9% of patients harboring FLT3-ITD underwent HSCT). With a median follow-up period of 3.5 years, the median 4-year OS rate was 24.2% (95% CI 19.8-30.1), and the 4-y EFS 22.9% (95% CI 18.6-28.2). When only patients with FLT3-ITD mutations were analyzed, the 4-y OS rate was 14.4% which was inferior to that of patients with wild-type FLT3 (27.5%; p=0.057) (Figure 1). The cumulative incidence of relapse at four years was 39.4% for patients with FLT3-ITD and 43.9% for those with wildtype FLT3 (p=0.5), while the non-relapse mortality (NRM) at four years was 28.1% and 32.4%, respectively. The four-year OS rate for patients with FLT3-ITD mutations who underwent HSCT was 49.1% (95% CI 38.9-62.1). Patients with FLT3-ITD were further subdivided according to the allelic ratio (AR) in those with low (<0.5) and high (³ 0.5), and no significant difference in OS was detected between the two groups. Of note that patients harboring FLT3-TKD mutations showed a better overall survival rate compared to those with wild-type FLT3 (HR 0.49, 95% CI 0.26-0.93, p=0.029), but this result needs to be interpreted cautiously due the small size of the cohort. Conclusion: our results show that the frequency of FLT3-ITD mutations and their association with worse prognosis was similar to those described in high-income countries previously to the introduction of TKI as the standard of care. Therefore, allogeneic HSCT remains a valid consolidation strategy for FLT3-mutated patients. However, less than half of the patients for whom HSCT was indicated were actually transplanted. Additionally, the allelic ratio of FLT3-ITD should no longer be considered a prognostic marker for intensively treated patients not receiving FLT3 inhibitors.

Gilteritinib: The Story of a Proceeding Success into Hard-to-Treat FLT3-Mutated AML Patients.

The traditionally dismal outcome of acute myeloid leukemia (AML) patients carrying the FMS-related tyrosine kinase 3 (FLT3) mutations has been mitigated by the recent introduction of tyrosine kinase inhibitors (TKI) into clinics, such as midostaurin and gilteritinib. The present work summarizes the clinical data that led to the use of gilteritinib in clinical practice. Gilteritinib is a second-generation TKI with deeper single-agent activity than first-generation drugs against both FLT3-ITD and TKD mutations in human studies. Moreover, the phase I/II dose-escalation, dose-expansion Chrysalis trial showed an acceptable safety profile of gilteritinib (diarrhea, elevated aspartate aminotransferase, febrile neutropenia, anemia, thrombocytopenia, sepsis, and pneumonia) and a 49% overall response rate (ORR) in 191 FLT3-mutated relapsed/refractory (R/R) AML patients. In 2019, the pivotal ADMIRAL trial showed that the median overall survival was significantly longer in patients treated with gilteritinib than among those receiving chemotherapy (9.3 vs. 5.6 months, respectively) and the ORR to gilteritinib was 67.6%, outperforming the 25.8% for chemotherapy arm and leading to the license for its clinical use by the US Food and Drug Administration. Since then, several real-world experiences have confirmed the positive results in the R/R AML setting. Finally, gilteritinib-based combinations currently under investigation, with several compounds (venetoclax, azacitidine, conventional chemotherapy, etc.) and some practical tips (maintenance after allogeneic transplantation, interaction with antifungal drugs, extramedullary disease, and onset of resistance), will be analyzed in detail in this review.

FMS-Like Tyrosine Kinase 3 Inhibitors in the Treatment of Acute Myeloid Leukemia: An Update on the Emerging Evidence and Safety Profile

FMS-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in acute myeloid leukemia (AML). Approximately 30% of the adult cases harbor an internal tandem duplication (FLT3-ITD) and 5-10% a tyrosine kinase domain (TKD) amino acid substitution (FLT3-TKD). The treatment paradigm of AML patients harboring FLT3 mutations (30%) has been modified by the discovery of tyrosine kinase inhibitors. First- and second-generation inhibitors classify FLT3 inhibitors according to FLT3 specificity: first-generation FLT3 inhibitors include sorafenib and midostaurin and second-generation inhibitors are represented by quizartinib, gilteritinib and crenolanib, among others. Activity of these inhibitors depends on their mechanism of receptor binding (active vs inactive conformation) and efficacy against the FLT3-ITD and -TKD mutations (type 1 inhibitors are active both on FLT3-ITD and TKD, whereas type 2 inhibitors are active only on FLT3-ITD). The FLT3 inhibitors sorafenib, midostaurin, quizartinib and gilteritinib have been tested in monotherapy in several settings including refractory or relapsed AML (R/R AML), post-transplant maintenance as well as in combination with intensive chemotherapy (ICT) or non-intensity regimens. The results of published randomized studies support the use of sorafenib in a post-transplant setting (SORMAIN trial), midostaurin in combination with ICT based (RATIFY trial) and gilteritinib for R/R AML (ADMIRAL trial). Gilteritinib in combination with hypomethylating agent as well as quizartinib are not supported by solid randomized trial results for their use in FLT3-mutated AML patients.

FLT3 inhibitors as maintenance therapy post allogeneic hematopoietic stem cell transplantation in acute myeloid leukemia patients with FLT3 mutations: A meta-analysis.

Acute myeloid leukemia (AML) patients with a Fms-like tyrosine kinase 3 (FLT3) mutation have a high incidence of relapse despite allogeneic hematopoietic stem cell transplantation (allo-HSCT) and a subsequent poor prognosis. FLT3 inhibitors (FLT3i) have been suggested to reduce the post-transplant relapse risk in recent studies. As more evidence is accumulated, we perform the present meta-analysis to assess the efficacy and safety of FLT3i as post-transplant maintenance therapy in AML patients. Literature search was performed in public databases from inception to December 31, 2021. Overall survival (OS), relapse-free survival (RFS), cumulative incidence of relapse (CIR), non-relapse mortality (NRM), graft-versus-host disease (GVHD) and adverse events were compared between FLT3i and control groups. Pooled hazard ratio (HR) or relative risk (RR) with corresponding 95% confidence interval (CI) were calculated. We identified 12 eligible studies with 2282 FLT3-mutated AML patients who had received HSCT. There was no between-study heterogeneity and a fix-effect model was used. Post-transplant FLT3i maintenance significantly prolonged OS (HR=0.41, 95%CI: 0.32-0.52, p < 0.001) and RFS (HR=0.39, 95%CI 0.31-0.50, p < 0.001), and reduced CIR (HR=0.31, 95%CI 0.20-0.46, p < 0.001) as compared with control. There were no significant risk differences in NRM (RR=0.69, 95%CI 0.41-1.17, p=0.169), acute GVHD (RR=1.17, 95%CI 0.93-1.47, p=0.175), chronic GVHD (RR=1.31, 95%CI 0.91-1.39, p=0.276) and grade ≥3 adverse events between both groups, except for skin toxicity (RR=5.86, 95%CI 1.34-25.57, p=0.019). Post-transplant FLT3i maintenance can improve survival and reduce relapse in FLT3-mutated AML patients and is tolerable.

Gilteritinib Activity in Refractory or Relapsed FLT3-Mutated Acute Myeloid Leukemia Patients Previously Treated By Intensive Chemotherapy and Midostaurin: A Study from the French AML Intergroup ALFA/Filo

Background The recent phase 3 ADMIRAL trial has shown that outcomes of relapsed/refractory (R/R) FLT3-mutated acute myeloid leukemia (AML) patients were improved by the single-agent FLT3 tyrosine kinase inhibitor gilteritinib as compared with salvage chemotherapy. In this trial, overall survival (OS) was significantly improved in the gilteritinib arm and complete remission (CR/CRi), after excluding those obtained after allogenic hematopoietic stem cell transplantation (Allo-HSCT) were 26.3% in the gilteritinib group. Methods Here, the real-world efficacy and safety of gilteritinib was assessed in an ambispective study that included 167 R/R FLT3-mutated AML patients (Cohort A) from the French early access program ELEGANCE between March 1st, 2019, and March 1st, 2021. Gilteritinib was administered to 27 patients in association with other treatments, while 140 received it as single agent (Cohort B), including 67 previously treated by intensive chemotherapy and midostaurin (Cohort C). Results Characteristics of the three cohorts at gilteritinib initiation are described in Table 1. Briefly, in the whole cohort A, there were 141 (84.4%) cases of de novo AML, FLT3 mutation was ITD in 104 (62.3%) and TKD in 33 (19.8%), whereas 36 (21.6%) patients were FLT3 wild-type at diagnosis, acquired mutation later in disease evolution and were FLT3 mutated at gilteritinib initiation. Finally, 6 (3.6%) patients had both FLT3-ITD and TKD mutations. The median FLT3-ITD/wt ratio was 0.50 (IQR, 0.12-0.70 and range 0.01-1.90), and the median size of ITD was 48 bp (IQR, 30-75, range 3-408). Most patients (136, 81.4%) had an intermediate cytogenetic risk, and 84 (50.3%) had an NPM1 co-mutation. The main differences in patient characteristics in this study, compared to ADMIRAL, were ECOG ≥ 2 (83.6% vs 16.6%), FLT3-TKD mutations (21.0% vs 8.5%), primary induction failure (15.0% vs 40.0%) and previous lines of treatment (beyond 2nd in 37.1% vs 0.0%). The rates of response to gilteritinib are described in Table 1. Moreover, CRc (CR + CR without hematological recovery + CR with incomplete platelet recovery) to gilteritinib as single agent in subgroups defined as per AML phase at gilteritinib initiation were 4.5% (N=22) for patients refractory after front-line, 37.5% (N=32) for those relapsing less than 6 months from CR/CRi, 26.7% (N=30) when relapse occurred after 6 months and 16.1% (N=56) for patients refractory after 1st relapse and beyond. Finally, 32 (19.2%), 25 (17.9%) and 15 (22.4%) patients received an Allo-HSCT after gilteritinib, in CRc for 21 (65.6%), 17 (68.0%) and 11 (73.3%) patients in cohorts A, B and C, respectively. In terms of safety, in the subgroup of patients treated with gilteritinib as single agent (Cohort B), 39 (28.3%) experienced a serious adverse event (SAE) during gilteritinib treatment: infection in 22 (56.4%), hemorrhage in 1 (2.6%), cardiovascular in 4 (10.3%), hepatic in 4 (10.3%), differentiation syndrome in 2 (5.2%) and other SAE in 6 (15.4%). In terms of health care resource consumption, the median duration of hospitalization during the first 6 months of treatment was 9.0 days (IQR 0-29, range 0-117) with a median of 7 RBC units (IQR 0-14, range 0-54) and of 5 platelet units (IQR 0-13.5, range 0-56) transfusions. After a median follow-up of 14.5 months, median OS and 12-month OS were 6.4 months (IQR, 3.2-14.7) and 31.4% (95%CI 22.4-39.7) in the 140 patients who received gilteritinib as single agent, and 7.8 months (IQR, 3.2-20.8) and 38.3% (95%CI 26.0-50.4) in the subgroup of 67 patients previously treated by intensive chemotherapy and midostaurin (Figure 1). Multivariate analyses disclosed female gender (HR 1.61, 95%CI 1.07-2.42, p=0.02), adverse cytogenetic risk (HR 2.52, 95%CI 1.24-5.13, p=0.01) and allo-HSCT after gilteritinib (HR 0.13, 95%CI 0.05-0.37, p<0.0001) as factors significantly and independently associated with OS. Conclusion Although patients in this study were more heavily pretreated, these real-world data reproduce ADMIRAL results and provide new insights in the outcome of patients previously treated by intensive chemotherapy and midostaurin and beyond the 2nd line of treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular Characterization of Clinical Response in Relapsed/Refractory Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndrome Patients Treated with Single Agent Emavusertib

Introduction Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are myeloid malignancies that exhibit a dynamic mutational landscape as the disease progresses. Genetic mutations in the splicing factors SF3B1 and U2AF1 drive overexpression of a highly active long isoform of interleukin-1 receptor-associated kinase 4 (IRAK4), which is critical in triggering inflammation, oncogenesis, and survival of cancer cells. Splicing factor mutations are frequently reported in AML and MDS and have been associated with disease progression and poor prognosis in relapsed/refractory (R/R) diseases. Emavusertib is a potent oral inhibitor of IRAK4 as well as Fms-like tyrosine kinase 3 (FLT3). The safety, clinical activity, and potential biomarkers of emavusertib in AML or high-risk MDS (HR-MDS) are investigated in the ongoing open-label, phase 1/2a TakeAim Leukemia trial (NCT04278768). Methods Eligible patients with R/R AML or HR-MDS were enrolled and treated with emavusertib. The exploratory objectives of the study are to assess the association between clinical activity and target-related biomarkers, gene expression signatures, and molecular subtypes. Bone marrow or peripheral blood were collected at the baseline and on treatment. To characterize the mutational profiles of enrolled patients and further define the targeted population, targeted next generation sequencing (NGS) of 68 genes was performed on genomic DNA from bone marrow or peripheral blood mononuclear cells. A minimum coverage of 100x, variant allele frequency (VAF) of >1%, and a VARSCAN-assigned variant p<0.001 were used as thresholds for single nucleotide variants (SNV) calling and presumed somatic driver mutations were identified as previously reported (Metzeler et al. Blood 2016). Mutations from patients’ molecular pathology reports were also documented. Results As of December 16, 2021, 49 patients have been treated with emavusertib monotherapy at one of dose levels of 200, 300, 400, or 500 mg BID, including 30 R/R AML and 19 R/R HR-MDS. The median number of prior anti-cancer therapies was 2 (range 1-5). This abstract is intended to add additional molecular findings to the patient subset presented in 2022 ASCO, of which 6 AML and 7 HR-MDS had splicing factor SF3B1 or U2AF1 mutations and 3 AML had FLT3 mutations, including 2 AML with both splicing factor and FLT3 mutations (hereafter referred to as "targeted mutations"). In the patients with targeted mutations, with available mutational profile data or molecular reports from sites, there were 6 responders: 1 CR, 1 CRh, and 4 mCR. In this responder population, the most common co-mutation was ASXL1. During emavusertib treatment, FLT3-mutated clones became undetectable in 2 of 3 (67%) FLT3-mutated AML patients. One of the 3 patients (33%) with a FLT3 mutation achieved CR and the remaining 2 patients achieved SD (1 with > 90% blast reduction). Additional gene mutations, such as ASXL1, RUNX1, CEBPA, ETNK1, ETV6, and SRSF2, became undetectable during emavusertib treatment. New mutations that emerged after ≥ 1 cycle of emavusertib treatment include NRAS, SETBP1, RUNX1, WT1, STAG2, KRAS, CCND2, DNMT3A, ASXL1, and NOTCH1. Analysis of genomic and transcriptomic profiles will be updated. Conclusion Emavusertib demonstrated anti-cancer activity in heavily pretreated AML and HR-MDS patients, especially in those with U2AF1, SF3B1, or FLT3 mutations. Our preliminary NGS data is suggestive of molecular responses and disease-modifying activity of emavusertib. Patients with targeted mutations responded to emavusertib, even in the presence of co-mutations of ASXL1 that are typically associated with poor prognosis. We will continue to explore potential genomic biomarkers of emavusertib sensitivity and resistance

Gilteritinib Maintenance Therapy Post-Allogenic Stem-Cell Transplantation Improves the Prognosis of Patients with FLT3-Mutated AML

Introduction FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) and -tyrosine kinase domain (TKD) mutation were common mutations in acute myeloid leukemia (AML). FLT3-ITD mutation adversely affected the outcome of hematopoietic stem-cell transplantation (SCT). While sorafenib and midostaurin as maintenance therapy after SCT prolonged relapse-free and overall survival (OS), there are very few reports of post-transplant gilteritinib maintenance therapy outside of clinical trials. Hence, to explore the efficacy of the gilteritinib maintenance therapy post-SCT, we here report the prognostic impact of post-SCT gilteritinib maintenance in a clinical setting. Methods We retrospectively analyzed 25 FLT3-mutated relapsed or refractory AML patients who received allogeneic SCT at our center from January 1, 2011 to April 30, 2022. FLT3 mutation was analyzed by a commonly used polymerase chain reaction (PCR)-based assay. Measurable residual disease (MRD) was assessed by Wilms tumor 1 (WT1) mRNA expression in peripheral blood, and MRD positivity was defined as WT-1 >50 copies/μg in this study. Relapse was defined as an increase of 5% blasts or more. Non-relapse mortality (NRM) was defined as death without relapse. Leukemia-free survival (LFS) was defined as the time from transplantation to leukemia relapse or death from any cause, which comes first. OS was defined as the time from transplantation to death from any cause. Results The median age was 52 years. Twenty-two patients had ITD mutation and 5 had TKD mutation (2 had both ITD and TKD mutation). At the time of the transplant, 20 patients were in remission (6 were MRD-negative and 14 were MRD-positive) and five were in non-remission. All patients achieved hematologic complete remission (CR) after SCT and no patients relapsed within 30 days post-SCT. After hematological engraftment, 14 patients started to receive gilteritinib as maintenance therapy and 11 patients did not. Between the two groups, the patients' characteristics including disease status at transplant, SCT procedures, and donor sources were well balanced except for the administration of FLT3-inhibitor prior to SCT (100% in patients with gilteritinib maintenance and 45.5% in those without gilteritinib maintenance, p = 0.003). Eleven patients (78.6%) in the gilteritinib group and eight (72.7%) patients in the non-gilteritinib group had residual MRD in hematological CR or were non-CR prior to SCT. The median time from SCT to the initiation of gilteritinib was 36 days. The median dose at gilteritinib initiation was 40mg (range 20-120 mg). One patient developed grade 2 skin acute graft-versus-host disease (GVHD) after gilteritinib administration and 4/14 patients (28.6%) developed moderate-severe chronic GVHD (cGVHD, two developed pulmonary cGVHD, one eye and mouse, and one skin) with a median onset of 10.5 months (range 3.1-30.5 months), while only one patient temporary discontinued gilteritinib until the improvement of cGVHD. Late engraftment failure during gilteritinib maintenance was not observed. In terms of survival benefit, patients with gilteritinib maintenance showed significantly longer LFS and OS than those without gilteritinib (1-y LFS; 100% vs. 36.4%, 95% CI; 100-100% vs. 11.2-62.7%, p = 0.0028, Figure A, and 1-y OS; 100% vs. 45.5%, 95% CI; 100-100% vs. 16.7-70.7%, p = 0.0075, Figure B). The cumulative incidence of AML relapse at one-year in patients with gilteritinib maintenance group was significantly lower than those without (0% vs. 51.5%, 95% CI; 0-0% vs. 24.9-86.0%, p = 0.005). Especially, among patients with MRD positive or non-CR prior to SCT (n=19), patients who received gilteritinib maintenance showed a lower cumulative incidence of AML relapse at one-year than those without gilteritinib maintenance (0% vs. 68.8%, 95% CI; 0-0% vs. 35.9-95.2%, p = 0.0028, Figure C). Patients without MRD prior to SCT did not relapse with or without gilteritinib maintenance. These results suggest that gilteritinib maintenance therapy contributes to preventing post-transplant relapse in patients who remain MRD positive at the time of SCT. Conclusion Gilteritinib maintenance therapy post-SCT showed survival benefit in a clinical setting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Phase I/II Study of Combination of ASTX727, Gilteritinib and Venetoclax in Patients with Relapsed/Refractory FLT3 Mutated Acute Myeloid Leukemia (AML) and Frontline FLT3 Mutated AML Patients Unfit for Chemotherapy

Background:FLT3 gene mutations occur in approximately 30% of patients (pts) with AML and are associated with worse survival and higher relapse risk. Gilteritinib, a selective potent oral FLT3 tyrosine kinase inhibitor (FLT3i), improves survival in pts with relapsed FLT3 mutated AML. ASTX727 is an oral fixed-dose combination of decitabine (35 mg) and cedazuridine (a cytidine deaminase inhibitor; 100 mg) approved for treating pts with myelodysplastic syndrome (MDS). We are evaluating the safety and efficacy of the combination of ASTX727, gilteritinib, and venetoclax (VEN) in pts with relapsed or refractory FLT3-mutated AML and older/unfit pts unable to receive chemotherapy. Methods: In the phase I, pts with R/R FLT3-mutated AML or high-risk (HR) MDS or chronic myelomonocytic leukemia (CMML) are eligible. FLT3-ITD and/or FLT3-TKD mutations were allowed. In the phase II, the study will also enroll newly diagnosed pts with FLT3-mutated AML ineligible for induction therapy. Pts are required to have an ECOG performance status ≤3, total bilirubin ≤2.5 x upper limit of normal (ULN), ALT/AST ≤3 x ULN, and creatinine clearance ≥30 mL/min. Hydroxyurea or 1 dose of cytarabine up to 1000 mg was allowed in pts with proliferative disease to lower white blood count to ≤ 25 x 109/L before initiation of study therapy. In the phase 1, we evaluated two dose levels of gilteritinib (80 mg and 120 mg daily) continuously with fixed dosages of ASTX727 (35/100 mg daily on days 1-5) and VEN (400 mg daily, dose adjusted for concomitant azoles, for up to 28 days). Cycles are repeated every 28 days for up to 24 cycles, followed by indefinite therapy with gilteritinib as maintenance. During cycle 1, VEN was given at a dose of 100 mg on day 1, 200 mg on day 2, and 400 mg daily onwards. Bone marrow (BM) aspiration was performed on day 21, and VEN was held in pts with BM blasts <5% or aplasia. During cycles 2-24, VEN was given at 400 mg daily (dose adjusted for concomitant azoles) on days 1-21 but dose reductions depending on tolerance were permitted. Gilteritinib continued at the enrolled dose in cycles 2-24. Results: Between 11/2021 and 6/2022, 8 patients (7 AML and 1 HR-CMML) have been enrolled in the phase 1 portion of the trial (Table 1). Their median age was 57 years (range, 38-83). Median number of prior lines of therapy was 1 (range, 1-10). Four pts (50%) had a previous allogeneic hematopoietic stem cell transplant (HSCT), 6 pts (75%) had prior treatment with hypomethylating agent (HMA)+VEN, and 3 pts (27%) received prior FLT3i. None received prior gilteritinib. Cytogenetics was diploid in 4 pts, complex in 3 pts, and miscellaneous in 1 pt. The median FLT3-ITD allelic ratio (AR) was 0.4 (range, 0.11-10.60), with 50% having a high AR (≥0.5). All pts had a FLT3-ITD mutation and no pt had a FLT3-TKD mutation. NPM1 co-mutation was present in 4 pts (50%), followed by DNMT3A (37%) and IDH2 (37%). Six pts have been enrolled on dose level 1 (gilteritinib 80 mg) and 2 pt on dose level 2 (gilteritinib 120 mg). No DLTs have been observed in either dose level. Myelosuppression was frequent, requiring dose adjustment of ASTX727 and/or VEN in cycle 2 onward in 5 pts. Grade 3 non-hematologic adverse events included: sepsis (25%), bacteremia (25%), pneumonia (13%), and other infection (25%). No tumor lysis syndrome, differentiation syndrome, or QTc prolongation was observed. The 30-day and 60-day mortality rates were 0% and 13%, respectively. The overall response rate (ORR) was 87% with 4 (50%) pts achieving CR/CRi (1 CR, 3 CRi) and 3 (37%) pts achieving MLFS. One pt withdrew from the study in cycle 1 and was not evaluable for response. The ORR in pts with prior HMA+VEN was 83% (3 CRi, 2 MLFS). Five of 7 responders (71%) achieved the best response within 1 cycle and 2 (29%) within 2 cycles. Pts received a median of 2 cycles of therapy (range, 1-6). With a median follow-up of 6 months, median overall survival was not reached (range, 1.2-8+ months), and the estimated 6-month overall survival is 70%. One responding pt proceeded to HSCT. Two pts have died, 1 in CRi due to an unknown cause, and 1 due to disease progression and sepsis. Conclusion: The combination therapy with ASTX727, VEN, and gilteritinib appears to be effective and safe in R/R FLT3 mutated AML and HR-MDS. Myelosuppression was frequent but manageable. Continued accrual is anticipated to define the optimal triplet regimen in both R/R and newly diagnosed FLT3-mutated AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Gilteritinib activity in refractory or relapsed FLT3-mutated acute myeloid leukemia patients previously treated by intensive chemotherapy and midostaurin: a study from the French AML Intergroup ALFA/FILO.

The real-world efficacy and safety of gilteritinib was assessed in an ambispective study that included 167 R/R FLT3-mutated AML patients. Among them, 140 received gilteritinib as single agent (cohort B), including 67 previously treated by intensive chemotherapy and midostaurin (cohort C). The main differences in patient characteristics in this study compared to the ADMIRAL trial were ECOG ≥ 2 (83.6% vs. 16.6%), FLT3-TKD mutation (21.0% vs. 8.5%), primary induction failure (15.0% vs. 40.0%) and line of treatment (beyond 2nd in 37.1% vs. 0.0%). The rates of composite complete remission, excluding those that occurred after hematopoietic stem cell transplantation (HSCT), were similar at respectively 25.4% and 27.5% in cohorts B and C. Median overall survival (OS) for these two groups was also similar at respectively 6.4 and 7.8 months. Multivariate analyses for prognostic factors associated with OS identified female gender (HR 1.61), adverse cytogenetic risk (HR 2.52), and allogenic HSCT after gilteritinib (HR 0.13). Although these patients were more heavily pretreated, these real-world data reproduce the results of ADMIRAL and provide new insights into the course of patients previously treated by intensive chemotherapy and midostaurin and beyond the 2nd line of treatment who can benefit from treatment in an outpatient setting.

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.

Hypomethylating agent and venetoclax with FLT3 inhibitor \u201ctriplet\u201d therapy in older/unfit patients with FLT3 mutated AML

In older/unfit newly diagnosed patients with FLT3 mutated acute myeloid leukemia (AML), lower intensity chemotherapy (LIC) in combination with either a FLT3 inhibitor or with venetoclax results in poor overall survival (median 8 to 12.5 months). We performed a retrospective analysis of 87 newly diagnosed FLT3 mutated AML patients treated on triplet (LIC + FLT3 inhibitor + Venetoclax, [N = 27]) and doublet (LIC + FLT3 inhibitor, [N = 60]) regimens at our institution. Data were collected from prospective clinical trials in 75% (N = 65) and 25% (N = 22) who received the same treatment regimens outside of a clinical trial. Triplet therapy was associated with significantly higher rates of complete remission (CR) (67% versus 32%, P = 0.002), CR/CRi (93% versus 70%, P = 0.02), FLT3-PCR negativity (96% versus 54%, P < 0.01), and flow-cytometry negativity (83% versus 38%, P < 0.01) than doublets. At the end of the first cycle, the median time to ANC > 0.5 (40 versus 21 days, P = 0.15) and platelet > 50 K (29 versus 25 days, P = 0.6) among responders was numerically longer with triplets, but 60-day mortality was similar (7% v 10%). With a median follow-up of 24 months (median 12 months for triplet arm, and 63 months for doublet arm), patients receiving a triplet regimen had a longer median overall survival (not reached versus 9.5 months, P < 0.01). LIC combined with FLT3 inhibitor and venetoclax (triplet) may be an effective frontline regimen for older/unfit FLT3 mutated AML that should be further validated prospectively.

Practical tips for managing FLT3 mutated acute myeloid leukemia with midostaurin

ABSTRACT Introduction FLT3 inhibitors have been recently introduced as novel treatment targets in patients with FLT3-mutated acute myeloid leukemia (AML). Midostaurin is an oral multikinase inhibitor that targets multiple receptor tyrosine kinases including FLT3 and has been approved for the treatment of AML with FLT3 mutations in patients candidates for intensive chemotherapy. This article presents an updated overall overview of the use of midostaurin in clinical practice. Areas covered Tests and examinations to be performed before the use of midostaurin, antifungal and antimicrobial treatment, as well as antifungal and antimicrobial prophylaxis are discussed. Practical tips for the treatment of QTc interval prolongation and heart failure are also presented. Expert opinion Midostaurin is the first agent showing significant survival benefit when combined with chemotherapy in FLT3-mutated AML patients. Optimal use of midostaurin should be a priority, being essential to know the interactions with other drugs like strong CYP3A4 inhibitors or inducers, which are particularly used in the concomitant treatment of AML patients and may increase toxicity or decrease therapeutic benefit. The active role of hematologists and nursing teams is crucial to ensure patient adherence to midostaurin treatment and to minimize adverse effects by administrating the optimal dose for each situation.

Phase II Study of Cladribine, Idarubicin, Cytarabine (CLIA) Plus Gilteritinib in Patients with FLT3 Mutated Acute Myeloid Leukemia (AML)

Phase II Study of Cladribine, Idarubicin, Cytarabine (CLIA) Plus Gilteritinib in Patients with FLT3 Mutated Acute Myeloid Leukemia (AML)

AML-468: Outcomes of FLT3-Mutated Acute Myeloid Leukemia Patients: A Single-Center, Real-World Experience

Background:Internal tandem duplications (ITDs) within the FMS-like tyrosine kinase 3 (FLT3) gene (FLT3-ITD) is a common driver mutation and confers a poor prognosis in patients (pts) with acute myeloid leukemia (AML) due to proliferative presentation, short remission durations and early relapse. The addition of midostaurin to induction therapy has become standard therapy in newly diagnosedFLT3-mutated (FLT3-mu) AML. Next-generation FLT3 inhibitors (FLT3i) as gilteritinib and quizartinib are more specific, more potent, and may have fewer toxicities associated with off-target therapies but are only available in clinical trials or for salvage therapy. Real-world experience in smaller academic institutions is scanty. Methods:We reviewedFLT3-ITD mu AML pts at our institution from October 2016-December 2019. FLT3i were given as a single agent or in combination with cytotoxic chemotherapy (CCT) or low intensity therapy (LIT) (hypomethylating agents). AllFLT3-ITD DNA was subjected to PCR amplification using primers targeting the juxtamembrane domain of FLT3, and products were analyzed by capillary electrophoresis. We analyzed the characteristics of these pts, responses to therapy, and outcomes. Results:Sixteen pts with newly diagnosedFLT3-ITD mu AML were identified. The median age was 47 years [range, 17-76 years] and the median follow-up was 9.3 months [range, 1.2-29.5 months]. The median time from AML diagnoses to FLT3 detection was 4 days [range, 3-5 days]. Patient characteristics are shown in Table 1. The most common co-occurring mutations wereNPM1(44%) andNRAS(31%); 2 pts (12%) had both mutations. Five pts (31%) received CCT and 11 pts (69%) received CCT+FLT3i. The most frequently used FLT3i was midostaurin 25 mg twice daily on days 8 through 21 (14 days) (10 pts, 63% of all pts); 1 pt received sorafenib (6% of all pts). The complete remission rate (CR) and the overall response rate (ORR) (CR+CR with incomplete hematologic recovery [CRi]) were 31% and 88%, respectively. The two pts who did not achieve a remission were older than 65 years. Among the pts who received CCT+FLT3i, CR and OOR were 50% and 100%, respectively. The median number of cycles to remission was 1 [range, 1-2 cycles], and the median number of consolidation cycles was 0 [range, 0-3 cycles]. Overall, the median duration of response in frontlineFLT3-ITD pts who did not receive allogeneic hematopoietic stem cell transplantation (Allo-HSCT) was 5 months [range, 1-9 months] (all of them received CCT+FLT3i). There was no statistically significant difference in response in pts with NPM1 mutation compared with pts with NRAS mutation (P=0.7), or with no additional mutations. Seven pts (44%) received an Allo-HSCT (6 after CCT+FT3i, and 1 after CCT alone): 2 (28%) from matched unrelated donor, 1 (14%) from a haploidentical transplant, 1 (14%) from dual cord transplant, and the remaining (44%) from matched related donors. Two patients relapsed after Allo-HSCT (28%). Both of these patients had received FLT3i before Allo-HSCT; one of the two relapsed pts received ivosidenib and azacitidine (IDH1 mutation) after Allo-HSCT, achieved remission then received the 2nd Allo-HSCT with subsequent remission. Two pts (29%) received maintenance therapy after Allo-HSCT, one with midostaurin, and the other with azacitidine + gilteritinib; both pts are still on remission (24 and 6 months after CR, respectively). All 7FLT3-ITD pts who did not receive Allo-HSCT relapsed. Gilteritinib was used as a single agent (n=3) or in combination with LIT (n=1) in these pts after relapse. CR rate was 25% and CRI 50%; one pt relapsed (33%). All pts who responded are still receiving gilteritinib. Conclusion:Combination of FLT3i based therapy with CCT is effective therapy inFLT3-ITD frontline pts in real-world setting. Duration of response is short in the absence of Allo-HSCT. Download : Download high-res image (299KB) Download : Download full-size image Disclosures Cortes:Arog:Research Funding;Pfizer:Consultancy, Research Funding;Amphivena Therapeutics:Research Funding;Daiichi Sankyo:Consultancy, Research Funding;Astellas:Research Funding;Telios:Research Funding;Novartis:Consultancy, Research Funding;BioPath Holdings:Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding;Merus:Research Funding;Bristol-Myers Squibb:Research Funding;Takeda:Consultancy, Research Funding;Sun Pharma:Research Funding;BiolineRx:Consultancy, Research Funding;Immunogen:Research Funding;Jazz Pharmaceuticals:Consultancy, Research Funding.

Poster: AML-468: Outcomes of FLT3-Mutated Acute Myeloid Leukemia Patients: A Single-Center, Real-World Experience

Poster: AML-468: Outcomes of FLT3-Mutated Acute Myeloid Leukemia Patients: A Single-Center, Real-World Experience

Histone Modification Patterns Using RPPA-Based Profiling Predict Outcome in Acute Myeloid Leukemia Patients

Background: Prognostic factors based on cytogenetic and molecular abnormalities combined with clinical features like age, performance status, or history of an antecedent hematological disorder, are currently used to predict clinical outcome in acute myeloid leukemia (AML). (Döhner H. et al. 2010) Although, sequencing of AML has identified many driver mutations in genes associated with epigenetic regulation, e.g. post-translational histone tail modifications. Altered activity of post-translational histone tail modifications has been shown to contribute to AML leukemogenesis via regulation of gene transcription. (Fong C.Y. et al. 2014) The functions and effects of modulating histone-modifying proteins (HMP) are already typically studied by altering an individual protein of interest. However in AML, it is the net consequence of the combined influences of all the HMP, rather than a single protein, that determines the net effect on the cell. A global analysis of the level and patterns of expression of multiple HMP and the effect of different patterns of expression on outcome and prognosis has not been investigated in AML patients.Methods: We analyzed 20 HMP by reverse phase protein array (RPPA) in a cohort of 205 newly diagnosed AML patients evaluated at the University of Texas M.D. Anderson Cancer Center. (Kornblau S.M. et al. 2009) Protein levels were correlated with patient and disease characteristics, including survival and mutational state.Results: Different protein clusters were identified and characterized by higher (more on) or lower (more off) expression of HMP, relative to normal CD34+ cells (figure 1A). We found that the more on state of HMP was associated with poorer overall survival (P= 0.007, figure 1B) and more chemo resistance (P < 0.001) compared to normal -like and a more off state. Furthermore, FLT3 mutated AML patients were significantly overrepresented in the more on state (P= 0.025). Identification of the markedly upregulated proteins: KDM1A, hnRNPK, NCL, SIRT1, ASH2L, WTAP in all, and BRD4 and NPM1 in some on state patients suggest targets to develop drugs against that may be the most efficacious when used individually or in combination.Conclusions: These findings showed for the first time that multiple HMP form recurrent patterns of expression and that these significantly correlate with prognosis, chemo resistance, and FLT3 mutational state in newly diagnosed AML patients.F igure legend 1:(A) RPPA-based heatmap containing 20 histone modification related proteins showing only 205 fresh AML patient samples. Clusters were determined by a progeny clustering based algorithm coupled with k-means, which computationally calculated the optimal number of five clusters. The five cluster colors along the top bar delineate the five fresh clusters. Cluster 1 (red) was defined as a normal -like pattern by principal component analysis, whereas clusters 2, 3, 6 and 7 were only seen with leukemic samples. Expression is normalized for each protein to range from the lowest (blue) to the highest (red). Cluster 2 and 7 were defined as the more on clusters and cluster 3 and 6 as the more off clusters.(B) Overall Survival of patients from the normal -like histone-modifying proteins (HMP) cluster (n=61), more on HMP clusters (n=131) and the more off HMP clusters (n=13) as determined in our cohort of newly diagnosed AML fresh patient samples by RPPA-based profiling. The survival of patients with the more on state of HMP was significantly reduced compared to the more off state and the normal- like HMP signatures. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The combination of CUDC-907 and gilteritinib shows promising in vitro and in vivo antileukemic activity against FLT3-ITD AML

About 25% of patients with acute myeloid leukemia (AML) harbor FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations and their prognosis remains poor. Gilteritinib is a FLT3 inhibitor approved by the US FDA for use in adult FLT3-mutated relapsed or refractory AML patients. Monotherapy, while efficacious, shows short-lived responses, highlighting the need for combination therapies. Here we show that gilteritinib and CUDC-907, a dual inhibitor of PI3K and histone deacetylases, synergistically induce apoptosis in FLT3-ITD AML cell lines and primary patient samples and have striking in vivo efficacy. Upregulation of FLT3 and activation of ERK are mechanisms of resistance to gilteritinib, while activation of JAK2/STAT5 is a mechanism of resistance to CUDC-907. Gilteritinib and CUDC-907 reciprocally overcome these mechanisms of resistance. In addition, the combined treatment results in cooperative downregulation of cellular metabolites and persisting antileukemic effects. CUDC-907 plus gilteritinib shows synergistic antileukemic activity against FLT3-ITD AML in vitro and in vivo, demonstrating strong translational therapeutic potential.

FLT3 Tyrosine Kinase Inhibitors for the Treatment of Fit and Unfit Patients with FLT3-Mutated AML: A Systematic Review.

FLT3-mutated acute myeloid leukemia accounts for around 30% of acute myeloid leukemia (AML). The mutation carried a poor prognosis until the rise of tyrosine kinase inhibitors (TKIs). New potent and specific inhibitors have successfully altered the course of the disease, increasing the complete response rate and the survival of patients with FLT3-mutated AML. The aim of this article is to review all the current knowledge on these game-changing drugs as well as the unsolved issues raised by their use for fit and unfit FLT3-mutated AML patients. To this end, we analyzed the results of phase I, II, III clinical trials evaluating FLT3-TKI both in the first-line, relapse monotherapy or in combination referenced in the PubMed, the American Society of Hematology, the European Hematology Association, and the Clinicaltrials.gov databases, as well as basic science reports on TKI resistance from the same databases. The review follows a chronological presentation of the different trials that allowed the development of first- and second-generation TKI and ends with a review of the current lines of evidence on leukemic blasts resistance mechanisms that allow them to escape TKI.

Multiplexed single\u2010cell mass cytometry reveals distinct inhibitory effects on intracellular phosphoproteins by midostaurin in combination with chemotherapy in AML cells

BackgroundFms-related tyrosine kinase 3 (FLT3) receptor serves as a prognostic marker and therapeutic target in acute myeloid leukemia (AML). Approximately one-third of AML patients carry mutation in FLT3, associated with unfavourable prognosis and high relapse rate. The multitargeted kinase inhibitor midostaurin (PKC412) in combination with standard chemotherapy (daunorubicin and cytarabine) was recently shown to increase overall survival of AML patients. For that reason, PKC412 has been approved for treatment of AML patients with FLT3-mutation. PKC412 synergizes with standard chemotherapy, but the mechanism involved is not fully understood and the risk of relapse is still highly problematic.MethodsBy utilizing the unique nature of mass cytometry for single cell multiparameter analysis, we have explored the proteomic effect and intracellular signaling response in individual leukemic cells with internal tandem duplication of FLT3 (FLT3-ITD) after midostaurin treatment in combination with daunorubicin or cytarabine.ResultsWe have identified a synergistic inhibition of intracellular signaling proteins after PKC412 treatment in combination with daunorubicin. In contrast, cytarabine antagonized phosphorylation inhibition of PKC412. Moreover, we found elevated levels of FLT3 surface expression after cytarabine treatment. Interestingly, the surface localization of FLT3 receptor increased in vivo on the blast cell population of two AML patients during day 3 of induction therapy (daunorubicin; once/day from day 1–3 and cytarabine; twice/day from day 1–7). We found FLT3 receptor expression to correlate with intracellular cytarabine (AraC) response. AML cell line cultured with AraC with or without PKC412 had an antagonizing phosphorylation inhibition of pAKT (p = 0.042 and 0.0261, respectively) and pERK1/2 (0.0134 and 0.0096, respectively) in FLT3high compared to FLT3low expressing cell populations.ConclusionsOur study provides insights into how conventional chemotherapy affects protein phosphorylation of vital signaling proteins in human leukemia cells. The results presented here support further investigation of novel strategies to treat FLT3-mutated AML patients with PKC412 in combination with chemotherapy agents and the potential development of novel treatment strategies.