European LeukemiaNet Guidelines Research Articles

AML-765 Refinement of Myelodysplasia-Related Gene Mutations Listed in the 2022 European LeukemiaNet (ELN-2022) Guidelines Enhances Prognostic Stratification in Acute Myeloid Leukemia

AML-765 Refinement of Myelodysplasia-Related Gene Mutations Listed in the 2022 European LeukemiaNet (ELN-2022) Guidelines Enhances Prognostic Stratification in Acute Myeloid Leukemia

Refinement of Myelodysplasia-Related Gene Mutations Listed in the 2022 European LeukemiaNet (ELN-2022) Guidelines Enhances Prognostic Stratification in Acute Myeloid Leukemia

Refinement of Myelodysplasia-Related Gene Mutations Listed in the 2022 European LeukemiaNet (ELN-2022) Guidelines Enhances Prognostic Stratification in Acute Myeloid Leukemia

Identification of Novel Potential Predisposing Variants in Familial Acute Myeloid Leukemia.

Myeloid neoplasms, including acute myeloid leukemia, have been traditionally among the less investigated cancer types concerning germline predisposition. Indeed, myeloid neoplasms with germline predisposition are challenging to identify because often display similar clinical and morphological characteristics of sporadic cases and have similar age at diagnosis. However, a misidentifications of familiarity in myeloid neoplasms have a critical impact on clinical management both for the carriers and their relatives. We conducted a family segregation study, in order to identify novel cancer predisposing genes in myeloid neoplasms and classify novel identified variants. We performed a thorough genomic analysis using a large custom gene panel (256 genes), the Myelo-Panel, targeted on cancer predisposing genes. In particular, we assessed both germline and somatic variants in four families, each with two siblings, who developed hematological neoplasms: seven acute myeloid leukemia and one Philadelphia-positive chronic myeloid leukemia. In each family, we identified at least one novel potentially predisposing variant, affecting also genes not included in the current European LeukemiaNet guidelines for AML management. Moreover, we suggest reclassification of two germline variants as pathogenic: likely pathogenic p.S21Tfs*139 in CEPBA and VUS p.K392Afs*66 in DDX41. We believe that predisposition to hematological neoplasms is still underestimated and particularly difficult to diagnosed. Considering that misidentification of familiarity in myeloid neoplasms have a critical impact on the clinical management both for the carriers and their relatives, our study highlights the importance of revision, in this clinical context, of clinical practices that should include thorough reconstruction of family history and in-depth genetic testing.

Real-world outcomes of intensive induction approaches in core binding factor acute myeloid leukemia.

Core-binding factor acute myeloid leukemia (CBF-AML) is characterized by the presence of inv(16)/t(16;16) or t(8;21) and is classified as a favorable risk by the 2022 European LeukemiaNet (ELN) guidelines. The CD33-targeting antibody-drug conjugate, gemtuzumab ozogamicin (GO), is commonly added to intensive chemotherapy (IC) in CBF-AML. We sought to compare outcomes in patients treated with IC with or without GO in CBF-AML. We included 200 patients with CBF-AML treated with IC across seven academic centers. Induction treatment regimens were categorized as IC alone, IC with GO, or IC with KIT inhibitor (dasatinib or midostaurin). Median follow-up for the whole cohort was 2.5 years. Three-year overall survival (OS) was 70% and 3-year event-free survival (EFS) was 51%. Patients treated with IC with GO experienced a 3-year EFS of 50% compared to those treated with IC alone who experienced a 3-year EFS of 47%, with no statistically significant difference (p=0.62). Similarly, those treated with IC with GO did not experience an improved OS compared to those treated with IC alone (p=0.67). Patients treated with IC with KIT inhibitor experienced a significantly improved 3-year EFS of 85% compared to those with IC with or without GO (p=0.04). We find in our study that there is no survival benefit in patients treated with IC with the addition of GO; improved EFS was seen in patients with CBF-AML treated with IC plus KIT inhibitors, consistent with outcomes noted in prospective studies utilizing this approach.

Long-term efficacy of high-dose imatinib in Hispanic patients without access to second-generation tyrosine kinase inhibitors treated in LATAM centers.

6512 Background: Treatment for patients with chronic myeloid leukemia (CML) had a benchmark with the introduction of imatinib, a tyrosine kinase inhibitor (TKI). Nevertheless, resistance due to genetic mutations in the BCR::ABL1 kinase receptor remains relatively common for many patients, leading to failure, lack of response, or suboptimal response. Ideally, to bypass genetic resistance, guidelines recommend switching to a second-generation TKI, but for many developing countries, socioeconomic barriers hinder the possibility of switching medication. Despite this scenario, scarce information is available to evaluate the clinical prognosis of these patients. Methods: We conducted a retrospective cohort analysis to compare the overall mortality of patients with CML who developed a suboptimal response to standard-dose imatinib and were treated with either high-dose imatinib or a second-generation TKI. We created a marginal structural model with inverse probability weighting and stabilized weights and depicted the survival curves and median using the Kaplan-Meier estimator. Our primary outcome was overall survival (OS) at 150 months, defined as the number of deaths once patients had reached treatment response based on parameters of the European Leukemia Net Guidelines for CML. Our secondary outcomes were disease-free survival (DFS) at 150 months, defined as the number of months after patients achieved either major molecular response or deep molecular response, and adverse events that included gastrointestinal, dermatologic, hematologic, and others. Results: The cohort included 148 patients, of which 32 received high-dose imatinib and 116 a second-generation TKI. While we found no difference in the 150-month risk in both OS and DFS (Table), patients receiving second-generation TKI had an increase in median survival of OS ( p-value = 0.009). No difference was found in the median survival of DFS ( p-value = 0.55). No difference was found in either hematologic, gastrointestinal, dermatologic, or other adverse events (p-values of 0.39, 0.94, 0.24, and 0.33, respectively). Conclusions: Ideally, patients who develop a suboptimal response to imatinib should be switched to a second-generation TKI. However, If impossible, our findings suggest that patients treated with high-dose imatinib have a similar OS and DFS prognosis to those receiving a second-generation TKI. [Table: see text]

Cytogenetic and epidemiological profile of chronic myeloid leukemia in Morocco.

Chronic myeloid leukemia (CML) is a neoplastic disease of genetic origin resulting from clonal proliferation of hematopoietic stem cells (HSCs). The reciprocal translocation t(9;22)(q34;q11) is the main chromosomal abnormality involved in this pathology, usually detected by conventional cytogenetics. This article aims to investigate the epidemiological, cytogenetic, therapeutic, and clinical characteristics of Moroccan patients with CML. This research represents the first large-scale study of CML patients in Morocco and was carried out at Institut Pasteur of Morocco. Bone marrow samples were processed for cytogenetic analysis, and karyotypes were described according to an international system of human cytogenetic nomenclature (ISCN 2016). Patients were studied according to their epidemiological characteristics, clinical information and cytogenetic results. For statistical calculations, R version 4.3.1 was used to analyze the data and calculate the statistical parameters. RStudio and Power BI were used for data visualization. The National Cancer Institute (NCI) Surveillance, Epidemiology, and End Results (SEER) method of incidence estimation was used to calculate our incidence. We received 826 patients (from 1992 to 2023) who were referred for suspected CML or who were undergoing treatment. Only 650 patients with confirmed CML were included in the study, all of whom underwent their first cytogenetic test. The median age of our patients was 45 years and the sex ratio was 1.03. At the time of diagnosis, 147 (30%) of the patients had clinical manifestations. Most patients were diagnosed in the chronic phase (94.5%). Nineteen complex variant translocations of the Philadelphia (Ph) chromosome were detected. At the time of diagnosis, 55 (11.5%) patients had ACAs, of which 30 (54.5%) were high-risk ACAs. Based on data from 174 patients treated with imatinib, the median time to complete cytogenetic response (CCyR) was 11 months, and at the last cytogenetic follow-up, 81 patients (46.6%) achieved CCyR, while 64 patients (36.8%) showed no response to treatment. Regarding adherence to European LeukemiaNet (ELN) guidelines, 58 patients (33%) were followed according to these guidelines, with optimal treatment in 8.6%, suboptimal treatment in 7% and treatment failure in 18%. The estimated incidence of chronic myeloid leukemia calculated is 0.6 cases per 100,000 in the Casablanca region. This study provides a detailed overview of CML in Morocco, highlighting important clinical, cytogenetic and therapeutic aspects despite some limitations. It also highlights the need to deepen our understanding of this complex disease for disease management in our specific context.

The Depth of the Molecular Response in Patients with Chronic Myeloid Leukemia Correlates with Changes in Humoral Immunity.

Background and Objectives: The effective treatment of chronic myeloid leukemia leads to the restoration of proper immune system function. We aimed to investigate fluctuations in circulating cytokines, angiogenic factors and complement components in patients with CML during the first year of treatment with TKI and correlate them with the degree of achieved molecular response. Material and Methods: We recruited 31 patients with newly diagnosed CML. Peripheral blood and bone marrow samples were obtained, and concentrations of serum proteins were measured using an immunology multiplex assay. Results: The study cohort was divided into two groups of optimal or non-optimal in accordance with the European Leukemia Net (ELN) guidelines. We found significantly higher concentrations of C1q, C4 and C5a in serum after 3 months of TKI treatment in patients who achieved optimal responses in the 6 months after diagnosis. The most alterations were observed during 12 months of therapy. Patients in the optimal response group were characterized by higher serum concentrations of TGF-β, EGF, VEGF, Angiopoietin 1, IFN-γ and IL-8. Conclusions: The later plasma concentrations of complement components were significantly increased in patients with optimal responses. The changes after 12 months of treatment were particularly significant. Similar changes in bone marrow samples were observed.

Appropriate use of the ELN guidelines based on results of real-world genetic analysis in Japanese patients with AML

Researchers in the field of acute myeloid leukemia have long sought to establish a prognostic stratification system for clinical use that combines multiple genetic mutations. In 2022, the European LeukemiaNet (ELN) proposed a new prognostic model incorporating new genetic mutations. However, Japanese National Health insurance only recently began covering clinical genetic analysis for AML. We established the Multi-center Collaborative Program for Gene Sequencing of Japanese AML (GS-JAML) to contribute to clinical practice by providing rapid genetic analysis results. Retrospective analysis of this research program revealed (1) the clinical significance of CEBPA-bZIP mutations, and (2) the clinical significance of DNMT3A mutations in NPM1 mutated AML.

Clinical characteristics and prognostic factors analysis of core binding factor acute myeloid leukemia in real world.

Chromosomal translocations involving core binding factor (CBF) genes account for 15% of adult acute myeloid leukemia (AML) cases in China. Despite being classified as favorable-risk by European Leukemia Net (ELN), CBF-AML patients have a 40% relapse rate. This study aims to analyze clinical characteristics and prognosis of CBF-AML, compare its subtypes (inv(16) and t(8;21)), and validate prognostic factors. Retrospective analysis of 149 AML patients (75 CBF-AML, 74 non-CBF) at Peking University First Hospital (March 2012-March 2022). CBF-AML patients have significantly lower disease-free survival (DFS) (p = 0.005) and higher non-relapse mortality (NRM) (p = 0.028) compared to non-CBF AML. inv (16) and t(8;21) show distinct co-occurring gene mutation patterns, with inv(16) being prone to central nervous system (CNS) leukemia. Multivariate analysis identifies age as a risk factor for overall survival (OS) and disease free survival (DFS), kinase mutation as a risk factor for DFS and Recurrence, while WT1 mutation as a risk factor for OS and non relapse mortality (NRM) risk in t(8;21) AML. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) improves prognosis in low-risk t(8;21). Prognosis of CBF-AML is poorer than ELN guidelines suggest. inv(16) and (8;21) are separate entities with relatively poor prognoses, requiring rational risk stratification strategies. Allo-HSCT may benefit low-risk t(8;21), but further research is needed for conclusive evidence.

Utility of Flowcytometry in the Diagnosis of Myelodysplasia in Routine Laboratories

INTRODUCTION Myelodysplastic syndromes (MDSs) cover a wide range of abnormalities, ranging from subtle morphological changes to overt findings such as prominently skewed morphology. The clinical picture is dominated by cytopenia of one or more lineages, defined by Haemoglobin<100g/L, absolute neutrophil count <1.8 x 10^9/L and platelets less than 100 x 10^9/L. The aberrant features, suggestive of MDS that can be picked up by flow cytometry can occasionally be missed on morphological features. In the presence of clonal haematopoiesis, these features detected by flow cytometry may help us to confidentially apply a label of MDS to an individual. MATERIALS AND METHODS All patients with suspected myelodysplastic syndrome and patients with acute myeloid leukemia (AML) <30% blasts. Profound dominance of myeloid blasts in a patient with AML automatically earns the patient the 2 points needed for the MDS label and the myeloblast dominance inevitably outnumbers B-cell progenitors, which will affect the Ogata scoring.Exclusion criteria: >2% monoclonal lymphocytes by flow cytometry<400 CD34+ events in the collected sample Parameters included: Ogata A simple 4-point scoring system by Ogata. Other features included as per European Leukemia Net guidelines on flow cytometry on MDS We included following nine parameters to be analysed in addition to the Ogata criteria: Myeloid progenitors: Increased expression of CD11bAbnormal expression of CD7 in progenitor cells (CD34/CD117 Positive cells) Monocytic Lineage: Decreased expression of CD11bDecreased expression of HLA-DRDecreased expression of CD14 Myeloid Lineage: Diminished expression of CD45Decreased expression of CD11bDecreased expression of CD13 or CD33 (Any one of them)Decreased expression of CD10 RESULTS In our analysis of normal patients, Ogata score was frequently positive in the category lymphocyte/ myeloblast CD45 ratio. This obviously may affect the sensitivity. In the series of 20 patients we analysed, we had only one patient with combined score of four or less (patient no: 12). Two patients with Ogata score of less than two fulfilled the flow cytometric criteria using the combined criteria (Patient no: 3 and 5). Since our numbers were low, we do not know whether the combined scoring system would gain more significance in a large cohort of patients. In one of the normal patients who presented with cytopenia with high MCV (patient no: 10) the morphological and flow cytometry features were not consistent with myelodysplasia. Moreover, the patient did not have clonal abnormalities. This patient could have classified as myelodysplasia by flow cytomeric criteria if Ogata score alone had been used. The sensitivity of the combined parameters in detecting myelodysplasia is 89.4% and specificity is 90.9%. CONCLUSION We devised this system based on the principles that, the Ogata scoring system is simple and reproducible. Moreover, its sensitivity can be improved by using nine additional markers based on WELLS criteria, which can be analysed based on mean fluorescence intensity (MFI) alone without much complexity. Our methodology was not demanding and it did not involve usage of special fluorescent dyes and hence can be reproduced in any routine diagnostic laboratories. In addition, we found that both sensitivity and specificity of Ogata scoring system has been improved by inclusion of the other parameters in this study. Our scoring system did not show any correlation with reference to the R-IPSS (Revised International Scoring System in myelodysplasia) at diagnosis. Many attempts have been made so far with extensive markers and application of Automated Intelligence strategies for the analysis of more complex combinations of parameters. However, a simplified, user friendly and cost effective method is the need of the hour.

Real-World Validation of the European Leukemia Network 2022 Risk Stratification in Acute Myelogenous Leukemia

Background: The 2017 European LeukemiaNet (ELN 2017) guidelines have become foundational for the risk stratification, prognostication, and treatment of acute myelogenous leukemia (AML). The updated 2022 European LeukemiaNet (ELN 2022) guidelines include additional cytogenetic abnormalities and genetic mutations for risk group assignment. It remains unknown how these updated guidelines compare for risk stratification and prognosis. Methods: We studied (N=287) adults with newly diagnosed AML from MedStar Georgetown University, MedStar Washington Hospital Center, and Baylor University Medical Center from 2012 to June 2023. Patients were stratified as favorable (fav), intermediate (int), or adverse (adv) risk according to ELN 2017 and ELN 2022 criteria. Overall survival (OS) was compared by age, race, and sex. Statistical analysis performed with SAS. Results: Median age was 65+16 years with a predominance of males (N=168, 59%). African-Americans (AA) composed 15% (N=44) of the cohort. Myelodysplastic-related mutations were present in 33 patients (15%), whereas the prevalence of epigenetic mutations was 25% (N=55). By ELN 2017 criteria, 19 (7%) patients were characterized as fav, 64 (23%) as int, and 198 (70%) as adv risk. Using ELN 2022 criteria, 18 (6%) patients were categorized as fav, 52 (19%) as int, and 211 (75%) as adv risk. Kaplan-Meier (KM) curves revealed a median overall survival of 200 days (d), 400 d, and 1532 d in the adv, int, and fav risk groups, respectively (log-rank p<0.001) by ELN 2017 criteria. Median OS was 379 d, 527 d, and 1552 d for the adv, int, and fav groups, respectively (log-rank p<0.001) in the ELN 2022 cohort. Stratifying by age <60 years, median OS was 390 d in the adv group and 832 d in the int group (log-rank p=.021)by ELN 2017 criteria,as compared to 350 d in the adv group and 757 d in the int group (log-rank p=.0012) by ELN 2022 criteria.Among those >60 years of age, median OS was 244 d and 349 d in the adv and int groups, respectively by ELN 2017 criteria (Anova p<0.25). In the ELN 2022 cohort, median OS among those >60 years of age was 284 d for the adv group and 209 days in the int group (Anova p<0.62). Among females, median OS in the adv group was 224 d and 342 d in the int group by ELN 2017 criteria (Anova p=0.4). In the ELN 2022 cohort, median OS among females was 214 d in the adv group and 421 d in the int group (Anova p=.13). Median OS among male patients in the ELN 2017 cohort was 324 d for those of adv risk and 623 d for those of int risk (Anova p<0.004). In the ELN 2022 cohort, median OS in males was 352 d and 563 d in the adv and int groups, respectively (Anova p<0.07) . In the ELN 2017 cohort, median OS was 259 d in AA patients as compared to 719 d in non-AA patients; this was not statistically significant (Anova p=0.16). Comparatively, in the ELN 2022 cohort, median OS for the AA patients was 153 d as compared to 212 d for the non-AA patient population (Anova p=0.86) .Conclusions: In this real-world dataset, the updated ELN 2022 guidelines reproduce differential survival for proposed favorable, intermediate, and adverse risk categories. When considering time to endpoint survival [Kaplan Meier curves] in our study, ELN 2022 seems to lack the ability to clearly discriminate among the adverse and intermediate risk subgroups. It is possible that patients seen in academic institutions not participating in FLT3 directed therapy trials retained inferior survival whereas similar patients fared better through early enrollment in trials using molecular targeted therapy. Finally, neither ELN 2017 nor ELN 2022 discriminate survival in AA populations for either adverse or intermediate subgroups. Efforts should be directed to develop novel risk stratification schemas to better encapsulate this vulnerable population.

Identification of Multivariable microRNA and Clinical Biomarkers Panels to Predict Imatinib Response in Chronic Myeloid Leukemia at Diagnosis

Imatinib Mesylate (IM) was once hailed as the magic bullet for chronic myeloid leukemia (CML) and remains a front-line therapy for CML to this day alongside other tyrosine kinase inhibitors (TKIs). However, TKI treatments are rarely curative and while they demonstrate efficacy in managing the disease, patients are often required to receive life-long treatment or risk experiencing relapse. Patients also frequently harbor innate TKI-resistance mechanisms to front-line IM therapy and without prior screening, patients can spend precious months on non-optimal treatment before transitioning to other therapeutic strategies. Thus, there is a growing interest in studying biomarkers in patients which can predict TKI response upon diagnosis. CML has unique microRNA (miRNA) expression profiles at different stages of the disease and in response to TKI-treatment. We previously generated global transcriptome profiles on treatment-naïve CD34 + CML cells with known subsequent imatinib (IM) responses and identified several differentially expressed miRNAs in IM-nonresponders as compared to IM-responders. We also reported that IM response could be predicted in treatment-naïve CD34 + cells by an in vitro colony forming cell (CFC) assay. Additionally, we previously analyzed patient data from the ENESTxtnd clinical trial to show that miRNA expression combined with CFC output data could accurately predict Nilotinib response in patients. In this study, we used a Cox Proportional Hazard (CoxPH) analysis to identify 22 out of a pool of 35 pre-validated miRNAs which were associated with IM nonresponse in CD34 + CML cells of 80 patients at diagnosis. These patients were later classified as IM-responders or IM-nonresponders based on the latest European LeukemiaNet (ELN) guidelines. Interestingly, a Welch t-test revealed 16 of these 22 IM-response associated miRNAs to be differentially expressed between IM-responders and IM-nonresponders. Between IM-responders and IM-nonresponders we also found 7 clinical parameters that were associated with IM-response of which 3 matched parameters had significantly different values including CFC assay outputs ( p=0.0015), Sokal scores ( p=0.0070) and white blood cell counts (WBC, p=0.0028). We then trained a machine learning model employing the random-forest (RF), gradient-boosting (GBM) and naïve-bayes (NB) algorithms with different combinations of the 16 miRNAs with and without the clinical parameters of these patients to identify panels with high predictive performance based on area-under-curve (AUC) values of receiver-operating-characteristic (ROC) and precision-recall (PR) curves. Notably, the multivariable panel consisting of both miRNAs and clinical features (AUC-ROC RF=0.83, GBM=0.83, NB=0.84, AUC-PR RF=0.68, GBM=0.67, NB=0.70) performed better than either miRNA (AUC-ROC RF=0.72, GBM=0.73, NB=0.78, AUC-PR RF=0.49, GBM=0.51, NB=0.4) or clinical (AUC-ROC RF=0.82, GBM=0.84, NB=0.84, AUC-PR RF=0.64, GBM=0.67, NB=0.64) panels alone. Interestingly, 2 miRNAs in this panel, miR-185 and miR-145, were also significant classifiers for our Nilotinib predictive study suggesting that expression patterns of these miRNAs may have predictive properties for multiple TKI responses. Thus, we show that predictive accuracy of biomarkers may be supplemented by inclusion of multivariable parameters, and our findings may inform future studies on developing predictive panels for more optimized treatment plans in the clinic.

Treatment Outcomes with Hypomethylating Agents and Venetoclax for AML in the Community Compared to an Academic Setting

Background: The treatment of acute myeloid leukemia (AML) in older and/or unfit patients consists of a combination of a hypomethylating agent (HMA), such as decitabine and azacitidine, with venetoclax, which can be safely administered in the ambulatory setting. In this study, we aimed to compare the outcomes of the treatment of patients with AML who received chemotherapy with venetoclax and an HMA in an ambulatory setting at an academic center or at a community practice with academic center support. Methods: This was a retrospective review of patients with de novo or relapsed/refractory AML treated with a combination of venetoclax and either decitabine or azacitidine between February 2018 and June 2023. Patients were included if they received treatment at an academic center or if they received their treatment at a community practice with ongoing consultation at an academic center. Drugs were administered per the standard of care and local practice. The primary objective was to evaluate the overall response rate (ORR). Secondary objectives were to evaluate differences in chemotherapy management between locations, potential socioeconomic disparities, and to identify adverse events that affect the dosing or administration of therapy. Results: A total of 129 patients received venetoclax plus HMA for AML for a minimum of one cycle as seen in table 1. Of these patients, 77 (60%) received treatment at an academic center, and 52 (40%) received treatment in the community setting. The average age at treatment initiation was 67 years. Disease risk was stratified according to the 2022 European LeukemiaNet (ELN) guidelines. While not statistically significant, there was a tendency for more patients at the academic center to have adverse risk disease (77.9% vs. 69.2%, p=0.364). They were also more likely to have had exposure to a previous HMA (n=10 vs. n=7, p=1) and prior AML treatment (n=20 vs. n=17, p=0.529). Additionally, patients were more likely to have been diagnosed with secondary AML in an academic setting (n=29 vs. n=18, p=0.868). In a multivariate Cox regression model, patients in the middle- and high-income groups, defined as a median household income greater than $52,000, had a 17% lower relative risk regarding survival probability than those in the lower-income group (HR=0.83, 95% CI 0.39-1.73, p=0.622). Overall, patients in the academic setting had a 33% higher relative risk regarding survival probabilities than the community-treated with covariate consideration of performance status, ELN risk, poverty status and exposure to prior-treatments (HR=1.33, 95% CI 0.78-2.26, p=0.285). The OS of all patients was 9.67 months (95% CI 8.33-12.7), with no significant difference between the groups as seen in figure 1. Additionally, the overall response rates (ORR) of the two groups were similar at 38.5% (n=20) and 35.1% (n=27) in community and academic settings, respectively. The complete remission (CR) rate in the community was 11%, while there was an increase of 18% in the academic setting. There were also increased CR rates in the academic compared to the community setting after cycle 1 (4.7 vs. 2.9%, p=0.33), cycle 2 (11.1 vs. 4.8%, p=0.858), and cycle 3 (18.2 vs. 0%, p=0.27). Adverse events in the study were similar except for increased rates of fatigue in the academic setting (76.6% vs. 55.8%, p=0.021). Only 67% of patients underwent a bone marrow biopsy after cycle 1 in the community versus 83% in the academic setting (p=0.101). Lastly, 67% of patients underwent dose reduction with the decitabine with venetoclax regimen in the community setting versus 31% in the academic setting (p=0.012). Conclusion: This study shows that the use of venetoclax plus HMA allows patients to safely receive AML treatment in the community with support from an academic center. Although there are some significant differences in management, such as increased rates of dose reduction in the community setting, the OS and ORR are similar. However, CR rates remain lower in the community setting. It is important to note that the patients in our study, who were treated at an academic center, had disease characteristics that were more resistant to treatment. Ultimately, this study identifies our efforts in creating community partnerships. Our goal is to further evaluate this partnership with quality improvement projects to bridge the gap in AML care between the community and academic practices.

Phase 2 Study of CPX-351 in Combination with Venetoclax in Patients with Newly Diagnosed, High Risk Acute Myeloid Leukemia

Background CPX-351 is a nanoscale liposome of a fixed 5:1 molar ratio of cytarabine and daunorubicin which has improved survival of secondary acute myeloid leukemia (AML) 1. Venetoclax (ven) is a BCL2 inhibitor which, in combination with azacitidine or cytarabine, has become the standard of care for elderly AML. Ven has shown synergism with chemotherapy, with high complete remission (CR) rates in newly diagnosed (ND) and relapsed / refractory (RR) AML 2. In this study, we aim to assess the efficacy and safety of CPX-351 and ven (CPX-ven) in RR and ND AML. Methods This was a phase 2 study with a lead-in safety phase. Enrolment was allowed if the blasts were ≥10%. Patients (pts) age ≥18 with RR AML were eligible for the safety lead-in phase (LIP) or arm A. Arm B enrolled pts age 18 - 69 with ND AML. Other inclusion criteria included adequate liver, renal and cardiac function, and ECOG performance status ≤2. Pts with CNS involvement or prior CPX-351 were excluded. The recommend phase 2 dose was CPX-351 (daunorubicin 44 mg/m 2 and cytarabine 100 mg/m 2 IV on D1, 3 and 5 of induction, and daunorubicin 22 mg/m2 IV on D1 and 3 during consolidation) combined with ven on D2-8 of each cycle. Ven was dosed at 300mg daily and reduced to 150mg or 50mg daily for pts on a moderate or strong CYP3A inhibitors, respectively. The primary outcome measure was complete remission (CR) or CR with incomplete count recovery (CRi). Here, we report the outcomes of arm B including pts with ND AML. Results 14 pts were enrolled on arm B. One pt withdrew and one was too early in treatment, therefore 12 were evaluable. Baseline characteristics are shown in Table 1. The median age was 59.5 years (range, 44 - 69); Four (33.3%) were female. All 12 (100%) pts were classified as adverse risk per the 2017 European Leukemia Net Guidelines. Two (16.7%) pts had treatment for a prior non-myeloid malignancy (therapy-related AML). Eight (66.7%) had an antecedent haematological disorder (secondary AML), of whom seven had treatment (median 1 line, range 1 - 2). Four (33.3%) pts had a complex karyotype, of whom three harbored a TP53 mutation. Nine (75.0%) pts achieved a response, including four CR (33.3%), five CRi (41.6%), and one induction mortality (8.3%) prior to disease assessment. 3/4 (75.0%) pts with complex cytogenetics (including 2/3 (66.7%) with a TP53 mutation) achieved a CR/CRi. Of the secondary AML pts with an evaluable response, 5/7 (71.4%) achieved a CR/CRi. Of the nine pts achieving CR/CRi, five (55.5%) were negative for minimal residual disease (MRD) by flow cytometry. The median number of cycles to CR/CRi was one (range 1 - 2). The median overall survival (OS) was 12.9 months (95% CI, 3.1 - not reached, NR, Figure 1). The median relapse-free survival (RFS) was 8.7 months (95% CI, 3.1 -NR). 7/9 (77.8%) of responding pts underwent an allogeneic stem cell transplant (SCT) after attaining CR/CRi. There were 90 adverse events (AE) in 10 (83.3%) pts. 80 (88.9%) were grade 1 or 2, 10 (11.1%) were grade ≥3. The most common AE was gastrointestinal with seven (7.8%) occurrences. Four (33.3%) pts developed grade ≥3 infections; of these, one (8.3%) died. The cause of death was neutropenic septic shock due to pneumonia. Conclusion CPX-ven is a feasible option for remission induction in pts with high-risk, newly diagnosed AML. The combination produced high rates of response and MRD negativity in a cohort of pts with adverse risk features, including pts with adverse karyotype and AML following prior HMA-based treatment, allowing them to proceed with SCT. Toxicities are as expected for an intensive chemotherapy regimen. Further studies are needed to confirm the efficacy of this combination.

Analysis of Post-Transplant Clinical Outcomes and Prognostic Factors in FLT3-ITD Mutated Acute Myeloid Leukemia Patients Under ELN 2022 Risk Stratification

Background In 2022 European LeukemiaNet (ELN) recommendations for diagnosis and management of acute myeloid leukemia (AML), one of the most important changes made to the genetic risk classification of AML is that the FLT3-ITD allelic ratio (AR) is no longer considered in the risk classification; consequently, AML with FLT3-ITD (without adverse-risk genetic lesions) are now categorized in the intermediate-risk group, irrespective of the allelic ratio or concurrent presence of NPM1 mutation. Aims To investigate the survival outcomes of FLT3-ITD mutated AML patients underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT), based on prognostic stratification defined by the 2017 and 2022 ELN guidelines, and to analyze the risk factors affecting transplant prognosis. Methods A total of 163 FLT3-ITD mutated AML patients underwent allo-HSCT were enrolled in this study. Bone marrow aspirates were obtained, cytogenetic and molecular characteristics were collected at the time of diagnosis. Genetic risk category was classified according to the 2017 and 2022 edition of the European LeukemiaNet (ELN) recommendations. HLA-haploidentical donor transplants were performed if no HLA matched sibling or unrelated donor was available. Patients received myeloablative conditioning with a modified Bu/Cy/Flu/Ara-c (busulfan/cyclophosphamide/fludarabine/cytarabine) or MCBC (melphalan/cladribine/busulfan/cyclophosphamide) preparative regimen. The therapeutic process and clinical outcomes were retrospectively analyzed. Results We assessed 163 adults with FLT3-mutated AML (median age 37 years) allografted between 2017 and 2022 in our clinical center. The median follow-up of alive patients was 24.9 months. The 2-year relapse-free survival (RFS) and overall survival (OS) were 67.2% and 71.9%, respectively. The 2-year incidences of relapse and non-relapse mortality were 27.9% and 12.5%, respectively. There was no significant difference in survival outcomes between the intermediate and high-risk groups after transplantation, both defined by the 2017 and 2022 ELN guidelines. High FLT3-ITD allelic ratio (≥0.5) at initial diagnosis was associated with elevated WBC counts ( P<0.001), but did not affect the response ratefollowing induction chemotherapy ( P=0.059) . Patients with high allelic ratio were more likely to benefit from maintenance therapy after transplantation ( P=0.048 for OS). In univariate analysis, presence of NPM1 mutation and the allelic ratio of FLT3-ITD did not affect any of the transplant outcomes. In multivariate analysis, MRD-negative at transplantation, occurrence of chronic GVHD and posttransplant maintenance therapy improved OS and RFS. Conclusion Allo-HSCT can overcome the poor prognosis in patients with FLT3-ITD mutated AML. Neither the presence of NPM1 mutation nor the FLT3-ITD allelic ratio affected survival. Prevention of disease relapse after transplantation remains a challenge. The main prognostic factors affecting transplant outcomes included pre-transplant MRD status and the application of maintenance therapy. Thus, introduction of FLT3 inhibitors as post-transplant maintenance therapy and the development of highly sensitive MRD assays will further reduce disease recurrence and improve survival.

Intensive Induction Chemotherapy with or without Gemtuzumab Ozogamicin Among Patients with Core-Binding Factor Acute Myeloid Leukemia

Background: Core-binding factor acute myeloid leukemia (CBF-AML) is characterized by the presence of inv(16)/t(16;16) or t(8;21), and is classified as favorable risk by the 2022 European LeukemiaNet (ELN) guidelines. High remission rates are observed in the setting of cytarabine- or anthracycline-based induction regimens. The CD33-targeted antibody-drug conjugate gemtuzumab ozogamicin (GO) is FDA-approved for use in newly-diagnosed CD33+ AML; a meta-analysis of 5 prospective randomized studies investigating intensive chemotherapy (IC) +/- GO demonstrated an improved overall survival (OS) with the addition of GO to IC for favorable-risk AML (Hills et al, Lancet Oncol 2014). The trials included in the meta-analysis did not restrict inclusion criteria to CBF-AML alone and there remains variation amongst IC regimens utilized in CBF-AML treatment. In addition, IC combined with the KIT inhibitor dasatinib has shown remission rates and promising overall survival in prospective studies (Paschka et al, Leukemia 2018; Marcucci et al, Blood Adv 2020). We set out to investigate the outcomes of patients (pts) with CBF-AML treated with IC +/- targeted agents (GO or KIT inhibitor) across 5 institutions that are part of the Consortium on Myeloid Malignancies and Neoplastic Diseases (COMMAND). Methods: Retrospective chart review was done to identify pts who were treated with IC with AML harboring inv(16)/t(16;16) or t(8;21) identified on cytogenetics, from January 2010 through April 2023, and were treated across 5 geographically-diverse academic institutions. Pt clinical characteristics, cytogenetics, molecular mutations, treatment, relapse, and OS data were collected. Relapse-free survival (RFS) and OS were both analyzed using Kaplan-Meier analysis. Results: One-hundred forty-nine consecutive pts with CBF-AML were identified, 76 (51%) with inv(16) and 73 (49%) with t(8;21). Median age of pts was 47 years (yrs) (range 18-80yrs), with 64% male pts, and 106 (71%) pts had an ECOG performance status of 0-1. One-hundred seven (72%) pts identified as non-Hispanic White, 22 (15%) identified as non-Hispanic Black, and 12 (8%) identified as Hispanic. Molecular testing via next-generation sequencing was available for 124 pts (83%); 49 (33%) pts had adverse-risk ELN cytogenetics or mutations when excluding CBF status, and 27 (18%) pts harbored KIT mutations. Other baseline pt characteristics are listed in Table 1A. All pts underwent induction with IC; 44 (30%) pts received IC + GO, 21 (14%) received IC + KIT inhibitor (14 received dasatinib, 7 received midostaurin), and the remaining 84 (56%) received IC without a targeted agent. One-hundred fourteen (77%) pts achieved a complete response (CR) to induction therapy, and 1 pt experienced induction-related mortality. Fluorescence in-situ hybridization (FISH) data was available in 82 pts at end of induction with 83% achieving FISH negativity. Measurable residual disease (MRD) testing was done by polymerase chain reaction (PCR) in 62 pts with 76% achieving MRD-negativity. One-hundred forty pts underwent consolidation with a median of 3 cycles of chemotherapy; 21 (15%) pts received GO with consolidation and 17 (12%) received a KIT inhibitor with consolidation. Forty-four (30%) pts underwent allogeneic stem cell transplantation (alloSCT), with 17 in first CR, and 27 after second-line or beyond therapy. With a median follow-up of 22.5 months, the observed 3-yr RFS was 51% (95% CI: 43-61%) and the 3-yr OS was 69% (95% CI: 61-78%). Pts treated with IC + KIT inhibitor had significantly improved 3-yr RFS of 85% (95% CI: 70-100%) compared to other pts, including those who received IC + GO who had a 3-yr RFS of 51% (95% CI: 37-70%) and those who received IC without targeted therapy who had a 3-yr RFS of 45% (95% CI: 35-58%) ( p = 0.02) ( Figure 1). Full survival statistics are shown in Table 1B and Figure 1. Conclusions: In this multi-center retrospective study of pts with CBF-AML undergoing induction with IC regimens with diverse national treatment practices, we find that the addition of GO to IC is not associated with improved outcomes. Pts treated with IC + KIT inhibitor had significantly improved RFS; of note, pts receiving a KIT inhibitor oftentimes continued on maintenance even after consolidation. These data suggest that there may be a role for targeted therapies other than GO to be explored in randomized prospective studies of intensive induction chemotherapy for CBF-AML.

Treatment Patterns Among Patients with Relapsed/Refractory Core-Binding Factor Acute Myeloid Leukemia after Intensive Induction Chemotherapy

Background: Core-binding factor acute myeloid leukemia (CBF-AML) is characterized by the presence of inv(16)/t(16;16) or t(8;21), and is classified as favorable risk by the 2022 European LeukemiaNet (ELN) guidelines. High remission rates are observed in the setting of cytarabine- or anthracycline-based induction regimens. However, for the subset of patients (pts) who relapse, outcomes after relapse were poor in prior single-center analysis with median survival of less than 1.5 years (Khan et al. Clin Leuk Lymph Myeloma 2017). There remains a need to better understand outcomes of pts who experience relapse after initial complete remission (CR), particularly after diverse intensive chemotherapy (IC) induction regimens, where variable practices show differential induction outcomes. We set out to investigate the outcomes of pts with CBF-AML in first relapse or with disease refractory to initial therapy, across 5 institutions that are part of the Consortium on Myeloid Malignancies and Neoplastic Diseases (COMMAND). Methods: Retrospective chart review was done to identify pts who were treated with intensive IC with inv(16)/t(16;16) or t(8;21) identified on cytogenetics, from January 2010 through April 2023, with disease in first relapse after initial CR or refractory to primary induction. Pts were treated across 5 geographically-diverse, academic institutions. Pt clinical characteristics, cytogenetics, molecular testing via next-generation sequencing (NGS), treatment, relapse, and overall survival (OS) data were collected. OS since time of disease relapse was analyzed using Kaplan-Meier analysis. Results: Fifty-four consecutive pts with relapsed/refractory (R/R) CBF-AML were identified, 24 (44%) with inv(16) and 30 (56%) with t(8;21). Median age of pts at initial diagnosis was 50 years (yrs) (range 20-80yrs), with 61% male pts. Molecular testing and/or NGS was available at initial diagnosis for 41 pts (76%); 12 (22%) pts had adverse-risk ELN cytogenetics or mutations when excluding CBF status, and 8/41 (20%) pts harbored KIT mutations. Other baseline pt characteristics are listed in Table 1. All pts underwent induction with IC; 14 (26%) pts received intensive induction with gemtuzumab ogozamicin (GO), 3 (6%) received intensive induction with a KIT inhibitor (dasatinib or midostaurin), and the remaining 37 (68%) received IC induction without a targeted agent. Forty-one (76%) pts achieved a CR to induction therapy. Measurable residual disease (MRD) testing by polymerase chain reaction (PCR) and/or fluorescence in situ hybridization (FISH) for CBF protein was available in 30 (56%) pts, with 16/21 (76%) achieving MRD-negativity by PCR and 20/26 (77%) by FISH. Forty-six pts underwent consolidation with a median of 4 cycles of chemotherapy (IQR 3-4), with 9 (17%) pts receiving GO with consolidation and 4 (7%) receiving a KIT inhibitor with consolidation. Median time to relapse was 10.1 months (IQR 8.0-13.6 months), and median OS after relapse was 9.2 months (IQR 4.4-22.1 months) for the whole cohort. Second line treatment data was available for 44 pts, with 29 (66%) receiving intensive 2nd line therapy vs 15 (34%) receiving lower intensity 2nd line therapy; 14 (32%) pts received venetoclax as part of their 2nd line therapy. There was no significant difference in 2-yr OS after relapse for those who received IC (50%, 95% CI: 34-72%) vs lower intensity therapy (24%, 95% CI: 8-72%) ( p=0.28) (Figure 1A). Twenty-eight (52%) pts who received 2nd line therapy for R/R disease proceeded to allogeneic stem cell transplantation (alloSCT), with a 2-yr OS of 57% (95%CI: 40-80%) for this group after time of relapse, compared to 15% (95% CI: 5-42%) for those who did not achieve CR after 2ndline therapy and/or proceed to transplant ( p<0.01) (Figure 1B). Conclusions: In this multi-center retrospective study of pts with R/R CBF-AML, we describe patterns of relapse after intensive induction chemotherapy, and do not observe a statistically difference in outcomes based on intensity of 2nd line therapy among pts with available treatment information. Median OS in the entire cohort was 9.2 months from diagnosis of R/R disease. Pts who proceeded to alloSCT after receiving 2nd line therapy had significantly improved long-term outcomes compared to those who did not proceed to alloSCT, highlighting the need for better therapies in the 2nd line and beyond to improve longer term outcomes for pts with CBF-AML.

The Co-Occurrence of bZIP in-Frame CEBPA-Mutation with Unfavorable Genetic Abnormalities Is Associated with a Poor Prognosis in Acute Myeloid Leukemia

In a previous study (SY Ahn et al, Cancer Res Treat 2023), we reported that the presence of only the basic leucine zipper in-frame mutation (bZIP in-f) CEBPA is associated with favorable outcomes in patients with CEBPAdouble mutations. However, when bZIP in-fCEBPAmutations were accompanied by certain mutations in chromatic/DNA modifiers, cohesion complex, and splicing genes, patients exhibited inferior overall survival (OS). FLT3-ITD or additional cytogenetic abnormalities is known to rarely co-occur with CEBPA mutation in patients with AML. In cases where both bZIP in-fCEBPA and FLT3-ITD coexist, there are conflicting aspects in risk stratification, and research on its prognostic significance is limited. Therefore, we aimed to evaluate the prognostic significances of the co-occurrence of bZIP in-fCEBPAmutation with unfavorable genetic abnormalities in acute myeloid leukemia. In 832 patients who were diagnosed with AML excluding acute promyelocytic leukemia and underwent intensive induction therapy, 98 (11.8%) had bZIP in-fCEBPA. Among 98 patients with bZIP in-fCEBPA, eight cases had FLT3-ITD (5 with low allelic ratio, 3 with high allelic ratio), 19 patients had unfavorable genetic mutations according to the 2022 European LeukemiaNet guidelines, and 10 patients had non-favorable cytogenetic abnormalities as detected in diagnostic samples. We observed no clinical differences in features such as age, sex, WBC count, bone marrow blast percentage at diagnosis based on the co-occurrence unfavorable genetic abnormalities (unfavorable-gene). Among the 98 patients with bZIP in-f CEBPA, 30 out of 37 patients with unfavorable-gene achieved complete remission (CR) after induction therapy, and 55 out of 61 without unfavorable genetic abnormalities showed CR (p=0.20). Allogeneic hematopoietic cell transplantation was proceeded at first CR in 21 out of 37 unfavorable-gene group, and 35 out of 61 bZIP in-fCEBPAonly group ( p=1.00). In the survival analysis, patients with FLT3-ITD, unfavorable genetic co-mutated, or non-favorable cytogenetic abnormalities with bZIP in-fCEBPA showed inferior OS (p=0.036) (Fig. A). Due to the limited number of patients for analysis, we classified them into two group: the unfavourable-gene group and bZIP in-fCEBPAonly group. We observed that the unfavourable-gene group had a poor OS compared to the bZIP in-fCEBPAonly group (HR 2.71, 95 CI; 1.32-5.55, p=0.007) (Fig.B). On multivariate analysis, unfavorable-gene group was only poorrisk factor for OS. Despite the inclusion of a relatively small number of patients, our study demonstrates that when co-occurrence of bZIP in-fCEBPA withunfavourable genetic abnormalities, it results in a poor prognosis. To confirm its clinical significance, further validation with a larger patient population is necessary.

Analytical Validation of a Duplex Sequencing AML Assay with a Limit of Detection Below 0.01% VAF for SNVs and Indels

Sensitive, specific, precise, and accurate detection of low frequency mutations in acute myeloid leukemia (AML) is critical to assess measurable residual disease (MRD). Next generation sequencing (NGS) assays are powerful tools for this, permitting interrogation of thousands of base positions across genomic regions of interest. However, PCR and sequencing errors in these assays limit accurate mutation detection at variant allele frequencies (VAF) below approximately 1%. Duplex sequencing (DS), an error-corrected NGS (ecNGS) method, greatly reduces such errors by relating original top and bottom DNA strands to make double stranded consensus sequences, thus enabling sensitive, specific, accurate, and precise detection of variants below 0.01% VAF. Here, we report analytical validation of an updated DuplexSeq™ AML assay, a 36-gene panel informed by 2022 European LeukemiaNet (ELN) recommendations combined with kitted library preparation reagents and a cloud-based analysis pipeline. The assay uniformly targets regions that harbor AML-associated mutations: 82% of targeted regions exhibit sequencing depth >80% of the panel-wide mean and >99.5% of targeted regions exhibit depth >20% of the panel-wide mean. These analytical studies also validate an improved “v2 chemistry” version of a DS library preparation kit. This improved chemistry provides an enzymatic fragmentation step that replaces time-consuming mechanical fragmentation methods while increasing the yield of duplex data for a given input mass of DNA sample and eliminating damage that results from mechanical fragmentation. A contrived human genomic DNA sample carrying 26 variants targeted by the panel, DNA extracted from AML-positive peripheral blood (PB) and bone marrow aspirate (BMA) specimens, and DNA extracted from healthy normal PB specimens were used as test samples to execute limit of detection (LoD), limit of blank (LoB), accuracy, linearity, and repeatability & reproducibility studies. In this assay a duplex depth threshold of ≥20,000x is applied; ≥91.8% of the 42,605 genomic base positions targeted by the panel are consistently (in 95% of analyzed libraries) above this threshold. The LoD study assayed samples carrying variants from 0.37% to 0.00055% VAF. The assay demonstrated excellent sensitivity, with variants down to 0.00055% VAF detected. A probit model was used to estimate the VAF at which 95% of variants would be detected, establishing an LoD of 0.008% VAF for single-nucleotide variants (SNVs) and insertions and deletions (indels) (see Figure 1). The LoB study assessed the background detection rate in DNA samples derived from healthy normal specimens. In >95% of sites surveyed, no variants were observed, establishing an assay LoB of 0 (0% VAF). Accuracy of the assay was determined by interrogating PB and BMA samples from AML-positive and -negative donors along with contrived test samples. Results were compared to data acquired via orthogonal methods. For variants above the LoD of 0.008% VAF, accuracy was high, with overall percent agreement (OPA) of 98.3%, positive percent agreement (PPA) of 100.0%, and negative percent agreement (NPA) of 98.3%. Data were linear from 0.0016% VAF to 96.1% VAF with R 2 = 0.95. All 6 expected FLT3-ITD variants, and all 5 expected NPM1 insertions were detected by the assay. Precision study data showed that the assay was highly repeatable, with OPA of 97.5%, PPA of 98.0%, and NPA of 97.3%. Reproducibility across 2 operators, 2 lots of reagents, and 3 independent runs of library preparation was also very high, with OPA of 97.5%, PPA of 98.8%, and NPA of 96.9%. Current ELN guidelines provisionally define NGS-MRD positivity as ≥0.1% VAF, but recent evidence suggests lower frequency variants may be informative. With an LoD of 0.008% VAF for SNVs and indels, and an LoB of 0, this novel AML assay comprising an updated gene panel coupled with improved duplex sequencing chemistry and software represents a highly sensitive, specific, accurate, and precise ecNGS assay for detecting variants associated with AML MRD.

Improvement in Survival of Patients with FLT3 Mutated Acute Myeloid Leukemia: Results from a Retrospective Canadian Cohort

Introduction: FLT3 internal tandem duplication (ITD) mutations have been historically associated with inferior outcomes in patients with acute myeloid leukemia (AML) and an intermediate risk status as per the European LeukemiaNet (ELN) 2022 guidelines. In 2017, the US FDA granted approval to midostaurin for frontline treatment and a year later, gilteritinib for use in relapsed/refractory (R/R) AML. These agents began to be widely used in our provincial leukemia program in early 2018 as per the label indications. In this study, we compared clinical outcomes for patients with FLT3-ITD mutated AML before and after this change in British Columbia. Methods: We identified patients in our program database diagnosed with FLT3-ITD mutated AML from January 1, 2010 to December 31, 2021. Only patients receiving intensive chemotherapy with cytarabine and anthracycline were included. Patients were divided into two eras: 2010-2017 (era 1) and 2018-2021 (era 2). The two eras were compared in terms of patient characteristics, genetics, rate of complete remission (CR) and CR with incomplete count recovery (CRi), receipt of allogeneic stem cell transplant (alloSCT) and the overall and relapse free survival (OS and RFS). OS was calculated from diagnosis to death from any cause and was not censored at the time of alloSCT. RFS was calculated from CR/CRi to relapse. Patients without an event during the study period were censored at the time of last follow-up. Categorical variables were compared using Chi-square and Fisher exact tests and continuous variable were compared using paired T tests. Kaplan-Meier survival method and log rank test were used to estimate and compare survival. Uni- and multivariate analyses were performed in a Cox proportional regression model and alloSCT was considered as a time dependent covariate. Results: 169 patients were included (era 1=101, era 2=68). The median duration of follow up of the entire cohort was 17 months (range 0.5-148.6 months) and follow-up of surviving patients (n=70) was longer in era 1 vs. era 2 (median 88 months vs. 32 months, p<0.001). The median age of the entire cohort was 60 years (range 26-79 years) and it was comparable in both eras. Most patients had normal cytogenetics (era 1: 76%; era 2: 69%). NPM1 was mutated in 69/101 (68%) and 38/68 (56%) patients in eras 1 and 2 respectively. Due to lack of genetic data for era 1, we did not risk stratify based on the recent ELN classification. The rate of CR/CRi was 58/101 (57%) vs. 40/68 (59%) after induction chemotherapy and 78/101 (77%) vs. 58/67 (87%) (p=0.09) at any time in eras 1 and 2 respectively. AlloSCT was performed in 58% and 72% of patients in the two cohorts. Midostaurin was used in 82% and gilteritinib was used in 26% of era 2 patients in the second era. OS was significantly longer in era 2 (median OS 73 months vs. 11 months, HR (death) 6.2, p=0.012) (Figure). The 2-year OS rate was higher in era 2 (57%) than in era 1 (39%). In patients achieving CR/CRi, median RFS was longer in era 2 (51 vs. 16 months, p=0.43), although this was not statistically significant. In univariate Cox regression analysis the following factors were associated with improved OS: use of midostaurin (HR 0.487, 95% CI 0.304-0.779, p=0.003), diagnosis in era 2 (HR 0.583, 95% CI 0.38-0.895, p=0.014) and receipt of alloSCT (HR 0.346,) 95% CI 0.219-0.545, p<0.001). Other parameters including age, sex, NPM1 status and cytogenetics were not significant. In multivariate analysis including era, midostaurin and alloSCT, only the receipt of alloSCT was associated with significantly improved OS (HR 0.362, 95% CI 0.229 - 0.572, p<0.001) whereas use of midostaurin (HR=0.465, CI 0.207-1.042, p=0.063) and era (HR 1.12, 95% CI 0.538-2.34, p=0.76) were not significant. Conclusions: Our results show thatOS for patients with FLT3-ITD AML has improved in patients diagnosed in a more recent treatment era. Patients diagnosed after 2018 were more frequently treated with AlloSCT and FLT3-inhibitors, which may have resulted in improvements in OS. Our findings support the assignment of FLT3-ITD AML to theintermediate risk category as per the current ELN guidelines. A limitation of this study is the differences in follow-up time between eras, which may influence the event rate between the groups.