Integrated Genomic Analysis Identifies UBTF Tandem Duplications As a Subtype-Defining Lesion in Pediatric Acute Myeloid Leukemia

Children with acute myeloid leukemia (AML) have a dismal prognosis due to a high relapse rate; however, the molecular basis leading to relapsed pediatric AML has not yet been fully characterized. To define the spectrum of alterations common at relapse, we performed integrated profiling of 136 relapsed pediatric AML cases with RNA sequencing (RNA-seq), whole-genome sequencing, and target-capture sequencing. In addition to well-characterized fusion oncoproteins, such as those involving KMT2A (n=36, 26.5%) or NUP98 (n=18, 13.2%), we also identified somatic mutations in UBTF (upstream binding transcription factor) in 12 of 136 cases (8.8%) of this relapsed cohort. Somatic alterations of the UBTF gene, which encodes a nucleolar protein that is a component of the RNA Pol I pre-initiation complex to ribosomal DNA promoters, have rarely been observed in AML. In our cohort, all alterations can be described as heterozygous in-frame exon 13 tandem duplications (UBTF-TD), either at the 3' end of exon 13 of UBTF or of the entire exon 13 (Fig. A). As we noticed limited detection in our pipeline as a result of complex secondary indels alongside the duplications, we established a soft-clipped read-based screening method to detect UBTF-TD more efficiently. Applying the screening to RNA-seq data of 417 additional pediatric AMLs from previous studies and our clinical service, we identified 15 additional UBTF-TDs, many of which have not been previously reported. At the amino acid level, UBTF-TDs caused amino acid insertions of variable sizes (15-181 amino acids), duplicating a portion of high mobility group domain 4 (HMG4), which includes short leucine-rich sequences. UBTF-TD AMLs commonly occurred in early adolescence (median age: 12.6, range: 2.4-19.6), and 19 of the total 27 cases had either normal karyotype (n=12) or trisomy 8 (n=7). UBTF-TD is mutually exclusive from other recurrent fusion oncoproteins, such as NUP98 and KMT2A rearrangements (Fig. B), but frequently occurred with FLT3-ITD (44.4%) or WT1 mutations (40.7%). The median variant allele fraction (VAF) of the UBTF-TD was 48.0% (range: 9.7-66.7%). In four cases with data at multiple disease time points, the identical UBTF-TDs were present at high allele fractions at all time points, suggesting that UBTF-TD is a clonal alteration. tSNE analysis of the transcriptome dataset showed that UBTF-TD AMLs share a similar expression pattern with NPM1 mutant and NUP98-NSD1 AML subtypes, including NKX2-3 and HOXB cluster genes (Fig. C) . Altogether, these findings suggest that UBTF-TD is a unique subtype of pediatric AML. To address the impact of UBTF-TD expression in primary hematopoietic cells, we introduced UBTF-TD and UBTF wildtype expression vectors into cord blood CD34+ cells via lentiviral transduction. UBTF-TD expression promotes colony-forming activity and cell growth, yielding cells with a persistent blast-like morphology (Fig. D). Further, transcriptional profiling of these cells demonstrated expression of HOXB genes and NKX2-3, similar to UBTF-TD AMLs in patients, indicating that UBTF-TD is sufficient to induce the leukemic phenotype. To investigate the prevalence of UBTF-TDs in larger de novo AML cohorts, we applied the above UBTF-TD screening method to the available de novo AML cohorts of TCGA (n=151, adult), BeatAML (n=220, pediatric and adult), and AAML1031 (n=1035, pediatric). We identified UBTF-TDs in 4.3% (45/1035) of the pediatric AAML1031 cohort, while the alteration is less common (0.9%: 3/329, p=0.002) in the adult AML cohorts (Fig. E). In the AAML1031 cohort, UBTF-TDs remain mutually exclusive with known molecular subtypes of AML and commonly occur with FLT3-ITD (66.7%) and WT1 (40.0%) mutations and either normal karyotype or trisomy 8. The presence of UBTF-TDs in the AAML1031 cohort is associated with a poor outcome (Fig. F, median overall survival, 2.3 years) and MRD positivity; multivariate analysis revealed that UBTF-TD and WT1 are independent risk factors for overall survival within FLT3-ITD+ AMLs. In conclusion, we demonstrate UBTF-TD defines a unique subtype of AMLs that previously lacked a clear oncogenic driver. While independent of subtype-defining oncogenic fusions, UBTF-TD AMLs are associated with FLT3-ITD and WT1 mutations, adolescent age, and poor outcomes. These alterations have been under-recognized by standard bioinformatic approaches yet will be critical for future risk-stratification of pediatric AML. Figure 1 Figure 1. Disclosures Iacobucci: Amgen: Honoraria; Mission Bio: Honoraria. Miller: Johnson & Johnson's Janssen: Current Employment. Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company.

The Clinical Features and Prognosis of NPM1/FLT3-ITD/DNMT3A Triple-mutated Acute Myeloid Leukaemia

Purpose: Previous studies have shown that patients with NPM1+/FLT3-ITD+ acute myeloid leukaemia (AML) have a poor prognosis, especially those with a high FLT3-ITD allelic ratio. However, no studies have confirmed a clear prognostic impact of DNMT3A on NPM1+/FLT3-ITD+ AML patients. Methods: Our study included a total of 165 patients with newly diagnosed non-acute promyelocytic leukaemia (non-APL) AML at The First Hospital of Lanzhou University between January 2018 and June 2021. Further bioinformatics analysis was performed using the Gene Expression Omnibus (GEO) database. Results: We retrospectively studied 165 patients newly diagnosed non-APL AML and identified 11 (6.7%) patients with NPM1/FLT3-ITD/DNMT3A triple mutations. The patients with triple-mutated AML had advanced age, higher white blood cell (WBC) counts, de novo AML, normal karyotypes, and poor survival, and all were in the M4/M5 French-American-British (FAB) category. Notably, half of the patients with triple-mutated AML had mature monocyte characteristics that were difficult to distinguish from chronic myelomonocytic leukaemia (CMML). We validated the prognosis of patients with triple-mutated AML by further bioinformatics analysis and found that the GNG4 gene, one of the hub genes, was related to triple-mutated AML patients' survival. Conclusion: Our data demonstrate that DNMT3A gene mutation has adverse prognostic significance in NPM1+FLT3-ITD+comutation AML patients.

Heterogeneous Definitions of Secondary Acute Myeloid Leukemia (AML) Yield Distinct Outcomes in Response to First-Line Treatment with Hypomethylating Agents (HMA) and Venetoclax (Ven)

Background: The combination of HMA and venetoclax is now standard of care for patients with AML who are not candidates for intensive chemotherapy. Elderly patients are more likely to have secondary AML (sAML), although the presence of an antecedent hematologic malignancy is often not apparent by history. Lindsley et al (Blood, 2015) showed that a somatic mutation in SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2 is >95% specific for sAML and associated with worse outcomes. While outcomes with HMA/ven in patients meeting standard criteria for sAML have recently been reported (Pullarkat, ASCO 2021), we set out to conduct a real-world analysis of sAML patients receiving HMA/ven, including those with a secondary mutation profile (SMP) as described by Lindsley et al. We hypothesized that-when treated with HMA/ven-outcomes of patients with SMP may be most similar to those with de novo AML. Methods: Patients diagnosed with AML at Stanford Cancer Institute from 4/2017-3/2021 and treated with front-line HMA/ven were retrospectively reviewed. These included patients previously treated with HMA monotherapy for an antecedent hematologic malignancy and those who had previously received ≤ 3 cycles of HMA monotherapy for AML. Responses were classified per the modified International Working Group response criteria. Overall survival (OS) was assessed for all patients, and for patients who had a complete response (CR) or CR with incomplete hematologic recovery (CRi), duration of response (DoR) was also assessed. Statistical analyses were performed in R using the logrank test, with hazard ratios (HR) computed using the Cox proportional hazards model. For multivariate analyses, p-values for a specific variable were calculated using Cox proportional hazards regression. Results: 82 patients met criteria for inclusion; 78 had valid response assessments and 49 (62.8%) had achieved a CR or CRi at first response assessment. Median age was 72 years, with 3 patients younger than 60. 62 patients were male, median ECOG performance status (PS) was 1, median Charlson Comorbidity Index (CCI) was 6, median time to death or end of follow-up from the start of treatment was 366 days, and 58% of patients had adverse risk AML per ELN guidelines. Fig 1a demonstrates demographics for de novo, sAML (excluding SMP), and patients with SMP AML. 13 patients met criteria for AML-MRC, 23 patients had prior history of antecedent hematologic malignancy (18 with MDS or CMML, 5 with MDS/MPN overlap or MPN), 12 had tAML, and 20 patients possessed a SMP and did not meet criteria for the other three categories of sAML. 14 patients with de novo AML were characterized by the absence of any of the above factors. Patients with de novo AML were less likely to have adverse risk disease (29% vs. 64% in others) and had lower CCI scores (mean 5.1 vs. 6.2) but had no significant differences in age, gender, follow-up time, or PS. There was no statistically significant difference in rates of CR/CRi between the different subgroups or the different types of sAML; 69% of patients with de novo AML, 79% of SMP patients, and 57% of patients with other types of sAML achieved a CR or CRi. However, SMP patients had response durations and OS patterns similar to patients with de novo AML (Fig 1b and 1c), and when grouped with de novo patients, both DoR (HR = 3.5, p = 0.047, Fig 1d) and OS (HR = 2.1, p = 0.042, Fig 1e) were significantly longer than those of the sAML patients. Neither DoR nor OS were significantly longer when the SMP patients were grouped with sAML patients (respectively: HR = 3.3, p = 0.22, Fig 1f; HR = 1.5, p = 0.37, Fig 1g). In multivariate Cox proportional regression adjusting for age, ELN risk category, CCI, and PS, worse OS for sAML patients was maintained relative to the SMP and de novo patients (HR 2.9, p = 0.036), although the difference in DoR was no longer significant (HR 4.4, p= 0.10). Conclusions: Patients meeting standard definitions of sAML had worse outcomes than those with de novo AML when treated with HMA/ven in a retrospective, real-world analysis. Although a secondary mutation profile as described by Lindsley et al may be helpful in identifying patients with sAML, when treated with HMA/ven, patients with this profile have outcomes that align more closely with those of patients with de novo AML. Figure 1 Figure 1. Disclosures Mannis: Astex, Forty Seven Inc/Gilead, Glycomimetics, and Jazz Pharmaceuticals: Research Funding; AbbVie, Agios, Astellas Pharma, Bristol Myers Squibb, Genentech, MacroGenics, Pfizer, and Stemline: Consultancy.

Venetoclax Combined with Daunorubicin and Cytarabine (DAV) As Induction Therapy in De Novo Young Adult Acute Myeloid Leukemia

Background: Anthracycline and cytarabine ("3+7") have been the standard induction therapy for acute myeloid leukemia (AML) for almost 4 decades. Only 60%-70% patients can achieve complete remission (CR) with "3+7" induction treatment in de nove AML. The novel induction regimens with higher CR rate are urgent needed. Venetoclax, a b-cell lymphoma 2 (BCL-2) inhibitor combining with hypomethylation agents (HMA) or low dose cytarabine has showed a high response rate and safe in elder AML patients [Dinardo CD, N Engl J Med. 2020; Dinardo CD, Lancet Oncol 2018; Wei AH, J Clin Oncol 2019]. Recently, venetoclax combined with FLAG-IDA induction achieved 90% CR rate in newly diagnosed adult AML (Dinardo CD, J Clin Oncol. 2021). Whether venetoclax combined with standard 3+7 regimen (daunorubicin + cytarabine) as induction therapy can further improve the CR rate in adult AML patients need to be investigated in a well-designed trial. Objective: To evaluate the efficacy and safety of "3+7" (daunorubicin and cytarabine) combined with venetoclax induction regimen (DAV regimen) in young adult patients with de novo AML. Design, setting and participants: Single-arm, prospective clinical trial conducted in the First Affiliated Hospital, Zhejiang University College of Medicine, China. Eligible patients (18-60 years old) with de novo AML (exclude acute promyelocytic leukemia) were enrolled since December 25, 2020, with final follow-up in July 31,2021. Interventions: Patients were treated with daunorubicin 60mg/m 2 on days 1-3 (d1-3) and cytarabine 100 mg/m 2/d by continuous intravenous infusion daily on d1-7, combined with venetoclax (100mg d4, 200mg d5, 400mg d6-11). Main outcomes and measures: The primary endpoint was the percentage of patients who achieved CR/CR with incomplete count recovery (CRi) after once cycle of DAV regimen. Secondary endpoints included minimal residual disease (MRD), overall survival (OS), event-free survival (EFS) and adverse events. Results: Thirty-two patients were enrolled. Median age was 40 years old (range, 19-59), with poor-risk in 25% (8/32) of patients (European LeukemiaNet 2017 risk). Other characteristics of patients were listed in Table 1. The CR rate were 90.6% (29/32) (Table 1). Seven out eight (87.5%) patients with poor-risk achieved CR. Measurable residual disease-negative composite CR was attained in 65.5% (19 out 29) of total patients achieved CR, and 71.4% (5 out 7) of poor-risk patients achieved CR (Table 1). Common adverse events (>30%) included fatigue, nausea, bleeding, febrile neutropenia, infection, neutropenia, anemia and thrombocytopenia. The main grade ≥ 3 hematologic toxicities during induction were neutropenia (100%), anemia (100%) and thrombocytopenia (100%). The main grade ≥ 3 nonhematologic toxicities during induction were infection (81.3%), bleeding (28.1%) and mucositis (3.1%) (Table 1). No tumor lysis syndrome was observed. After a median follow-up of 118.5 days, no patient relapsed or died, and 24.1% (7/29) received allogeneic hematopoietic stem-cell transplantation in CR1. Conclusions: The novel combination of "3+7" (daunorubicin and cytarabine) with venetoclax (DAV regimen) was effective and well tolerated in young adult patients with de novo AML, with high CR rate and deep remission. Trial registration: The trial was registered in the Chinese Clinical Trial Register, number ChiCTR2000041509. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Real-World Study of CPX-351 Treatment Outcomes for Acute Myeloid Leukemia (AML) in England

Introduction: CPX-351 (US: Vyxeos ®; Europe: Vyxeos ® Liposomal) is a dual-drug liposomal encapsulation of daunorubicin and cytarabine in a synergistic 1:5 molar ratio. Since November 2018, the National Institute for Health and Care Excellence (NICE) has recommended its use for adults with newly diagnosed, therapy-related AML (t-AML) or AML with myelodysplasia-related changes (AML-MRC) due to either prior myelodysplastic syndrome (MDS)/chronic myelomonocytic leukemia (CMML) or de novo AML with myelodysplasia-related cytogenetic changes. The key aims of this study were to utilize the Cancer Analysis System (CAS) database available through the National Cancer Registration and Analysis Service (NCRAS) to describe the demographics and clinical characteristics of adults with AML in England who have received CPX-351, as well as to estimate overall survival (OS) and survival within stratifications of interest. Methods: The NCRAS systematically collects and curates population-level data about cancer diagnoses, treatments, and outcomes across England. Adults (aged ≥18 years) diagnosed with AML and treated with CPX-351 were included in this study. A diagnosis of t-AML or AML-MRC between January 2013 and March 2020 was determined either directly using International Classification of Diseases for Oncology, Third Edition (ICD-O-3) codes or indirectly using non-specific ICD-O-2, ICD-O-3, or ICD-10 AML codes in combination with either prior systemic anticancer therapy or radiotherapy (t-AML) or a prior diagnosis of MDS or CMML (AML-MRC; other AML-MRC subtypes could not be specifically identified and are included within the de novo AML subgroup). OS was measured from the date of diagnosis; a separate analysis of OS landmarked from the date of hematopoietic cell transplant (HCT) was also performed. Within this preliminary analysis, no OS adjustments have been made to account for any COVID-19-related deaths. Results: A total of 172 patients with AML who were treated with CPX-351 were identified: 37 (22%) had t-AML, 57 (33%) had AML-MRC, and 78 (45%) had de novo AML. At diagnosis, the mean (standard deviation) age was 62.8 years (10.1), with 49/172 (28%) patients aged <60 years; 66% of patients were male; 87% were white; and most had an Eastern Cooperative Oncology Group performance status of 0 or 1 (68%). Six (3%) patients had received azacitidine treatment for a prior malignancy. To date, 43/172 (25%) patients had undergone HCT overall, including 43/97 (44%) patients with ≥3 months of follow-up. The cut-off date for OS was December 31, 2020, giving a median (interquartile range) follow-up of 11.2 months (3.6, 16.9). Overall, 91 patients had died, with an estimated median OS (95% confidence interval [CI]) of 16.6 months (11.0, not estimable) and probability of survival (95% CI) at 1 and 2 years of 0.54 (0.47, 0.62) and 0.39 (0.30, 0.50), respectively (Figure 1). Early mortality rates were 7% at 30 days and 15% at 60 days. When OS was landmarked from the date of HCT, median OS was not reached, with a probability of survival (95% CI) at 1 year of 0.74 (0.62, 0.89; Figure 2). When stratified by age, estimated median OS (95% CI) was not reached for patients aged <60 years and 12.8 months (8.9, 17.6) for patients aged ≥60 years. In a treatment patterns analysis that evaluated second-line treatments after CPX-351, 68 patients died without salvage therapy and 64 were alive without receiving subsequent therapy by the end of the study period. The most common salvage treatments were fludarabine, cytarabine, idarubicin, and granulocyte-colony stimulating factor (FLAG-Ida; n = 15), daunorubicin plus cytarabine (DA)-based therapy (n = 6), and azacitidine alone (n = 7). Of the 43 patients who received an HCT, 6 (14%) underwent HCT following salvage therapy. Conclusions: This is the largest study to date examining the real-world outcomes for patients with AML who were treated with CPX-351. The estimated median OS of 16.6 months is consistent with reported real-world outcomes for CPX-351 in French and Italian studies. Median OS has not been reached in patients aged <60 years or when landmarked from the date of HCT. Once the CAS database has been updated, these analyses will be repeated to increase follow-up and patient numbers and to determine the impact of COVID-19 on OS following CPX-351 treatment. Figure 1 Figure 1. Disclosures Legg: Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Doubleday: IQVIA Inc., which was contracted by Jazz Pharmaceuticals for the conduct of this analysis: Current Employment. Reich: IQVIA Inc., which was contracted by Jazz Pharmaceuticals for the conduct of this analysis: Current Employment. Lambova: IQVIA Inc., which was contracted by Jazz Pharmaceuticals for the conduct of this analysis: Current Employment. Medalla: Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.

Crebbp Alterations Are Associated with a Poor Prognosis in De Novo AML

Introduction: The translocation, t(8;16)(p11;p13), results in the fusion between KAT6A and CREBBP and has been associated with a poor prognosis in both pediatric and adult acute myeloid leukemia (AML). This lesion has therefore been re-classified as high risk on the active Phase 3 Children's Oncology Group (COG) trial for de novo AML, AAML1831 (NCT04293562). Less is known about the prognostic significance of CREBBP sequence variants. Methods: CREBBP variant status was determined in patients with AML enrolled on 4 successive COG trials for de novo pediatric AML (NCT00003790, NCT00070174, NCT01407757, NCT01371981). Fusions involving CREBBP were prospectively obtained via conventional cytogenetics and retrospectively confirmed via RNAseq. Insertions and deletions (indels) leading to frameshift mutations and single nucleotide variants (SNVs) were retrospectively interrogated via next generation sequencing. Results: Of 2216 patients (age: 0-29.8 years), 55 (2.5%) patients had an alteration involving CREBBP. Sixteen (29%) of these were a fusion involving CREBBP (CREBBP/fus), with KAT6A being the most common translocation partner (n=15) and the remaining translocation involving ANK1. The remaining 39 patients (71%) had a CREBBP mutation (CREBBP/mut), including 19 with an indel (CREBBP/indel) leading to a frameshift mutation and 20 with a SNV (CREBBP/SNV). We compared clinical and biologic characteristics between the three cohorts. CREBBP/fus patients were significantly younger than CREBBP/indel and CREBBP/SNV patients (median ages of 2.6 vs. 7.8 vs. 11.9 years; p=0.027). There was a higher prevalence of t(8;21)/RUNX1-RUNX1T1 in CREBBP/indel patients compared to CREBBP/SNV patients (42.1% vs. 5%; p=0.008). In contrast, CREBBP/SNV patients were more likely to be associated with a normal karyotype (40% vs. 5.3%; p=0.02). There was a similar prevalence of co-occurring high-risk lesions in CREBBP/indel (n=5; CBFA2T3-GLIS2, KMT2A-AFF1, KMT2A-MLLT4, MLLT10-PICALM, NUP98-HOXA9) and CREBBP/SNV (n=7; DEK-NUP214, ETV6-FOXO1, FUS-ERG, NUP98-NSD1, ETV6-MNX1, FLT3-ITDx2) patients. There was otherwise no difference between presenting WBC count, FLT3-ITD, NPM1, CEBPA, remission rates or MRD status after Induction 1 therapy. Patients with any CREBBP alteration had a significantly worse 5-year event free survival (EFS) compared to patients without (25.9% vs. 45.2%; p=0.002) and this inferior EFS overlaps with contemporarily defined high-risk patients (Figure 1a). Evaluation of outcomes based on type of alteration demonstrated a similar 5-year EFS of 33.3% and 23.1% between CREBBP/fus and CREBBP/mut patients, respectively (Figure 1b; p=0.832). This poor EFS was maintained in the CREBBP/indel patients with a co-occurring t(8;21) (n=8, 5-year EFS 12.5%). When patients with co-occurring high-risk lesions were excluded from analysis, the remaining CREBBP/mut (n=27) patients maintained their poor EFS (29.6%). Despite their poor EFS, CREBBP/mut patients had an analogous overall survival (OS) to non-CREBBP patients (57.4% vs. 62.3%; p=0.499, Figure 1c), demonstrating that these patients could be successfully salvaged following relapse. In contrast, all patients with CREBBP/fus that relapsed subsequently died from their disease (OS 33.3%). Conclusions: In a large study of CREBBP alterations in pediatric patients with de novo AML, we show that these patients have a dismal EFS, regardless of alteration type. Further, despite enrichment of t(8;21), the favorable prognosis typically conferred by this alteration was abrogated by the co-occurrence of CREBBP/indel. Similarly, by excluding patients with co-occurring high-risk lesions from analysis, we show that these poor outcomes persist in a cohort of patients that would otherwise be considered low risk. Translocations between CREBBP and KAT6A in patients over 90 days of age are considered high risk on the active COG phase 3 trial. Given the inferior EFS and high salvage rates associated with other CREBBP alterations, intensification of upfront treatment, including hematopoietic stem cell transplant, should be considered in this population. The authors would like to acknowledge Astellas Pharma Global Development, Inc. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Efficacy and Safety of FLAG-Ida As Frontline Therapy in a Pediatric AML Population: A Single Institution Experience

Children with acute myeloid leukemia (AML) are commonly treated using a daunorubicin-cytarabine ±etoposide (DA/ADE) backbone as developed by the Medical Research Council (MRC) AML 12 trial and since adopted by the Children's Oncology Group (COG). This regimen is an anthracycline-intensive regimen, incorporating mitoxantrone in the consolidation phase, with a stated cumulative cardiotoxic exposure of ~444 mg/m 2 doxorubicin equivalents, though recent data suggests mitoxantrone is more cardiotoxic than previously considered (~10x more cardiotoxic than doxorubicin instead of 3x). The MRC AML15 trial randomized DA/ADE versus fludarabine-cytarabine-idarubicin (FLAG-Ida) and found superior event-free survival (EFS) from FLAG-Ida, with fewer cycles of intensive chemotherapy (4 vs 5), and less than half the cardiotoxic exposure, potentially reducing acute and long-term morbidity. Experience with FLAG-Ida as frontline therapy in children with de novo AML is limited. Following MRC 15, minimal residual disease (MRD) monitoring has become standard of care for risk stratification in AML, but MRD data for the FLAG-Ida regimen in children has not yet been reported. We collected data from 30 pediatric patients (age 0.3-20.0 years) with newly diagnosed de novo AML (no preceding myelodysplastic syndrome, secondary AML, or prior malignancy) and no underlying genetic disease (e.g., Trisomy 21, Fanconi Anemia, Kostmann Syndrome, Shwachman-Diamond Syndrome) treated at our institution with MRC 15-style FLAG-Ida between 2014 and 2021 (Table 1). Each case was characterized by cytogenetics, chromosomal microarray analysis (since 2015, in 29/30), and a proprietary molecular testing panel (since 2017, in 21/30); AML risk category was assigned at diagnosis using the contemporary COG classification (AAML1831). Following Induction I and II, MRD was measured in each patient by multiparameter flow cytometry using a 'difference from normal' approach (MRD+ defined by contemporary COG threshold, MRD≥0.05%). Patients with poor disease response (MRD+) or high-risk cytogenetics proceeded to hematopoietic stem cell transplantation (HSCT) in first remission. Patients routinely received antimicrobial prophylaxis with sulfamethoxazole-trimethoprim, an azole or echinocandin, and levofloxacin. The study was approved by the Institutional Review Board. Following Induction I, 28/30 (93%) patients were MRD negative, and 30/30 (100%) patients following Induction II. Only 1/20 (5%) patients without high-risk AML required HSCT for slow-responding disease; 1/30 (3%) patients were unable to proceed to consolidation (prolonged myelosuppression). No patient developed treatment-related mortality (TRM) during pre-HSCT chemotherapy (2 subsequent to HSCT). Cardiac evaluation in surviving patients was normal (LVSF>28%) post-therapy in 23/24 (96%). Median survival for patients alive at last follow-up was 2.3 years with overall 3-year EFS and overall survival (OS) of 73±9% and 80±8%, respectively (Figure 1A). Survival for patients in the low-risk subset is depicted (Figure 1B, 1C); all low-risk patients were alive at last follow-up. FLAG-Ida was well tolerated and effective in our pediatric AML population with no TRM, excellent early disease response by MRD, and encouraging albeit early EFS and OS. This compares favorably with the adult MRC AML 15 experience as well as with the COG DA/ADE regimen, particularly for MRD response (in AAML0531, ~30% patients were MRD+ following Induction I [Brodersen et al 2020]). These data support our continued use of FLAG-Ida in pediatric AML alone or in combination with targeted and/or molecular therapies. Further study of FLAG-Ida as frontline therapy in children with AML is warranted. Figure 1 Figure 1. Disclosures Gaynon: Takeda pharmaceuticals: Other: member of DSMC committee. Orgel: Jazz Pharmaceuticals: Consultancy.

Retrospective Analysis of the Outcomes of Patients with Relapsed/Refractory Acute Myeloid Leukemia Included in a Patient Named Program of Gemtuzumab Ozogamicin

Introduction: In 2010 the French Health Agency opened a compassionate patient named program of gemtuzumab ozogamicin (GO, Mylotarg®) in relapsed/refractory (R/R) patients with acute myeloid leukemia (AML). Of note, since 2012, it was recommended to use GO at the dose of 3 or 6 mg/m 2 in addition to chemotherapy. We conducted a retrospective trial (NCT03287128) to evaluate the efficacy and the safety of GO-based regimen in R/R adult AML patients. Patients and methods: We retrospectively collected data of patients older than 18 years treated with GO-based regimen for AML in first relapse or for refractory AML, defined by failure after a prior standard intensive chemotherapy, in 18 French centers between December 15, 2011 and November 10, 2016. The primary objective was to assess the response to GO-based regimen. Patients were considered in response if reaching complete remission (CR), CR without platelet recovery (CRp) or CR with incomplete hematological recovery (CRi). Secondary objectives were the cumulative incidence of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and the safety of the use of GO-based regimen. Results: Three hundred and thirty-five adult patients with R/R AML were included. Median age was 58 years (20 to 80 years). At diagnosis, cytogenetics was favorable in 50 (17%) patients, intermediate in 173 (59%) and adverse in 60 (20%). ELN distribution was favorable: 35%, intermediate: 42% and unfavorable: 23%. NPM1 mutation was present in 29% of patients and FLT3 mutation in 23%. Most patients had de novo AML (84%). Two hundred and thirty-eight patients (79%) were in first relapse and 65 (21%) had a refractory AML. The time between first diagnosis of AML and treatment with GO-based regimen was 4 to 16 months (median 9.4 months). Most patients (88%) received GO in combination with various intensive chemotherapy scheme including "7+3" with anthracycline/cytarabine (n=39 patients), intermediate and high-dose cytarabine (n=68), cytarabine in continuous intravenous infusion (n=78), mitoxantrone/cytarabine (n=49) and fludarabine/cytarabine and/or amsacrine and/or etoposide chemotherapy (n=35). Median follow-up time was 11 months. Among the 305 patients, 191 responded to GO-based regimen: 110 (36%) were in CR, 62 (20%) were in CRp and 19 (6%) in CRi for an overall response rate (CR+CRp+CRi, ORR) of 63%. In multivariate analysis, response was associated with age <50 years, de novo AML and relapse status. Among the 191 responders, 110 received additional courses of chemotherapy, 69 with GO. Main reason to not receive additional course (with or without GO) was allo-HSCT project. In the whole population, median overall survival (OS) after day 1 of treatment with GO was 11.2 months. In the population of responders, median OS after response was 20.4 months. In multivariate analysis, longer survival was associated with age < 50 years, de novo AML and favorable ELN group. Cumulative incidence of relapse at 24 months after response was 46%. One hundred and forty-seven patients received allo-HSCT, including 122 responders after GO-based regimen and 25 patients in treatment failure. Cumulative incidence of allo-HSCT at 18 months was 48%. Four-year OS was 48% in transplanted patients versus 19% in non-transplanted patients (Figure 1). Regarding safety of GO-based regimen, early deaths occurred within <30 days after the first dose of GO in 14 patients, and within <60 days in 35 patients. Myelosuppression was observed in all patients. Mean duration of thrombocytopenia <100 G/L was 35 days in responders. Bleeding grade 3 or more was observed in 22 patients (7%). Infection grade 3 or more was observed in 112 patients (30%). Sinusoidal obstruction syndrome (SOS) after GO treatment was reported in 6 patients, resolving in 4 of them. Four cases of fatal SOS were reported after allo-HSCT. Toxic deaths, i.e., not related to worsening leukemia, were reported in 20 patients after the first course of chemotherapy, 3 after additional courses and 33 after allo-HSCT. Conclusion. Our study is the first to report efficacy data in the real-world setting of R/R AML adult patients treated with GO-based regimen. In our cohort of 305 patients, response rate was 63% and GO-based regimen appears as a valuable bridge-to-transplant option. Safety analysis showed toxicities consistent with the known safety profile of GO and chemotherapy. Figure 1 Figure 1. Disclosures Lambert: ASTELLAS: Consultancy; CELGENE/BMS: Consultancy. Pautas: PFIZER: Consultancy; ABBVIE: Consultancy. Raffoux: ASTELLAS: Consultancy; PFIZER: Consultancy; ABBVIE: Consultancy; CELGENE/BMS: Consultancy. Legrand: Servier: Consultancy. Gastaud: PFIZER: Consultancy; CELGENE/BMS: Consultancy; ABBVIE: Consultancy; GSK: Consultancy. Pigneux: Amgen: Consultancy; Sunesis: Consultancy, Research Funding; BMS Celgene: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Mathilde: SERVIER: Consultancy; ABBVIE: Consultancy. Dombret: Amgen: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; Jazz Pharmaceuticals: Honoraria, Research Funding; Novartis: Research Funding; Pfizer: Honoraria, Research Funding; Servier: Research Funding; Abbvie: Honoraria; BMS-Celgene: Honoraria; Daiichi Sankyo: Honoraria. Rousselot: Incyte, Pfizer: Consultancy, Research Funding. Castaigne: PFIZER: Consultancy.

Efficacy, Toxicity and Cost of Venetoclax-Based Combinations for the Treatment of Acute Myeloid Leukemia: Real-World Evidence from a Canadian Academic Center

Introduction: Venetoclax (ven) in combination with azacitidine (aza) or low-dose cytarabine (LDAC) has demonstrated efficacy for the first-line treatment of acute myeloid leukemia (AML) patients who are deemed unfit for intensive induction chemotherapy. The efficacy of ven-based treatment for relapsed or refractory (r/r) AML has not been prospectively evaluated. We have used off-label ven-based combinations to treat r/r AML patients and de novo AML in otherwise fit patients to avoid prolonged hospitalization. The objective of this study was to review the efficacy, toxicity and medication costs associated with ven-based treatments for AML in a Canadian university hospital. Methods: After local IRB approval, we conducted a retrospective chart review of all patients who received ven-based treatments, outside of a clinical trial, for AML at Hopital Maisonneuve-Rosemont. Supportive care included tumor lysis syndrome (TLS) prophylaxis, antiviral, antifungal and antibacterial prophylaxis. Results: 40 patients received 41 ven-based treatments between November 2017 and July 2021. Most patients had r/r AML (n=25, including 17 in first relapse after allogeneic hematopoietic cell transplantation), while 16 patients had de novo AML (10 deemed fit for intensive chemotherapy). Median age was 62 years old. Median duration of ven-based treatment was 4 cycles and median follow-up was 140 days after ven initiation. Ven-aza was used for 33 patients. ven-LDAC was used for the first 7 patients. One patient received ven-gilteritinib. Posaconazole was the main antifungal agent used, with ven dose reduced to 70 mg daily (25 patients). The complete remission (CR) and CR with incomplete hematological recovery (CRi) rate was 46%, higher for de novo vs r/r AML (56% vs 40%). Most patients achieved blast clearance with treatment: CR+CRi+morphologic leukemia-free state (MLFS) rate 63% (75% de novo and 56% r/r). Median overall survival was 258 days (376 days for ven-aza treated patients). All 8 patients (de novo n=5, r/r n=3) with NPM1 or IDH1/2 mutations achieved blast clearance (CR+CRi 87%), while 22 patients with adverse-risk AML as defined by 2017 ELN risk stratification had a lower yet respectable response rate (CR+CRi 36%). Ven-LDAC had no activity in advanced disease, with no response for 6 r/r patients who all had adverse-risk AML, and is no longer used in our institution. The majority of patients were able to receive treatment on an outpatient basis after a brief hospital stay during ven ramp-up. Five patients remained hospitalized for the entire first cycle. Four patients died from infectious causes during the first cycle (2 unfit patients with first line ven-aza, 2 r/r patients with ven-LDAC). Two cases of suspected TLS requiring treatment delays occurred, with no clinical TLS. Venetoclax dose reductions for hematological toxicity were frequently required (51% for all patients, 73% of patients that achieved a response to ven). The average medication cost per cycle of ven-aza was 4 394 $ CAN and was 6 765 $ CAN per cycle for ven-LDAC. Conclusion: In this retrospective real-life review, ven-based treatment produced response rates in line with published prospective evidence among de novo AML patients, and a very clinically meaningful response rate for r/r AML, where therapeutic options are limited and outcomes with conventional chemotherapy dismal. The presence of NPM1 or IDH1/2 mutations was predictive of high response rates. Dose reductions were often required for cytopenias but non-hematological toxicities were limited. Although associated with significant cost, ven-aza represents a safe and effective treatment option for r/r AML which can successfully be delivered in an ambulatory setting. Figure 1 Figure 1. Disclosures Bouchard: Otsuka: Consultancy; Pfizer: Consultancy; Jazz Pharmaceuticals: Consultancy. Bambace: AbbVie: Consultancy; Excelthera: Research Funding; Kiadis: Research Funding. Bernard: Kiadis: Research Funding; Excelthera: Research Funding; BMS: Consultancy; Taiho: Consultancy. Hebert: BMS-Celgene: Research Funding. Bergeron: Jazz Pharmaceuticals: Consultancy; Amgen: Consultancy; Servier: Consultancy; BMS: Consultancy; AbbVie: Consultancy; Pfizer: Consultancy. OffLabel Disclosure: Venetoclax to treat relapsed or refractory acute myeloid leukemia