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Treatment results for patients with newly diagnosed FMS-like tyrosine kinase 3 (FLT3)-mutated (FLT3mut+) acute myeloid leukemia (AML) ineligible for intensive chemotherapy are disappointing. This multicenter, open-label, phase 3 trial randomized (2:1) untreated adults with FLT3mut+ AML ineligible for intensive induction chemotherapy to receive gilteritinib (120 mg/d orally) and azacitidine (GIL + AZA) or azacitidine (AZA) alone. The primary end point was overall survival (OS). At the interim analysis (August 26, 2020), a total of 123 patients were randomized to treatment (GIL + AZA, n = 74; AZA, n = 49). Subsequent AML therapy, including FLT3 inhibitors, was received by 20.3% (GIL + AZA) and 44.9% (AZA) of patients. Median OS was 9.82 (GIL + AZA) and 8.87 (AZA) months (hazard ratio, 0.916; 95% CI, 0.529-1.585; P = .753). The study was closed based on the protocol-specified boundary for futility. Median event-free survival was 0.03 month in both arms. Event-free survival defined by using composite complete remission (CRc) was 4.53 months for GIL + AZA and 0.03 month for AZA (hazard ratio, 0.686; 95% CI, 0.433-1.087; P = .156). CRc rates were 58.1% (GIL + AZA) and 26.5% (AZA) (difference, 31.4%; 95% CI, 13.1-49.7; P < .001). Adverse event (AE) rates were similar for GIL + AZA (100%) and AZA (95.7%); grade ≥3 AEs were 95.9% and 89.4%, respectively. Common AEs with GIL + AZA included pyrexia (47.9%) and diarrhea (38.4%). Gilteritinib steady-state trough concentrations did not differ between GIL + AZA and gilteritinib. GIL + AZA resulted in significantly higher CRc rates, although similar OS compared with AZA. Results support the safety/tolerability and clinical activity of upfront therapy with GIL + AZA in older/unfit patients with FLT3mut+ AML. This trial was registered at www.clinicaltrials.gov as #NCT02752035.

Clonal evolution in acute myeloid leukemia (AML) originates long before diagnosis and is a dynamic process that may affect survival. However, it remains uninvestigated during routine diagnostic workup. We hypothesized that the mutational status of bone marrow dysplastic cells and leukemic blasts, analyzed at the onset of AML using integrated multidimensional flow cytometry (MFC) immunophenotyping and sorting (FACS) with next-generation sequencing (NGS), could reconstruct leukemogenesis. Dysplastic cells were detected by MFC in 285 of 348 (82%) newly-diagnosed AML patients. Presence of dysplasia according to MFC and WHO criteria had no prognostic value in the elderly. NGS of dysplastic cells and blasts isolated at diagnosis identified three evolutionary patterns: stable (n=12/21), branching (n=4/21) and clonal evolution (n=5/21). In patients achieving complete response, integrated MFC and FACS with NGS showed persistent measurable residual disease (MRD) in phenotypically normal cell types, as well as the acquisition of genetic traits associated with treatment resistance. Furthermore, whole-exome sequencing of dysplastic and leukemic cells at diagnosis and of MRD uncovered different clonal involvement in dysplastic myelo-erythropoiesis, leukemic transformation and chemoresistance. Altogether, we showed that it is possible to reconstruct leukemogenesis in approximately 80% of newly diagnosed AML patients, using techniques other than single-cell multiomics. ACKNOWLEDGEMENTS: The authors acknowledge the patients, caregivers, and the biobank of the University of Navarra. This work was supported by grants from the Área de Oncología del Instituto de Salud Carlos III, Centro de Investigacion Biom ´ edica en ´ Red (CIBER-ONC) (CB16/12/00369, CB16/12/00233, CB16/12/ 00489, and CB16/12/00284), Instituto de Salud Carlos III/Subdireccion General de Investigaci ´ on Sanitaria (FIS numbers PI16/ ´ 01661, PI16/00517, and PI19/01518), and the Plan de Investigacion´ de la Universidad de Navarra (PIUNA 2014-18). This work was supported internationally by the Cancer Research UK, FCAECC, and AIRC under the Accelerator Award Program (EDITOR).

This open-label, randomized, phase 3 trial (NCT02577406) compared enasidenib, an oral IDH2 inhibitor, with conventional care regimens (CCR) in patients aged =60 years with late-stage, mutant-IDH2 acute myeloid leukemia (AML) relapsed/refractory (R/R) to 2 or 3 prior AML-directed therapies. Patients were first preselected to a CCR (azacitidine, intermediate-dose cytarabine, low-dose cytarabine, or supportive care), and then randomized (1:1) to enasidenib 100 mg/day or CCR. The primary endpoint was overall survival (OS). Secondary endpoints included event-free survival (EFS), time to treatment failure (TTF), overall response rate (ORR), hematologic improvement (HI), and transfusion independence (TI). Overall, 319 patients were randomized to enasidenib (n=158) or CCR (n=161). Median age was 71 years. Median (range) enasidenib exposure was 142 days (3-1270) and CCR was 36 days (1-1166). One enasidenib (0.6%) and 20 CCR (12%) patients received no randomized treatment, and 30% and 43%, respectively, received subsequent AML-directed therapies during follow-up. Median OS with enasidenib vs CCR was 6.5 vs 6.2 months (HR [hazard ratio] 0.86; P=.23); 1-year survival was 37.5% vs 26.1%. Enasidenib meaningfully improved EFS (median 4.9 months, vs 2.6 months with CCR; HR 0.68; P=.008), TTF (median 4.9 vs 1.9 months, HR 0.53; P

The need for allogeneic hematopoietic stem cell transplantation (allo-HSCT) in adults with Philadelphia chromosome–negative (Ph−) acute lymphoblastic leukemia (ALL) with high-risk (HR) features and adequate measurable residual disease (MRD) clearance remains unclear. The aim of the ALL-HR-11 trial was to evaluate the outcomes of HR Ph− adult ALL patients following chemotherapy or allo-HSCT administered based on end-induction and consolidation MRD levels. Patients aged 15 to 60 years with HR-ALL in complete response (CR) and MRD levels (centrally assessed by 8-color flow cytometry)

Effective treatment options are limited for patients with acute myeloid leukemia (AML) who cannot tolerate intensive chemotherapy. Adults age ≥18 years with newly diagnosed AML ineligible for intensive chemotherapy were enrolled in this international phase 3 randomized double-blind placebo-controlled trial. Patients (N = 211) were randomized 2:1 to venetoclax (n = 143) or placebo (n = 68) in 28-day cycles, plus low-dose cytarabine (LDAC) on days 1 to 10. Primary end point was overall survival (OS); secondary end points included response rate, transfusion independence, and event-free survival. Median age was 76 years (range, 36-93 years), 38% had secondary AML, and 20% had received prior hypomethylating agent treatment. Planned primary analysis showed a 25% reduction in risk of death with venetoclax plus LDAC vs LDAC alone (hazard ratio [HR], 0.75; 95% confidence interval [CI], 0.52-1.07; P = .11), although not statistically significant; median OS was 7.2 vs 4.1 months, respectively. Unplanned analysis with additional 6-month follow-up demonstrated median OS of 8.4 months for the venetoclax arm (HR, 0.70; 95% CI, 0.50-0.98; P = .04). Complete remission (CR) plus CR with incomplete blood count recovery rates were 48% and 13% for venetoclax plus LDAC and LDAC alone, respectively. Key grade ≥3 adverse events (venetoclax vs LDAC alone) were febrile neutropenia (32% vs 29%), neutropenia (47% vs 16%), and thrombocytopenia (45% vs 37%). Venetoclax plus LDAC demonstrates clinically meaningful improvement in remission rate and OS vs LDAC alone, with a manageable safety profile. Results confirm venetoclax plus LDAC as an important frontline treatment for AML patients unfit for intensive chemotherapy. This trial was registered at www.clinicaltrials.gov as #NCT03069352.

Although hypomethylating agents (HMAs) are frequently used in the frontline treatment of older acute myeloid leukemia (AML) patients, little is known about their effectiveness in relapsed or primary treatment–refractory (RR)-AML. Using an international multicenter retrospective database, we studied the effectiveness of HMAs in RR-AML and evaluated for predictors of response and overall survival (OS). A total of 655 patients from 12 centers received azacitidine (57%) or decitabine (43%), including 290 refractory (44%) and 365 relapsed (56%) patients. Median age at diagnosis was 65 years. Best response to HMAs was complete remission (CR; 11%) or CR with incomplete count recovery (CRi; 5.3%). Additionally, 8.5% experienced hematologic improvement. Median OS was 6.7 months (95% confidence interval, 6.1-7.3). As expected, OS differed significantly by best response, with patients achieving CR and CRi having a median OS of 25.3 and 14.6 months, respectively. In multivariate analysis, the presence of ≤5% circulating blasts and a 10-day schedule of decitabine were associated with improved response rates, whereas the presence of >5% circulating blasts and >20% bone marrow blasts were associated with decreased OS. A significant subset of RR-AML patients (16%) achieved CR/CRi with HMAs and experienced a median OS of 21 months. Outside of a clinical trial, HMAs represent a reasonable therapeutic option for some patients with RR-AML.

A previous report of the Programa de Estudio y Tratamiento de las Hemopatías Malignas (PETHEMA) Group showed that a risk-adapted strategy combining all-trans retinoic acid (ATRA) and anthracycline monochemotherapy for induction and consolidation in newly diagnosed acute promyelocytic leukemia results in an improved outcome. Here we analyze treatment outcome of an enlarged series of patients who have been followed up for a median of 65 months. From November 1999 through July 2005 (LPA99 trial), 560 patients received induction therapy with ATRA plus idarubicin. Patients achieving complete remission received 3 courses of consolidation followed by maintenance with ATRA and low-dose chemotherapy. The 5-year cumulative incidence of relapse and disease-free survival were 11% and 84%, respectively. These results compare favorably with those obtained in the previous LPA96 study (P = .019 and P = .04, respectively). This updated analysis confirms the high antileukemic efficacy, low toxicity, and high degree of compliance of a risk-adapted strategy combining ATRA and anthracycline monochemotherapy for consolidation therapy.

Numerous recurrently mutated genes have been described in the last years for acute myeloid leukemia (AML). Nevertheless, only a few of them (NPM1, CEBPA, FLT3), and usually individually analyzed, have been systematically incorporated into clinical laboratories to stratify prognosis and guide risk-adapted therapy. So far, technology made impossible their simultaneous study, however, development of high throughput techniques as "next generation sequencing" (NGS) allows a parallel assay. Recently, our group designed a NGS panel (custom panel) interrogating the complete coding sequence of 87 genes potentially involved in AML. Interactome analysis revealed that 13 genes were highly implicated in AML pathogenesis. Although NGS has been broadly utilized in the investigational scope, its application to the routine diagnosis is still far from become a standard. The aim of this study is to validate the clinical applicability to routine laboratories of a hotspot NGS panel that includes these 13 genes and other potentially actionable targets. We included 62 "de novo" AML patients in which we tested the Ion Ampliseq AML community panel (Life Technologies) (IAAC panel) in the Ion PGM/Proton platforms. This panel includes hotspots of ASXL1 (exon 12), BRAF (V600E), CBL (exons 8-9), FLT3 (codons 676, 830-850), IDH1 (exon 4), IDH2 (exon 4), JAK 2 (exon 14), KIT (exons 8, 10, 11 and 17), KRAS (exons 2-4), NRAS (exons 2-4), PTPN11 (exons 3,7,8,13), RUNX1 (exons 3-8) y WT1 (exons 7 and 9), and the entire coding sequence of CEBPA, DNMT3A, GATA, TET2 and TP53. The design does not include the FLT3-ITD region. This panel requires only a total of 40 ng of DNA, far less than the custom panel (250 ng), which is very convenient in a clinical laboratory, where the input sample can be limited. The hotspot NGS panel detected 153 variants in 62 patients (2.47 mutations/patient). Mean read depth and uniformity were 1580 and 94.89%, respectively. The IAAC panel detected 25 NPM1 mutations, 20 TET2 and DNMT3A mutations, 14 in CEBPA, 12 in TP53, 11 in RUNX1, 8 in FLT3, 7 in IDH2, 6 in ASXL1, GATA2 and NRAS, 5 in PTPN11 and KRAS, 3 in WT1, 2 in CBL and 1 mutation in IDH1, KIT, and BRAF. Only 4 patients (6%) remained wild-type for these genes after the analysis with the IAAC panel. These samples had previously been analyzed by conventional molecular biology techniques (CMBT) for FLT3-D835, NPM1-T288, DNMT3A-R882 and CEBPA. The IAAC panel found 100% of these previously known mutations plus 5 extra mutations that were negative by CMBT. These mutations were reconfirmed by Sanger sequencing. Therefore, the IAAC panel detects more mutations than CMBT. To further assess the suitability of the hotspot panel, we selected a subset of 25 patients that had been also analyzed by the custom panel (Sure Desing Tool (Agilent) and an Illumina platform). This allowed us to analyze the complete coding sequence of the genes included in the IAAC panel and thus look for mutations outside the hotspots. A total of 53 variants were found with the custom panel. Fifty of these variants (95%) were also detected with the IAAC panel. The remaining 3 variants (5%) were located outside the hotspot regions. Additionally, the IAAC panel detected 13 variants in the overlapping regions that were not found with the custom panel. This could be explained because when focusing on recurrent regions of particular genes, it is possible to increase the mean read depth and therefore reach higher sensibility, which can be achieved with the IAAC panel but not with the custom panel. In conclusion, the Ion Ampliseq AML community panel detects mutations currently analyzed in most of clinical laboratories with validated prognostic relevance (NPM1 and CEBPA) in one assay, with low sample input requirement and with 100% sensibility compared with CMBT. In addition, this panel is able to find out alterations in these genes that are lost by CMBT. Moreover, other mutations with probable diagnostic or prognostic value and/or potential therapeutic targets are also studied and identified with high sensibility. Only a few changes are excluded of the covered regions. Therefore, IAAC panel is useful for routine diagnosis; however, detection of FLT3 internal tandem duplications is not possible, which limits its clinical utility. Disclosures No relevant conflicts of interest to declare.

Abstract 1469 The Wilms Tumor 1 (WT1) gene was first described as a tumour suppressor gene, but its accurate role in leukemia development has not been completely elucidated. Some authors support the role of WT1 as a prognostic marker in acute myeloid leukemia (AML) based on the assessment of its expression at the mRNA level. However, the prognostic value of the main isoforms of WT1 has been less studied. The aim of this study was to develop a specific quantitative assay to estimate the ratio of expression of the four major WT1 isoforms (A, 5-/KTS-; B, 5+/KTS-; C, 5-/KTS+; D, 5+/KTS+) and to evaluate their prognostic impact. WT1 expression was analyzed in bone marrow samples from 108 patients with AML at diagnosis (65 male/46 female, median age: 61 yr, range: 17 – 91). Likewise, peripheral blood samples of 20 healthy donors and 6 samples of cord blood CD34+ cell selection were analyzed as normal controls. We performed a new method to quantify the ratios of the four major isoforms of WT1. Briefly, to amplify all isoforms within a PCR reaction, specific WT1 primers flanking exon 4 to exon 10 were used in cDNA samples, followed by capillary electrophoresis with laser-induced fluorescence analysis on an ABIPRISM 310 DNA Analyzer (Applied Biosystems, Foster City, CA) and lastly analyzed with the Gene Mapper 4.2 software (Applied Biosystems). The amount of each isoform was calculated by the area under the curve. Subsequent comparisons of isoform ratios were made by standardized calculation of percentage. All values are given as the mean of duplicate PCRs. In parallel, RQ-PCR for total WT1 detection was performed as previously described by Barragan et al. (Haematologica 2004; 89: 926–933). GUS gene was used as housekeeping gene. Eighteen patients (17%) did not express WT1, while 90 patients (83%) overexpressed WT1 above background levels. The median value of each WT1 isoform was: 18% (range: 2 – 73) for A isoform; 16% (range: 7 – 63) for B isoform; 24% (range: 2 – 52) for C isoform; and 33% (range: 3 – 55) for D isoform. None of healthy donors had detectable WT1 levels in peripheral blood. All samples of CD34+ cells expressed the four isoforms of WT1: 21% (range: 2 – 26) for A isoform; 16% (range: 1 – 64) for B isoform; 24% (range: 1 – 47) for C isoform; and 36% (range: 25 – 44) for D isoform. These data reveal that, in our series, the most predominant isoform was +5/+KTS, both in AML and in cord blood CD34+ cell selection samples. There were no significant differences when comparing the proportion of each isoform between the cord blood CD34+ cell selection samples and the cohort of AML patients. There was not significant correlation between the overexpression of total WT1 with the ratio of each isoform, and we were unable to demonstrate that the overexpression of WT1 is due to a particular isoform overexpression. A significant lower event-free survival (EFS) was observed in those patients overexpressing total WT1, taking a cut-off value of 3000 WT1 copies/ GUS copies × 104 (75th percentile, P =.001). However, when the same cut-off as well as the median value for each one of the isoforms was used, we found no significant differences in EFS and in overall survival. To sum up, none of the isoforms were correlated with overexpression of total WT1 or survival. We were unable to find differences between the expression of each isoform of WT1 in CD34+ cells from normal cord blood and in AML patients. Further studies including larger controls need to be carried out. Disclosures: No relevant conflicts of interest to declare.

Background: Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML) characterized by specific morphological and clinical features. However, its relative incidence among patients with AML is not yet established. Objectives: To define the relative incidence of APL in a large series of patients diagnosed with AML in a single institution and to identify the clinical-biological features characterizing the APL patients within the target population of AML. Methods: From 1976 to 2007, 1205 patients (median age 56 years, range 15–98) were consecutively diagnosed with AML in our institution. One hundred patients (8%) were considered AML secondary to solid neoplasia or chemotherapy, while 143 (12%) were secondary to myelodysplastic or myeloprolypherative syndromes (MDS/MPCS). The APL diagnosis was achieved through cytological studies supported by cytogenetics, and since 1995 by molecular techniques. Inmunophenotype analysis, using flow cytometry or inmunohistochemistry, was available in 808 (67%) patients. Positivity was defined as more than 20% blasts expressing a specific antigen. Results: Overall, 170 patients (14%) were diagnosed of APL. Of them, 43 (25%) were classified as M3 variant. The relative incidence of APL within AML secondary to solid neoplasia or chemotherapy and de novo AML was 13% y 16%, respectively (p=ns). In contrast, the relative incidence of APL within AML secondary to MDS/MPCS was 0.7% (p75% (94% vs 27%, p70% (92% vs 49%, p Conclusion: In this large series of patients diagnosed with de novo or secondary AML, the relative incidence of APL was 14%, being exceptional the APL secondary to MDS/MPCS. To our knowledge, this is the larger hospital registry establishing the relative incidence of APL in a target AML population.