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The relapse rate for children with acute myeloid leukemia is nearly 40% despite aggressive chemotherapy and often stem cell transplant. We sought to understand how environment-induced signaling responses are associated with clinical response to treatment. We previously reported that patients whose AML cells showed low G-CSF-induced STAT3 activation had inferior event-free survival compared to patients with stronger STAT3 responses. Here, we expanded the paradigm to evaluate multiple signaling parameters induced by a more physiological stimulus. We measured STAT3, STAT5 and ERK1/2 responses to G-CSF and to stromal cell-conditioned medium for 113 patients enrolled on COG trials AAML03P1 and AAML0531. Low inducible STAT3 activity was independently associated with inferior event-free survival in multivariate analyses. For inducible STAT5 activity, those with the lowest and highest responses had inferior event-free survival, compared to patients with intermediate STAT5 responses. Using existing RNA-sequencing data, we compared gene expression profiles for patients with low inducible STAT3/5 activation with those for patients with higher inducible STAT3/5 signaling. Genes encoding hematopoietic factors and mitochondrial respiratory chain subunits were overexpressed in the low STAT3/5 response groups, implicating inflammatory and metabolic pathways as potential mechanisms of chemotherapy resistance. We validated the prognostic relevance of individual genes from the low STAT3/5 response signature in a large independent cohort of pediatric AML patients. These findings provide novel insights into interactions between AML cells and the microenvironment that are associated with treatment failure and could be targeted for therapeutic interventions. Supplementary Information The online version contains supplementary material available at 10.1007/s12094-021-02621-w.

Purpose: The current standard-of-care for front-line therapy for acute myeloid leukaemia (AML) results in short-term and long-term toxicity, but still approximately 40% of children relapse. Therefore, there is a major need to accelerate the evaluation of innovative medicines, yet drug development continues to be adult-focused. Furthermore, the large number of competing agents in rare patient populations requires coordinated prioritisation, within the global regulatory framework and cooperative group initiatives. Methods: The fourth multi-stakeholder Paediatric Strategy Forum focused on AML in children and adolescents. Results: CD123 is a high priority target and the paediatric development should be accelerated as a proof-of-concept. Efforts must be coordinated, however, as there are a limited number of studies that can be delivered. Studies of FLT3 inhibitors in agreed paediatric investigation plans present challenges to be completed because they require enrolment of a larger number of patients than actually exist. A consensus was developed by industry and academia of optimised clinical trials. For AML with rare mutations that are more frequent in adolescents than in children, adult trials should enrol adolescents and when scientifically justified, efficacy data could be extrapolated. Methodologies and definitions of minimal residual disease need to be standardised internationally and validated as a new response criterion. Industry supported, academic sponsored platform trials could identify products to be further developed. The Leukaemia and Lymphoma Society PedAL/EUpAL initiative has the potential to be a major advance in the field. Conclusion: These initiatives continue to accelerate drug development for children with AML and ultimately improve clinical outcomes.

PURPOSE Effective regimens are needed for children with relapsed acute myeloid leukemia (AML). AAML1421 is a phase I/II study of CPX-351, a liposomal preparation of daunorubicin and cytarabine. AAML1421 sought to determine the recommended phase II dose (RP2D) of CPX-351 and the response rate after up to 2 cycles of therapy. PATIENTS AND METHODS Children > 1 and ≤ 21 years of age with relapsed/refractory AML were eligible for dose finding; those in first relapse were eligible for the efficacy phase. Dose-limiting toxicity (DLT) assessment occurred during cycle 1. Two cycles of therapy were offered (cycle 1: CPX-351; cycle 2: FLAG [fludarabine 30 mg/m2/dose on days 1-5; cytarabine 2,000 mg/m2/dose on days 1-5; and granulocyte-colony stimulating factor 5 µg/kg/dose, days 1-5 and day 15 through absolute neutrophil count > 500/µL]). Response was assessed after each cycle. RESULTS Thirty-eight patients enrolled: 6 in the dose-finding phase and 32 in the efficacy phase. During dose finding, 1/6 patients experienced a DLT (grade 3 decrease in ejection fraction). The RP2D was 135 units/m2 on days 1, 3, and 5. Toxicities of grade ≥ 3 during cycle 1 included fever/neutropenia (45%), infection (47%), and rash (40%). There was no toxic mortality. Best responses included 20 complete response (CR; 54%), 5 CR with partial recovery of platelet count (CRp; 14%), and 5 CR with incomplete blood count recovery (14%). Twenty-one of 25 with CR/CRp had no detectable residual disease (RD; 84%) by flow cytometry. Hematopoietic stem cell transplantation (HSCT) was used as consolidation in 29/30 responders (96.7%); 20/25 (80%) had no RD before HSCT. CONCLUSION The RP2D of CPX-351 is 135 units/m2/dose on days 1, 3, and 5. Toxicity was manageable, and protocol therapy was effective. Response rates are superior to prior published North American cooperative group clinical trials for children with AML in first relapse.

Patients with inv(16)/CBFB-MYH11 AML are considered favorable risk, however, nearly one-third relapse despite intensive therapy. Despite efforts to define risk groups within this favorable risk cohort, CBFB-MYH11 AML patients continue to be treated as a uniform cohort. Through transcriptome sequencing of 186 patients with inv(16) AML, we demonstrate that fusion junction breakpoints (exon 5-exon 33 versus other) are highly associated with outcome. The presence of exon 17 KIT mutations provides additional prognostic significance. Additionally, we provide insights into the transcriptional landscapes that differentiate these distinct CBFB-MYH11 AML subtypes. Children's Oncology Group trials include CCG-2961 (registered at www.clinicaltrials.gov as NCT00002798), AAML03P1 (NCT00070174), AAML0531 (NCT00372593), and AAML1031 (NCT01371981).

Despite a remarkable increase in the genomic profiling of cancer, integration of genomic discoveries into clinical care has lagged behind. We report the feasibility of rapid identification of targetable mutations in 153 pediatric patients with relapsed/refractory or high-risk leukemias enrolled on a prospective clinical trial conducted by the LEAP Consortium. Eighteen percent of patients had a high confidence Tier 1 or 2 recommendation. We describe clinical responses in the 14% of patients with relapsed/refractory leukemia who received the matched targeted therapy. Further, in order to inform future targeted therapy for patients, we validated variants of uncertain significance, performed ex vivo drug-sensitivity testing in patient leukemia samples, and identified new combinations of targeted therapies in cell lines and patient-derived xenograft models. These data and our collaborative approach should inform the design of future precision medicine trials. Significance: Patients with relapsed/refractory leukemias face limited treatment options. Systematic integration of precision medicine efforts can inform therapy. We report the feasibility of identifying targetable mutations in children with leukemia and describe correlative biology studies validating therapeutic hypotheses and novel mutations. See related commentary by Bornhauser and Bourquin, p. 1322. This article is highlighted in the In This Issue feature, p. 1307

BACKGROUND: Novel therapies are urgently needed for pediatric patients with relapsed acute myeloid leukemia. METHODS: To determine if the histone deacetylase inhibitor panobinostat could be safely given in combination with intensive chemotherapy, we performed a phase I trial in which 17 pediatric patients with relapsed or refractory acute myeloid leukemia received panobinostat (10 mg/m(2), 15 mg/m(2), or 20 mg/m(2)) prior to and in combination with fludarabine and cytarabine. RESULTS: All dose levels were tolerated, with no dose-limiting toxicities observed at any dose level. Pharmacokinetic studies demonstrated that exposure to panobinostat was proportional to the dose given, with no associations between pharmacokinetic parameters and age, weight, or body surface area. Among the nine patients who had sufficient (>2%) circulating blasts on which histone acetylation studies could be performed, seven demonstrated at least 1.5-fold increases in acetylation. Although no patients had a decrease in circulating blasts after single-agent panobinostat, eight of the 17 patients (47%) achieved complete remission, including five of six patients treated at dose level 3. Among the 8 complete responders, 6 (75%) attained negative minimal residual disease status. CONCLUSIONS: Panobinostat can be safely administered with chemotherapy and results in increased blast histone acetylation, suggesting that it should be further studied in acute myeloid leukemia. This trial is registered with ClinicalTrials.gov (NCT02676323).

Background Advances in risk stratification have improved the 3-year disease-free survival (DFS) and overall survival (OS) of low-risk pediatric acute myeloid leukemia (LR-AML) to approximately 70 % and 85 % respectively. LR-AML is defined by favorable cytogenetic/molecular features and/or optimal early response to therapy. However, cumulative anthracycline exposure in contemporary Children’s Oncology Group (COG) regimens approach a doxorubicin equivalent exposure of 540 mg/m2; with rates of non-infection related left ventricular systolic dysfunction (LVSD) approaching 15 %. This is a major cause of toxicity in these patients and precludes the further use of anthracyclines in the relapsed setting; therefore, strategies that reduce cardiotoxicity while maintaining excellent outcomes are needed. Patients and methods Twenty-seven pediatric patients with LR-AML were treated with an anthracycline-reduced approach (Aflac-AML regimen) between 2011 and 2016. Patients received four courses of therapy including three high-dose cytarabine containing courses and a cumulative doxorubicin equivalent exposure of 390 mg/m2, a 28 % reduction in anthracycline dosing compared to current COG regimens. Results The 3-year DFS and OS was 70.0 % and 85.5 % respectively, from end of Induction I (first chemotherapy cycle) with a median follow-up of 3.2 years. These survival outcomes are comparable to current LR-AML regimens. Only two patients developed non-infection related LVSD during therapy and more importantly, none developed LVSD after completion of therapy. Conclusion These findings suggest that LR-AML outcomes can be maintained using a reduced anthracycline chemotherapy regimen, resulting in lower cardiac toxicity. This new chemotherapy backbone is now being tested prospectively (NCT04326439) to further validate its use in pediatric LR-AML.

Purpose: Treatment failure from drug resistance is the primary reason for relapse in acute lymphoblastic leukemia (ALL). Improving outcomes by targeting mechanisms of drug resistance is a potential solution. Patients and Methods: We report results investigating the epigenetic modulators decitabine and vorinostat with vincristine, dexamethasone, mitoxantrone, and PEG-asparaginase for pediatric patients with relapsed or refractory B-cell ALL (B-ALL). Twenty-three patients, median age 12 years (range, 1–21) were treated in this trial. Results: The most common grade 3–4 toxicities included hypokalemia (65%), anemia (78%), febrile neutropenia (57%), hypophosphatemia (43%), leukopenia (61%), hyperbilirubinemia (39%), thrombocytopenia (87%), neutropenia (91%), and hypocalcemia (39%). Three subjects experienced dose-limiting toxicities, which included cholestasis, steatosis, and hyperbilirubinemia (n = 1); seizure, somnolence, and delirium (n = 1); and pneumonitis, hypoxia, and hyperbilirubinemia (n = 1). Infectious complications were common with 17 of 23 (74%) subjects experiencing grade ≥3 infections including invasive fungal infections in 35% (8/23). Nine subjects (39%) achieved a complete response (CR + CR without platelet recovery + CR without neutrophil recovery) and five had stable disease (22%). Nine (39%) subjects were not evaluable for response, primarily due to treatment-related toxicities. Correlative pharmacodynamics demonstrated potent in vivo modulation of epigenetic marks, and modulation of biologic pathways associated with functional antileukemic effects. Conclusions: Despite encouraging response rates and pharmacodynamics, the combination of decitabine and vorinostat on this intensive chemotherapy backbone was determined not feasible in B-ALL due to the high incidence of significant infectious toxicities. This study is registered at http://www.clinicaltrials.gov as NCT01483690.

PURPOSE To determine whether dexrazoxane provides effective cardioprotection during frontline treatment of pediatric acute myeloid leukemia (AML) without increasing relapse risk or noncardiac toxicities of the chemotherapy regimens. PATIENTS AND METHODS This was a multicenter study of all pediatric patients with AML without high allelic ratio FLT3/ITD treated in the Children’s Oncology Group trial AAML1031 between 2011 and 2016. Median follow-up was 3.5 years. Dexrazoxane was administered at the discretion of treating physicians and documented at each course. Ejection fraction (EF) and shortening fraction (SF) were recorded after each course and at regular intervals in follow-up. Per protocol, anthracyclines were to be withheld if there was evidence of left ventricular systolic dysfunction (LVSD) defined as SF < 28% or EF < 55%. Occurrence of LVSD, trends in EF and SF, 5-year event-free survival (EFS) and overall survival (OS), and treatment-related mortality (TRM) were compared by dexrazoxane exposure. RESULTS A total of 1,014 patients were included in the analyses; 96 were exposed to dexrazoxane at every anthracycline course, and 918 were never exposed. Distributions of sex, age, race, presenting WBC count, risk group, treatment arm, and compliance with cardiac monitoring were similar for dexrazoxane-exposed and -unexposed patients. Dexrazoxane-exposed patients had significantly smaller EF and SF declines than unexposed patients across courses and a lower risk for LVSD (26.5% v 42.2%; hazard ratio, 0.55; 95% CI, 0.36 to 0.86; P = .009). Dexrazoxane-exposed patients had similar 5-year EFS (49.0% v 45.1%; P = .534) and OS (65.0% v 61.9%; P = .613) to those unexposed; however, there was a suggestion of lower TRM with dexrazoxane (5.7% v 12.7%; P = .068). CONCLUSION Dexrazoxane preserved cardiac function without compromising EFS and OS or increasing noncardiac toxicities. Dexrazoxane should be considered for cardioprotection during frontline treatment of pediatric AML.

TO THE EDITOR: Despite improvements in treating childhood leukemia, relapses remain the primary cause of death.[1][1][⇓][2][⇓][3]-[4][4] On relapse, leukemic cells are more resistant to chemotherapy. Thus, there is an urgent need to incorporate new strategies to treat childhood leukemia.

Background Relapse free survival of pediatric AML remains only 60%. Current standard myelosuppressive therapy has been maximized, so novel therapies with minimal toxicities are needed to improve outcomes. Previously, we found atovaquone (AQ), an FDA-approved drug that treats pneumocystis jiroveci pneumonia (PJP), reduces AML burden - by suppressing oxidative phosphorylation (OXPHOS) - in xenograft mice. Clinically achievable concentrations of AQ for anti-PJP are 40-80µM, but the anti-leukemia effects are observed as low as 10µM (Stevens et al, Bld Adv, 2019). This makes AQ an ideal drug to incorporate into AML treatment. AQ is a daily administered oral medication, and plasma levels depend on patient compliance, absorption, and entero-hepatic recirculation, which can be compromised due to the patient population and adverse events (AE) of chemotherapy. Here we investigated the feasibility of incorporating AQ into standard pediatric AML treatment. Methods Patients with de novo AML were enrolled at two children's hospitals in the USA. Daily administration of AQ at established PJP prophylaxis dosing was combined with standard chemotherapy for AML, based on the Medical Research Council (MRC) backbone of cytarabine 100mg/m2 q12h x 10 days, and daunorubicin 50mg/m2/dose on days 1, 3, and 5. As it was unclear if our AQ dosing would provide adequate PJP prophylaxis, it was left to provider discretion to give additional PJP protection. AQ compliance, AEs (per NCI CTCAE v5), parent/caregiver ease of administration score (scale: 1-5, 1=very difficult, 5=very easy to administer) and peripheral blood/bone marrow pharmacokinetics (PK) were collected during Induction 1. Real time AQ plasma concentration results were not provided. To address feasibility of achieving adequate levels, all gastrointestinal (GI) AEs ≥ grade 2 were collected, in addition to grade 4 or greater AEs. Patients who took at least 85% of planned doses and missed less than 2 consecutive doses of AQ were eligible for analyses. Correlative biology studies assessed AQ induced apoptosis at 30uM, effects on OXPHOS and relevant signaling activities. Patient derived xenografts (PDX) were established and treated with AQ. This trial is registered with ClinicalTrials.gov (NCT03568994). Results A total of 26 pediatric AML patients enrolled (ages 8 months - 19.7 years, mean 10.7 years); 24 patients were evaluable for this study. Two patients had Grade ≥ 3 GI toxicities that prohibited enteral administration so they were excluded from AQ PK and ease of administration analyses. We found that 14/24 (58%) patients achieved plasma levels above the target anti-leukemia concentration (10µM) by day 11. At the end of induction, 19/24 (75%) patients achieved plasma levels above 10µM, but only 7/24 (29%) patients achieved adequate levels for PJP prophylaxis (40µM). Only 1 patient achieved levels above 40µM throughout the trial [FIG A]. Mean ease of administration score was 3.8. For the youngest patients (x ≤ 2.6years), the average score was 3.4 which was not significantly different from older patients (ANOVA, p > 0.05) [FIG B]. Ease of administration scores showed no association with plasma levels (Pearson's correlation, p > 0.05). Finally, correlative biology studies in patient samples demonstrated robust AQ-induced apoptosis, OXPHOS suppression, and prolonged survival in a PDX model receiving AQ [FIG C]. Conclusion Our data demonstrate the feasibility of combining AQ with traditional chemotherapy for pediatric AML. Patients of all ages were able to tolerate AQ and no AEs were attributable to AQ administration. The target anti-leukemic concentration of AQ in the plasma (> 10uM) was frequently achieved, but concentrations of > 40uM at standard dosing were rare. Low plasma levels of AQ did not correlate with the presence of GI related AEs or weight loss, so plasma levels should be monitored to ensure sufficient PJP prophylaxis. Our correlative biology results support suppression of OXPHOS as the primary mechanism of action by which AQ exerts its anti-leukemia effect, and AQ may be an active anti-leukemia agent for pediatric AML patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Detection of residual disease is a critical component of modern, risk-adapted therapy for Acute Myeloid Leukemia (AML). However, the genetic and phenotypic diversity of AML has made the development of a universal assay for disease assessment particularly challenging. While purely mutation-based tests promise high sensitivity, they are not broadly applicable given molecular heterogeneity and complex clonal evolution. Single-cell approaches, such as multiparameter flow cytometry (MFC), are more broadly applicable and increasingly accepted as the standard in clinical care. However, the limited number of leukemia-specific cell-surface markers and high numbers of shared markers between malignant myeloid blasts and healthy progenitors make MFC data extremely challenging to interpret. Motivated to develop a broadly applicable assay that can provide a more confident assessment of residual disease, we developed a platform using droplet-partitioned single-cell RNA sequencing accompanied by a computational pipeline specifically tailored to quantify residual disease after allogeneic HCT (alloHCT). With bone marrow samples from an 11-year-old patient with suspected post-alloHCT relapse of AML, we interrogated three methods of sample processing, 1) RBC lysis, 2) Ficoll-centrifugation, and 3) Ficoll-centrifugation combined with CD34+ immunomagnetic selection. The samples were further split to separately capture the 3' or 5' end of polyadenylated transcripts. The six resulting libraries were sequenced using standard short-read sequencing, and reads were demultiplexed and counted using common workflows. Data from the samples were combined, and sub-populations were visualized using UMAP (see Figure). This study demonstrated the feasibility of real-time single-cell sequencing for clinical utility. It is possible to process, capture, and sequence a patient's sample in approximately three working days (A). By integrating our data with single-cell expression profiles from an atlas of healthy human bone marrow, we were able to identify cells with gene-expression programs distinct from those of normal hematopoietic cells (B). With these integrated data, we could clearly identify populations of cells that embed away from healthy atlas cells (yellow circle, B), defining a different than normal single-cell profile. This "malignant" profile also included several genes whose expression is usually restricted to healthy hematopoietic progenitors (Panel C), suggesting these cells had a severely dysregulated transcriptome. As this patient was post-alloHCT, we interrogated the abundance of single-nucleotide-polymorphisms (SNPs) in the sequence data. We quantified these SNPs in single cells to distinguish each cell as either of donor or recipient origin using a method we have previously validated for genotyping RNA sequence in single cells. We clearly demonstrate that those cells identified as "different than normal" have a distinct SNP profile suggesting they are of recipient origin. Further analysis revealed that this malignant population was highly enriched for a population of cells expressing a previously described set of "AML-restricted genes" (Huang, B. et al., ASH 2021). (Panel E). Finally, from the Ficoll-processed sample, we quantified a level of 9.8% residual disease (243 malignant cells from a total of 2487). Notably, the number of abnormal myeloid progenitors determined by MFC was 2.0% which increased to 13% on a subsequent marrow sample drawn one week later. Incidentally, we observed only minimal differences across the two single-cell sequencing chemistries (3' vs. 5'). Taken together, our data strongly argue that droplet-based, single-cell RNA sequencing is a feasible and powerful tool for the ascertainment of residual disease in AML. Given the robust nature of the platform and the ability to incorporate SNP integration into the analytic pipeline, it allows confident detection of residual disease in the post-alloHCT setting. By combining genomic quantification of transcripts with the power of SNP-based genotyping all at the level of the single cells, we believe this technology can substantially improve our diagnosis of post-alloHCT AML relapse. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

BackgroundDecitabine is a deoxycytidine nucleoside derivative inhibitor of DNA-methyltransferases, which has been studied extensively and is approved for myelodysplastic syndrome in adults but with less focus in children. Accordingly, we conducted a phase 1 multicenter, randomized, open-label study to evaluate decitabine pre-treatment before standard induction therapy in children with newly diagnosed AML to assess safety and tolerability and explore a number of biologic endpoints.ResultsTwenty-four patients were fully assessable for all study objectives per protocol (10 in Arm A, 14 in Arm B). All patients experienced neutropenia and thrombocytopenia. The most common grade 3 and 4 non-hematologic adverse events observed were gastrointestinal toxicities and hypophosphatemia. Plasma decitabine PK were similar to previously reported adult data. Overall CR/CRi was similar for the two arms. MRD negativity at end-induction was 85% in Arm A versus 67% in Arm B patients. DNA methylation measured in peripheral blood over the course of treatment tracked with blast clearance and matched marrow aspirates at day 0 and day 21. Unlike end-point marrow analyses, promoter methylation in blood identified an apparent reversal of response in the lone treatment failure, one week prior to the patient’s marrow aspirate confirming non-response. Decitabine-induced effects of end-induction marrows in Arm A were reflected by changes in DNA methylation and gene expression comparison with matched paired marrow diagnostic aspirates.ConclusionsThis first-in-pediatrics trial demonstrates that decitabine prior to standard combination chemotherapy is feasible and well tolerated in children with newly diagnosed AML. Pre-treatment with decitabine may represent a newer therapeutic option for pediatric AML, especially as it appears to induce important epigenetic alterations. The novel biological correlates studied in this trial offer a clinically relevant window into disease progression and remission. Additional studies are needed to definitively assess whether decitabine can enhance durability responses in children with AML. This trial was registered at www.clinicaltrials.gov as NCT01177540.

Introduction: Pediatric acute myeloid leukemia (AML) is a molecularly heterogeneous group of diseases lacking therapy options that would improve overall survival. Venetoclax (VEN) is an oral inhibitor for selective targeting of B-cell lymphoma 2 (BCL2), which is highly expressed in most patients (pts) with AML and has demonstrated promising efficacy in pediatric pts with AML when combined with chemotherapy (CTx) (Karol SE, et al. Lancet Oncol. 2020;21:551-560). Here, we present safety, efficacy, and preliminary genomic results from pediatric pts with relapsed/refractory (R/R) AML receiving VEN + CTx. Methods: This phase 1 open-label, 2-part, multicenter study (NCT03236857) enrolled pts Results: As of June 2020, 36 pts with R/R AML were enrolled and received VEN monoTx (n=3) and VEN + CTx (n=33: VEN + decitabine [VEN-DEC, n=5], azacitidine [VEN-AZA, n=19], or low- [VEN-LDAC, n=1] or high-dose cytarabine [VEN-HDAC, n=8]) (Table). The primary reason for VEN discontinuation was progressive disease (n=19); median duration of VEN therapy was 3.1 months (range 0.2-9.3). All pts experienced adverse events (AEs); 3 pts (n=1 VEN-DEC, n=2 VEN-AZA) had fatal AEs considered unrelated to VEN. The most common grade 3/4 AEs were febrile neutropenia (58%) and hypokalemia (33%). The overall objective response rate (ORR) was 25% (9/36); median duration of response was 0.8 month (95% CI, 0.5, 3.6). The best ORR was seen with VEN-HDAC (4/8, 50%) with 1 complete response (CR), 1 CR without platelet recovery, 1 CR with incomplete marrow recovery (CRi), and 1 partial response (PR); 2 pts achieved minimal residual disease negativity and 2 pts proceeded to transplant. The ORR with VEN-AZA was 26% (5/19), with 3 CR/CRi and 2 PR. No responses were seen with VEN monoTx or VEN + other CTx. The genomic landscape of biomarker-evaluable pts was highly heterogeneous (Figure A). Mutations of genes involved in epigenetic modification (MYH11, IDH2, ASXL1, SETBP1, TET2, and NSD1) and transcription regulation (GATA1, WT1, RUNX1, and CEBPA) were the most common, in 58% and 48% of pts, respectively. Analysis of the recurring mutations found in ≥2 pts revealed that responses to VEN-AZA were seen in pts with IDH2 (1/4), MYH11 (2/6), RUNX1 (1/3), or FLT3 (1/3) mutations, and responses to VEN-HDAC were seen in pts with JAK2 (1/4) or GATA1 (1/3) mutations. Pts with WT1 (3/6) and PTPN11 (3/4) mutations responded to both regimens. Pts with TP53 (n=2) or ETV6 (n=3) mutations and PML-RARA (n=2) or KMT2A rearrangements (n=8) did not respond to any treatment. Gene expression profiling revealed that BCL-xL expression was significantly higher compared with BCL2;MCL1 levels were the highest (Figure B). There was no association between expression of these genes and response. Mutations were seen in BCL2 and MCL1 (n=1 each), but not in BCL-xL. Conclusions: VEN + CTx was well tolerated in pediatric pts with R/R AML, with no unexpected toxicities. Preliminary efficacy was seen in pts receiving VEN-AZA or VEN-HDAC: ORR 26% and 50%, respectively. VEN + CTx resulted in responses in pts harboring mutations across a range of functional classifications; however, some alterations may confer resistance. Due to the limited number of pts harboring each mutation and the overall heterogeneity of the genomic landscape, these findings need to be evaluated in a larger population, and warrant further investigation. Disclosures Karol: AbbVie Inc.: Other: Unrelated to this study, St. Jude has received a charitable contribution from AbbVie, Inc. The charitable contribution is not being used for clinical or research activities, including any activities related to this study. . Bittencourt:Jazz Pharmaceuticals: Consultancy, Other: travel, accommodations, expenses; Novartis: Consultancy. Morgenstern:EUSA Pharma: Consultancy, Other: travel support; Bayer: Consultancy; Clarity Pharmaceuticals: Consultancy; BMS: Other: Institutional Research Funding; Boehringer Ingelheim: Consultancy; Roche: Consultancy. Macy:Merck: Other: Institutional Research Funding; Pfizer: Other: Institutional Research Funding; Bayer: Other: Institutional Research Funding; AbbVie Inc.: Other: Institutional Research Funding; Roche: Other: Institutional Research Funding; Johnson & Johnson: Current equity holder in publicly-traded company. Khaw:Amgen: Other: Institutional Research Funding; Bristol-Myers Squibb: Other: Institutional Research Funding; AbbVie Inc.: Other: Institutional Research Funding; Novartis: Other: travel, accommodation, expenses; Walter and Eliza Hall Institute of Medical Research.: Patents & Royalties: Recipient of a share in royalty payments . Cooper:Celgene: Other: Spouse was an employee of Celgene (through August 2019). Ramsingh:Genentech: Current Employment, Current equity holder in publicly-traded company; Roche: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Tong:AbbVie, Inc.: Current Employment, Other: may hold stock or other options. Unnebrink:AbbVie: Current Employment, Other: may hold stock or other options. Vishwamitra:AbbVie, Inc.: Current Employment, Other: may hold stock or other options. Dunbar:Abbvie: Current Employment, Current equity holder in publicly-traded company. Prine:AbbVie: Current Employment, Other: may hold stock or other options. Palenski:AbbVie: Current Employment, Other: may hold stock or other options. Place:Novartis: Consultancy, Other: Institutional Research Funding; AbbVie: Consultancy. OffLabel Disclosure: Venetoclax is a BCL-2 inhibitor that is FDA approved for some indications. Venetoclax for treatment of pediatric AML is not an approved indication.

Background: Somatic mutations in the IDH genes are common in acute myeloid leukemia (AML). Mutations occur at active site arginine residues in IDH1 (R132) and IDH2 (R140, R172). IDH inhibitors, ivosidenib (IDH1) and enasidenib (IDH2), have shown improved clinical outcomes in patients with relapsed/refractory IDH-mutant AML and are approved for use in this setting, while investigations in combination with chemotherapy are underway in de novo AML. The prognostic significance of IDH mutations remains controversial. We hypothesize that refining our understanding of IDH-mutated AML will contribute to risk-adapted treatment strategies, including optimal use of IDH-targeted agents. The objective of our study was to identify characteristics that affect outcome in de novo IDH-mutated AML across the age spectrum utilizing a large cohort of patients enrolled on several pediatric and adult trials. Methods: The total cohort (N=3588) included patients age Results: The prevalence of IDH mutations among the entire cohort was 8.6% (N=276). Analysis according to mutation type demonstrated that IDH2 mutations comprised 57% (N=158) and IDH1 mutations 43% (N=118). The prevalence of IDH mutations was strongly correlated with increased age (Fig 1A); according to the age-defined cohorts was 4.0% (N=82) in younger, 15.2% (N=126) in intermediate, and 20.3% (N=65) in older patients (p Conclusion: Analysis of this large patient cohort provides the most comprehensive description of IDH mutations in AML across the age spectrum. We confirm age-associated prevalence of IDH mutations and frequent co-occurrence with NPM1 mutation in all ages and in further combination with DNMT3A mutation in intermediate-aged adults. We definitively demonstrate that IDH mutation status is not an independent prognostic determinant of outcome in any age group. Co-occurrence of NPM1 and IDH mutations favorably impacts outcome in patients < 60 years of age with AML, particularly in the absence of DNMT3A mutation. Our data support that IDH inhibitors may be of particular interest in older adults and in patients Disclosures Othus: Marck: Consultancy; Daiichi Sankyo: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; Glycomimetics: Membership on an entity's Board of Directors or advisory committees. Radich:Jazz: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Amgen: Consultancy; Bristol-Myers Squibb: Consultancy. Abdel-Wahab:Merck: Consultancy; Janssen: Consultancy; Envisagenics Inc.: Current equity holder in private company; H3 Biomedicine Inc.: Consultancy, Research Funding. Tallman:UpToDate: Patents & Royalties; ADC Therapeutics: Research Funding; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; Glycomimetics: Research Funding; Rafael: Research Funding; Amgen: Research Funding; Bioline rx: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; KAHR: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Cellerant: Research Funding; Orsenix: Research Funding. Atallah:Jazz: Consultancy; Genentech: Consultancy; Abbvie: Consultancy; Takeda: Consultancy, Research Funding; Pfizer: Consultancy; Novartis Pharmaceutical Corporation: Consultancy. Luger:Daiichi-Sankyo: Honoraria; Pfizer: Honoraria; Bristol-Myers Squibb: Honoraria; Acceleron: Honoraria; Agios: Honoraria; Loxo Oncology: Honoraria; Onconova: Research Funding; Kura: Research Funding; Biosight: Research Funding; Ariad: Research Funding; Hoffman La Roche: Research Funding. Levine:Novartis: Consultancy; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy; Astellas: Consultancy; Janssen: Consultancy; Prelude Therapeutics: Research Funding; Amgen: Honoraria; Gilead: Honoraria; Lilly: Consultancy, Honoraria; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding. Cooper:Celgene: Other: Spouse was an employee of Celgene (through August 2019).

Introduction: Venetoclax (VEN) is an orally administered selective inhibitor of the B-cell lymphoma 2 (BCL2) antiapoptotic protein. VEN combined with chemotherapy (CTx) has demonstrated encouraging responses in a variety of hematologic malignancies. Here, we present safety, efficacy, and preliminary genomics results from our phase 1 study of VEN administered with CTx in pediatric patients (pts) with relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL). Methods: This open-label, phase 1, 2-part study enrolled pts Results: As of June 2020, 25 pts with ALL were enrolled and received VEN + CTx; CTx regimens included dexamethasone and/or vincristine and/or peg-asparaginase (D/V/P, n=16) or cytarabine and/or etoposide and/or peg-asparaginase (C, n=9). Pts had a median of 3.5 (range 1.0-9.0) prior therapies; the median duration of VEN therapy was 2.1 mo (range 0.4-4.6). Most common reasons for VEN discontinuation were progressive disease (28%; D/V/P: n=1; C: n=6) or continuation to transplant (28%; D/V/P: n=6; C: n=1). Eight (32%; D/V/P: n=4; C: n=4) pts had fatal adverse events (AEs), all unrelated to VEN. Most common grade 3/4 AEs were febrile neutropenia (52%) and anemia (44%); 1 pt (D/V/P)completed ramp up, but reported laboratory tumor lysis syndrome with CTx combination. A best objective response rate (ORR) of 56% was seen with VEN + D/V/P: 4 complete responses (CR), 4 CR with incomplete marrow recovery (CRi), and 1 CR without platelet recovery (CRp). ORR of 11% (CRi: n=1) was seen with VEN + C (Table). In pts who achieved CR/CRi/CRp, MRD Genetic analysis of evaluable pts (VEN + D/V/P: n=12; VEN + C: n=6) demonstrated a highly heterogeneous genomic landscape (Figure A) including a range of somatic mutations and structural variants, comprising translocations, rearrangements, and chromosomal abnormalities. Analysis of mutations found in ≥2 pts revealed that responses were seen in VEN + D/V/P-treated pts with CREBBP, KMT2A, KMT2D, RB1, PTPN11, and PDGFRB mutations. Of the pts who had CREBBP mutations, 2/3 obtained CR. Pts with mutations in RB1, PTPN11, and PDGFRB (n=2 each) also achieved CR or CRi. Of the pts with KMT2A or KMT2D mutations detected by our sequencing platform, 1/2 and 2/4 achieved CR, respectively. Furthermore, of the 6 pts with KMT2A translocations identified locally, 3 obtained CR with VEN + D/V/P. Baseline gene expression profiling revealed that BCL-xL and MCL1 levels were slightly higher in comparison to BCL-2 (Figure B). There was no significant correlation between these gene expression levels and response. No mutations were detected in BCL2, BCL-xL, or MCL1. Conclusions: VEN + CTx was well tolerated, with no unexpected toxicities in pediatric pts with R/R ALL. Promising preliminary efficacy was observed in these refractory pts receiving VEN + D/V/P (ORR 56%). Our genomic profiling suggests that responses with VEN + CTx occur in pediatric ALL with a variety of mutations, including those with KMT2A rearrangements. However, due to the limited sample size and the overall heterogeneity, further investigation in a larger pt cohort is warranted to determine which mutations confer resistance or sensitivity to VEN. Disclosures Place: Novartis: Consultancy, Other: Institutional Research Funding; AbbVie: Consultancy. Karol:AbbVie: Other: Unrelated to this study, St. Jude has received a charitable contribution from AbbVie, Inc. The charitable contribution is not being used for clinical or research activities, including any activities related to this study. . Gambart:Jazz Pharmaceuticals: Other: travel, accommodations, expenses; Eisai: Other: travel, accommodations, expenses. Cooper:Celgene: Other: Spouse was an employee of Celgene (through August 2019). Fraser:Novartis, Amgen: Consultancy. Cario:Jazz Pharmaceuticals: Consultancy, Other: travel support; Novartis: Consultancy, Other: travel support. O'Brien:Celgene: Research Funding; Jazz: Research Funding; Amgen: Research Funding; BMS: Research Funding; AbbVie: Research Funding; Pfizer: Honoraria, Research Funding; Incyte: Research Funding. Gerber:AbbVie, Loxo, Novartis: Other: Institutional Research Funding. Reinhardt:Behring: Other: Institutional Research Funding; Novo Nordisk: Other: Institutional Research Funding; Biotest: Other: Institutional Research Funding; Roche: Consultancy, Other: Institutional Research Funding; bluebird bio: Consultancy; Celgene: Consultancy, Other: Institutional Research Funding; Jazz: Consultancy, Other: Institutional Research Funding; Novartis: Consultancy, Other: Institutional Research Funding; AbbVie: Consultancy. Ramsingh:Genentech: Current Employment, Current equity holder in publicly-traded company; Roche: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Tong:AbbVie, Inc.: Current Employment, Other: may hold stock or other options. Unnebrink:AbbVie: Current Employment, Other: may hold stock or other options. Vishwamitra:AbbVie, Inc.: Current Employment, Other: may hold stock or other options. Dunbar:Abbvie: Current Employment, Current equity holder in publicly-traded company. Prine:AbbVie: Current Employment, Other: may hold stock or other options. Palenski:AbbVie: Current Employment, Other: may hold stock or other options. Loh:Pfizer: Other: Institutional Research Funding; Medisix Therapeutics: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Venetoclax is a BCL-2 inhibitor that is FDA approved for some indications. Venetoclax for treatment of pediatric ALL is not an approved indication.

Introduction: Venetoclax is a selective BCL2 inhibitor approved for the treatment of CLL and AML in adults and is currently being evaluated in several hematologic and solid malignancies. While plasma pharmacokinetics (PK) of venetoclax is well documented, data regarding the accumulation of venetoclax into the central nervous system (CNS) are limited. Venetoclax has a molecular weight of 868.44 which was hypothesized to limit its passage through the tight junctions of the blood brain barrier (BBB). Moreover, venetoclax is a substrate of the P- glycoprotein (P-gp) and breast cancer resistance protein (BCRP) efflux transporters, expressed by endothelial cells at the BBB, which presents an extra hurdle for penetration into the CNS. In this analysis we characterized the passage of venetoclax into the CNS using PK samples collected during a phase 1 study (NCT03236857) of pediatric patients with relapsed and refractory acute leukemia receiving venetoclax in combination with chemotherapy. Methods: PK samples from cerebrospinal fluid (CSF) were collected at screening and if a lumbar puncture was performed as standard of care during treatment. Plasma PK samples were collected throughout the study. CSF and plasma concentrations of venetoclax were determined using liquid-liquid extraction followed by liquid chromatography (LC) with tandem mass spectrometric detection (MS/MS). The lower limit of quantitation for venetoclax was 2.14 ng/mL in plasma and 0.1ng/mL in CSF. Results: There was a total of 66 samples from 33 patients with relapsed or refractory AML or ALL. The venetoclax concentration in CSF ranged between Conclusion: To our knowledge this is the first study reporting venetoclax CSF pharmacokinetics in the AML and ALL setting. The lower disposition observed in humans is contrary to our expectation, given the significantly higher expression of P-gp in mice BBB compared to humans and suggests that other factors are involved in venetoclax disposition to the CSF. The ability of venetoclax to cross the blood brain barrier may explain the reported activity of venetoclax in treatment of hematologic malignancies with CNS involvement1,2. References: Reda G, Cassin R, Dovrtelova G, et al. Venetoclax penetrates in cerebrospinal fluid and may be effective in chronic lymphocytic leukemia with central nervous system involvement. Haematologica. 2019;104(5):e222-e223. doi:10.3324/haematol.2018.213157 Beziat G, Gauthier M, Protin C, et al. Venetoclax with high-dose methotrexate and rituximab seem effective and well-tolerated in the treatment of central nervous system involvement of chronic lymphocytic leukemia: A case report. Clin Case Rep. 2020;8(2):269-273. Published 2020 Jan 10. doi:10.1002/ccr3.2580 Disclosures Salem: AbbVie Inc.: Current Employment, Other: may hold stock or other options. Badawi:AbbVie Inc.: Current Employment, Other: may hold stock or other options. Place:Novartis: Consultancy, Other: Institutional Research Funding; AbbVie: Consultancy. Palenski:AbbVie: Current Employment, Other: may hold stock or other options. Arrendale:AbbVie Inc.: Current Employment, Other: may hold stock or other options. Kim:AbbVie, Inc.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Other: may hold stock or other options. Cooper:Celgene: Other: Spouse was an employee of Celgene (through August 2019). Menon:AbbVie Inc.: Current Employment, Other: may hold stock or other options.

Background Cytogenetic and molecular aberrations are important prognostic factors in pediatric acute myeloid leukemia (AML). NUP98 translocations with more than 30 different partner genes have been identified in pediatric AML. The 2 most common fusions, NUP98-NSD1 and NUP98-KDM5A, have been shown to have distinct characteristics and are both associated with adverse outcomes. Although NUP98 fusions with less common fusion partners have been identified, the biological and clinical implications of these variants are unknown. Methods To determine the biological and clinical implications of the less common "other" NUP98 translocations (NUP98-X), we evaluated the clinical characteristics and transcriptome and genome sequencing data from 2396 children and young adults with AML within 4 consecutive Children's Cancer Group (CCG) and Children's Oncology Group (COG) trials CCG-2961, AAML03P1, AAML0531 and AAML1031. All NUP98-X translocations were confirmed by RNA sequencing. Results Of the 2396 patients screened, 164 patients (6.8%) had a NUP98 translocation. We identified 20 patients with a NUP98-X fusion (0.83%) and compared them with those with NUP98-NSD1 (n=110, 4.5%), NUP98-KDM5A(n=34, 1.4%), and a reference cohort without NUP98 translocations (n=2232). Translocation partners identified in the NUP98-X group were HOXA9 (n=4), HOXD13 (n=3), PHF15 (n=2), PHF23 (n=2), and single cases of BPTF, BRWD3, DDX10, HMGB3, HOXA13, KAT7, PRRX1, SET and TOP1. Besides the distinct characteristics of NUP98-NSD1 and NUP98-KDM5A, the NUP98-X group showed high inter-patient variance in clinical characteristics compared to our reference cohort. NUP98-X patients showed a clear bimodal age distribution with half of the patients being in the older age category and a similar number in the category We evaluated the impact of NUP98-X translocations in response to the initial induction therapy. The morphological complete remission (CR) rate after course 1 was 65% in the NUP98-X cohort versus 76% in the reference cohort (p=0.266). NUP98-NSD1 patients had an inferior CR rate (36%, p Conclusion NUP98-X translocated pediatric AML patients represent a rare cohort with a high variability in both translocation partners and other clinical characteristics. Despite this heterogeneity, the OS of these patients is comparable to high-risk pediatric AML patients, which justifies a high-risk stratification of these patients and emphasizes the need for developing new treatment strategies. Further research within large patient cohorts is needed to investigate the biologic and clinical characteristics of these rare translocations and potential differences between the different NUP98-X fusion partners. Disclosures Kaspers: AbbVie: Ended employment in the past 24 months; Helsinn Healthcare: Ended employment in the past 24 months; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Janssen R&D: Ended employment in the past 24 months. Cooper:Celgene: Other: Spouse was an employee of Celgene (through August 2019).

Acute myeloid leukemia (AML) remains a therapeutic challenge with high mortality rates despite intensive and myeloablative therapies. Structural and sequence alterations have been linked to outcomes in pediatric AML and have been used for risk-based therapy allocation with modest success. Given the vast heterogeneity of AML, conventional cytogenetic and mutational (cytomolecular) biomarkers have not yielded a robust prognostic model: nearly one-third of pediatric patients deemed "low risk" relapse and, inversely, approximately one-third of those in "high risk" categories have favorable outcomes. AML studies in adults previously identified a leukemia stem cell score (LSC17) that was highly prognostic across five independent cohorts comprised of adult patients with diverse AML subtypes (n = 908). We reasoned that incorporating a similar scoring system in pediatric AML would lead to improved prognostic risk models. To assess for the effects of LSC17 on pediatric AML, we leveraged transcriptome sequencing data from bone marrow aspirates and peripheral blood collected from 1,503 children, adolescents, and young adults with AML at the time of diagnosis. Patients were enrolled on one of three upfront phase III Children's Oncology Group trials spanning the past three decades: CCG-2961, AAML0531, and AAML1031. In aggregate, patients with a high LSC17 score had an event free survival (EFS) of 36.9 ± 3.5% at 5 years from diagnosis compared to 55.3 ± 3.7% for those with low LSC17 scores (p < 0.0001). (Figure 1A) LSC17 scores were also associated with adverse overall survival (OS): 51.9 ± 3.9% versus 73.8 ± 3.5% (p < 0.0001) (data not shown). Intriguingly, we found that LSC17 scores significantly cluster within fusion groups and that median LSC17 scores closely correlate with survival based on fusion status (Figure 1B). Thus, when the impact of LSC17 scores was evaluated in the context of established cytomolecular risk groups, LSC17 scores were no longer predictive of outcome (Figure 1C). We then asked whether LSC gene expression data could be utilized to generate a more robust risk classification schema in the context of disease defining structural variants. Importantly, AMLs diagnosed in children, adolescents, and young adults are associated with frequent driver gene fusion alterations that also play an important role in risk stratification and transcriptional landscape (Figure 1D). We went on to confirm that AML fusion groups occupy distinct transcriptional stages of hematopoietic stem cell and myeloid progenitor maturation based on gene set enrichment analysis (GSEA) using normal hematopoiesis transcriptome experiments as their reference (data not shown). To develop more predictive biomarkers related to stemness, we used the 54 original LSC genes identified by Ng S, et al. and performed linear regression based on a least absolute shrinkage and selection operator (LASSO) algorithm to fit a Cox regression model for patients within each fusion group. The study population was divided into discovery (n = 752) and validation (n = 752) cohorts using stratified randomization based on fusion status (RUNX1-RUNX1T1, CBFB-MYH11, KMT2A, NUP98, CBFA2T3-GLIS2, and Other/None). In the discovery cohort, we identified distinct LSC signatures that best distinguished outcome cohorts in patients with conventional high/standard risk disease (KMT2A, NUP98, and Other/None fusions) (Figure 1E). For patients deemed favorable risk (RUNX1-RUNX1T1 and CBFB-MYH11 or core binding factor/CBF), LSC signatures were not reliably predictive based on "leave one out" cross validation. Therefore, we performed multivariable analysis incorporating clinical, mutational, and transcriptional signatures to determine the factors that best discriminated outcomes with CBF AML, and found GLIS2-like transcriptional signatures were most predictive. These cytomolecular and LSC (CM-LSC) biomarkers were then combined to build a robust risk determination model that was then validated in an independent cohort (Figure 1F). This study demonstrates that a 54 LSC gene expression panel can enhance the predictive power of conventional cytomolecular markers and can more effectively partition patients into risk groups. Figure 1 Disclosures Cooper: Celgene: Other: Spouse was an employee of Celgene (through August 2019).

10018 Background: Outcomes for children with relapsed/refractory (R/R) AML and MDS are poor and new therapies are needed. Pevonedistat is an inhibitor of the NEDD-8 activating enzyme, a key regulator of the ubiquitin proteasome system that is responsible for protein turnover, cell growth and survival. In preclinical models, pevonedistat was synergistic with cytarabine (AraC) and azacitidine (aza). The combination of pevonedistat + aza in adults with AML demonstrated improved responses compared to either single agent. We evaluated the feasibility, toxicity and pharmacokinetics (PK) of pevonedistat in combination with aza, fludarabine, AraC (Aza-FLA) in children with R/R AML and MDS. Methods: Pevonedistat 20 mg/m2, IV days 1, 3, 5, the recommended adult dose, was administered in combination with aza (75 mg/m2, days 1-5), fludarabine (30 mg/m2, days 6-10), and AraC (2000 mg/m2, days 6-10). Intrathecal AraC was administered at the start of therapy and additional doses given to patients with CNS leukemia. If < 33% of the initial 6 enrolled patients experienced dose limiting toxicity (DLT) during cycle 1 the regimen would be considered tolerable and 6 additional patients could enroll to further assess tolerability and PK. Pevonedistat PK was determined during cycle 1 following doses 1 and 5. Response was evaluated after cycle 1. Results: A total of 12 patients were enrolled, median age was 13 years (range 1-21). All patients received prior chemotherapy, median number of prior regimens was 2 (range 1-5) and 3 (25%) patients had prior hematopoietic stem cell transplant. Diagnoses were AML NOS (n = 10, 83%), acute monocytic leukemia (n = 1), and therapy related AML (n = 1). One of the initial 6 patients had DLTs (hypertension, GGT elevation, and proteinuria); pevonedistat 20 mg/m2 + Aza-FLA was considered tolerable. Six additional patients were enrolled, two had DLTs (weight loss, hypoxia). Overall, 3/12 (25%) of patients experienced DLTs. As expected, using the intensive Aza-FLA backbone, myelosuppression, electrolyte abnormalities, and hepatic transaminase elevation were common. Day 1 PK parameters (n = 12, mean±SD) were: Cmax= 223±91 ng/mL, AUC0-24h= 892±216 ng/hr/mL, T1/2=4.3±1.2 hours, CL = 23.2±6.9 L/hr/m2. PK parameters were similar following doses 1 and 5, for patients < 12 (n = 6) and ≥ 12 (n = 6) years, and to adult PK profiles. Ten patients were evaluable for response. The overall response rate was 30% (95% CI: 7,75) with 3 patients achieving a CR with incomplete hematologic recovery (CRi). Conclusions: Pevonedistat 20 mg/m2 combinedwith Aza-FLA was tolerable in children with R/R AML. The toxicity of the regimen was similar to other intensive AML regimens. PK parameters were similar among the two age groups and were comparable to adults. Within the confines of a phase I study, there was limited anti-leukemic activity of the combination of pevonedistat +Aza-FLA in R/R AML. Clinical trial information: NCT03813147.

Purpose Blinatumomab is a bispecific T-cell engager antibody construct targeting CD19 on B-cell lymphoblasts. We evaluated the safety, pharmacokinetics, recommended dosage, and potential for efficacy of blinatumomab in children with relapsed/refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Methods This open-label study enrolled children < 18 years old with relapsed/refractory BCP-ALL in a phase I dosage-escalation part and a phase II part, using 6-week treatment cycles. Primary end points were maximum-tolerated dosage (phase I) and complete remission rate within the first two cycles (phase II). Results We treated 49 patients in phase I and 44 patients in phase II. Four patients had dose-limiting toxicities in cycle 1 (phase I). Three experienced grade 4 cytokine-release syndrome (one attributed to grade 5 cardiac failure); one had fatal respiratory failure. The maximum-tolerated dosage was 15 µg/m2/d. Blinatumomab pharmacokinetics was linear across dosage levels and consistent among age groups. On the basis of the phase I data, the recommended blinatumomab dosage for children with relapsed/refractory ALL was 5 µg/m2/d for the first 7 days, followed by 15 µg/m2/d thereafter. Among the 70 patients who received the recommended dosage, 27 (39%; 95% CI, 27% to 51%) achieved complete remission within the first two cycles, 14 (52%) of whom achieved complete minimal residual disease response. The most frequent grade ≥ 3 adverse events were anemia (36%), thrombocytopenia (21%), and hypokalemia (17%). Three patients (4%) and one patient (1%) had cytokine-release syndrome of grade 3 and 4, respectively. Two patients (3%) interrupted treatment after grade 2 seizures. Conclusion This trial, which to the best of our knowledge was the first such trial in pediatrics, demonstrated antileukemic activity of single-agent blinatumomab with complete minimal residual disease response in children with relapsed/refractory BCP-ALL. Blinatumomab may represent an important new treatment option in this setting, requiring further investigation in curative indications.

Immunotherapy with the adoptive transfer of T cells redirected with CD19-specific chimeric antigen receptors (CARs) for B-lineage acute lymphoblastic leukemia (ALL) can salvage >80% of patients having relapsed/refractory disease. The therapeutic index of this emerging modality is attenuated by the occurrence of immunologic toxicity syndromes that occur upon CAR T-cell engraftment. Here, we report on the low incidence of severe cytokine release syndrome (CRS) in a subject treated with a CAR T-cell product composed of a defined ratio CD4:CD8 T-cell composition with a 4-1BB:zeta CAR targeting CD19 who also recieved early intervention treatment. We report that early intervention with tocilizumab and/or corticosteroids may reduce the frequency at which subjects transition from mild CRS to severe CRS. Although early intervention doubled the numbers of subjects dosed with tocilizumab and/or corticosteroids, there was no apparent detrimental effect on minimal residual disease-negative complete remission rates or subsequent persistence of functional CAR T cells compared with subjects who did not receive intervention. Moreover, early intervention therapy did not increase the proportion of subjects who experience neurotoxicity or place subjects at risk for infectious sequelae. These data support the contention that early intervention with tocilizumab and/or corticosteroids in subjects with early signs of CRS is without negative impact on the antitumor potency of CD19 CAR T cells. This intervention serves to enhance the therapeutic index in relapsed/refractory patients and provides the rationale to apply CAR T-cell therapy more broadly in ALL therapy. This trial was registered at www.clinicaltrials.gov as #NCT020284.

CPX-351 (Vyxeos®; daunorubicin and cytarabine liposome for injection), a dual-drug liposomal encapsulation of daunorubicin and cytarabine at a 1:5 synergistic ratio, is approved by the US FDA and EMA for the treatment of adults with newly diagnosed therapy-related AML or AML with myelodysplasia-related changes. Recently, CPX-351 was used in pediatric patients (pts) with relapsed AML (JCO 2020, ASH 2019, ASH 2018). A population pharmacokinetic (PK) analysis of plasma concentrations of cytarabine and daunorubicin following IV administration of CPX-351 was performed to assess sources of variability in PK and to determine if age-based dose adjustments may be warranted, particularly in pediatric pts. The PK population consisted of 250 pts with advanced hematologic malignancies from 7 studies and included 46 (18%) pediatric pts (1-17 y) and 204 (82%) adults (≥18 y). The population included 148 (59%) males, mainly of white origin (82%). Nonlinear mixed-effect modeling was performed using NONMEM®. Model evaluation and selection were assessed using a standard model discrimination process that included statistical criteria (eg, objective function value) and pertinent graphical representations of goodness-of-fit. Separate PK models were developed for cytarabine and daunorubicin; intrinsic and extrinsic factors were evaluated as covariates. The intrinsic factors included body weight, body mass index, age, sex, race, white blood cell count, and markers of renal function (creatinine clearance, serum creatinine) and hepatic function (bilirubin, aspartate and alanine aminotransferases, alkaline phosphatase). The extrinsic factors included study type (adult vs pediatric/young adult) and formulation (frozen vs lyophilized). Based on previous population PK models developed for adults, the PK models for cytarabine and daunorubicin used 2-compartment structural models, with drug input into the central compartment, first-order distribution between the central and peripheral compartments, and first-order elimination from the central compartment. Two-compartment structural models with body surface area (BSA) as an allometric scalar provided minimum bias in estimates of systemic clearance (CL) and volume of distribution for the central compartment (Vc). Based on the final population PK models, the estimates of CL and Vc in adults were 0.101 L/h and 4.76 L, respectively, for cytarabine and 0.140 L/h and 4.04 L for daunorubicin. The population estimates of CL and Vc for cytarabine in children, adolescents, and young adults ( All tested covariates were eventually excluded from the population PK models, except BSA, bilirubin, study type, and formulation, which have a small effect on PK and are not expected to result in detectable changes in clinical safety or efficacy. Age was evaluated both as a continuous and categorical variable (1-5, 6-11, and 12-17 y) and was not a significant covariate for cytarabine and daunorubicin CL and Vc. Study type was identified as a significant covariate for CL and Vc for cytarabine and daunorubicin, which was possibly due to a change in analytical site for pediatric studies. Mean AUC0-48 values of cytarabine in pts aged 1-5, 6-11, and 12-17 y (135 U/m2) were similar (2767, 2783, and 2806 μg·h/mL, respectively) and approximately 40% higher than that observed in pts ≥18 y (100 U/m2, 1928 μg·h/mL), proportional to the 35% higher dose in pediatric pts. Mean AUC0-48 values of daunorubicin in pts aged 1-5, 6-11, and 12-17 y (135 U/m2) were similar (967, 896, and 982 μg·h/mL, respectively) and approximately 40% higher than that observed in pts ≥18 y (100 U/m2, 615 μg·h/mL), proportional to the 35% higher dose in pediatric pts. The results of this population PK analysis indicated exposures to CPX-351 in pediatric pts were not affected by age and were similar to those in adults when administered at the same BSA-normalized dose. Disclosures Wang: Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Cooper:Celgene: Other: Spouse was an employee of Celgene (through August 2019). Absalon:Jazz Pharmaceuticals: Research Funding. OffLabel Disclosure: Yes, in this study, CPX-351 was also evaluated in pediatric AML

Introduction: CD74 is a type II transmembrane protein expressed on antigen-presenting cells and an MHC class II chaperone. It has been associated with tumor progression and metastasis in solid tumors, and its expression has been suggested to serve as a prognostic factor in many cancers, with higher relative expression associated with oncogenesis. As the expression of CD74 has been associated with response to bortezomib in multiple myeloma, we inquired whether such correlation might be seen in pediatric AML. We prospectively evaluated CD74 expression by difference from normal (ΔN) flow cytometry and correlated expression with clinical characteristics and outcome as part of Children's Oncology Group protocol AAML1031 that randomized patients younger than 30 years of age with de novo AML to standard treatment with (Arm B) or without (Arm A) bortezomib. The addition of bortezomib to standard chemotherapy increased toxicity but did not improve survival. Given the association of CD74 with B-lymphoid neoplasms and bortezomib's known efficacy in B-cell neoplasms and multiple myeloma, we hypothesized that within Arm B, the patients with CD74 expression would have a more favorable outcome. Methods: In total, 1,139 newly diagnosed pediatric patients with de novo AML were randomized to standard chemotherapy (n=561, Arm A) or standard chemotherapy with bortezomib (n=578, Arm B). All patients received the identical chemotherapy backbone with either four intensive chemotherapy courses or three courses followed by allogeneic hematopoietic stem cell transplantation for high-risk patients. To qualify for this correlative study, 991 patients satisfied 2 criteria: (1) submitting a bone marrow aspirate for ΔN at diagnosis and (2) providing consent for correlative biology studies. All diagnostic specimens were centrally and prospectively evaluated for the expression of CD74 by ΔN. AML was considered to be CD74-positive if the MFI was more than two times above background autofluorescence and more than 40% of the leukemia was above background autofluorescence. Results: Among 991 patients, 263 were CD74-positive (26.5%) by ΔN, with similar prevalence in Arm A (27.9%) and Arm B (25.2%). Correlation of CD74 expression with clinical characteristics showed that those with CD74 expression had higher median age (p For patients in Arm A (no bortezomib), the differences in OS (66.1% vs 61.0%, p=0.138) and EFS (48.9% vs 41.7% p=0.088) were not significant between those that were CD74-positive and those that were CD74-negative (Figure 1). However, patients in Arm B receiving bortezomib that were CD74-positive showed a significant improvement in OS (75.3% vs 62.5%, p=0.006) and EFS (53.6% vs 44.3%, p=0.028) compared to those who were CD74-negative (Figure 1). Comparison of the outcomes for CD74-positive patients with and without bortezomib exposure showed a difference in OS of 66.1% vs. 75.3% for those in Arm A vs. Arm B but did not reach significance (p=0.155). Multivariable analysis for OS yielded a hazard ratio of 0.67 (95% CI: 0.44 - 1.02) and p=0.061, approaching, but not reaching, statistical significance. Conclusions: These data demonstrate that CD74 expression is associated with more favorable disease characteristics and survival. Patients receiving bortezomib that were CD74-positive showed a superior response to therapy compared to patients who did not express CD74, by both OS and EFS, suggesting that CD74-positive childhood AML patients stand to benefit from bortezomib therapy. Bortezomib may induce a mechanistic response in CD74-positive AMLs similar to that in bortezomib-treated B-cell neoplasms and/or multiple myeloma, where bortezomib has proven to be beneficial. Disclosures Cooper: Celgene: Other: Spouse was an employee of Celgene (through August 2019). Pollard:Syndax Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

CPX-351 (Vyxeos®; daunorubicin and cytarabine liposome for injection), a dual-drug liposomal encapsulation of daunorubicin and cytarabine at a synergistic 1:5 molar ratio, is approved by the US FDA and EMA for the treatment of adults with newly diagnosed therapy-related AML or AML with myelodysplasia-related changes. Separate studies at Cincinnati Children's Hospital (CCH) and by the Children's Oncology Group (COG) evaluated the efficacy and safety of CPX-351 in pediatric patients (pts). CPX-351 was administered by IV infusion on Days 1, 3, and 5 for the first induction only. The CCH study included 2 dose levels, 100 and 134 units/m2, with 100 units/m2 identified as the recommended phase 2 dose (RP2D); pts with multiple relapsed or refractory AML were included in the exposure-response (E-R) analyses. The COG study established an RP2D of 135 units/m2; the majority of pts in the E-R analyses had first-relapsed AML. E-R analyses were conducted to evaluate the relationship between plasma exposures to CPX-351 and efficacy or safety endpoints. Plasma PK parameters of cytarabine and daunorubicin, including area under the curve up to 48 h post dose on Day 5 Cycle 1 (AUC48), maximum concentration on Day 5 Cycle 1 (Cmax), and concentration at 48 h post dose on Day 5 Cycle 1 (C48), were derived from a previously developed population PK model for pediatric and adult pts. The efficacy endpoints were complete remission (CR), CR+CRp (partial platelet count recovery), or CR+CRp+CRi (incomplete hematologic recovery). Age, sex, bone marrow blast, white blood cell, and platelet counts at baseline were included in the covariate analysis. The safety endpoints included grade ≥3 TEAEs. From the studies, both pediatric and adult pts were included in the E-R analyses for efficacy. Overall, the E-R efficacy population included 43 (53%) pediatric pts (1-17 y) and 38 (47%) adults (≥18 y); the majority were male (57%) and of white origin (63%). Of the 81 pts included in the analysis, 28 (31%), 16 (25%), and 1 (2%) pt achieved CR, CRp, and CRi, respectively; 39 (48%) pts had progressive disease or no response. Various models were used to describe the relationship between probability of response (CR, CRi, or CRp) and exposure parameters (AUC48, Cmax, and C48). C48 of cytarabine was associated with the best statistical goodness-of-fit in the logistic regression model. The effect of cytarabine C48 on the probability of response was statistically significant (P = 0.0144; odds ratio = 1.057). These results suggest the odds of response increased by ~6% for each 1-unit increment of cytarabine C48 (ie, 1 μg/mL). The estimated probability of response was separated into exposure quartiles: 33% for Q1, 49% for Q2, 59% for Q3, and 83% for Q4. The cytarabine C48 was mainly observed in Q1 and Q2 for the 100 units/m2 dose and in Q3 and Q4 for the 134-135 units/m2 doses. In a covariate analysis, none of the covariates tested explained the variability in response. AUC48 of cytarabine and daunorubicin and C48 of daunorubicin were also significantly correlated with probability of response to CPX-351. This was expected, as the PK of daunorubicin and cytarabine are highly correlated when administered as CPX-351. Logistic models were also developed to describe the relationship between probability of response and exposure parameters. Similarly, cytarabine C48 was a significant predictor of the probability of CR or CRi. However, no logistic models were found to describe the relationship of probability of CR+CRp (or CR only) and exposure parameters. Although pt populations of the CCH and COG studies were different, a similar correlation was shown for exposures and efficacy in the CCH study, where higher exposures were associated with higher response rates. Grade ≥3 TEAEs observed in pediatric pts were separated into exposure quartiles for Cmax of cytarabine or daunorubicin. The probabilities of grade ≥3 TEAEs for quartiles of cytarabine Cmax were 77% for Q1, 85% for Q2, 85% for Q3, and 71% for Q4. Similar results were observed with daunorubicin. In these post hoc E-R analyses, higher CPX-351 doses were associated with higher plasma exposures, which led to a higher probability of response (CR+CRp+CRi) in pediatric pts with relapsed or refractory AML. There was no correlation of plasma exposures to grade ≥3 TEAEs. These analyses should be interpreted with caution due to the small sample size; however, they support use of the higher dose of 135 units/m2. Disclosures Wang: Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Absalon:Jazz Pharmaceuticals: Research Funding. Faderl:Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Cooper:Celgene: Other: Spouse was an employee of Celgene (through August 2019). OffLabel Disclosure: Yes, in these studies, CPX-351 was evaluated in pediatric AML

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is a subtype of T-acute lymphoblastic leukemia (T-ALL) arising from a primitive precursor. We present a unique case of an infant with ETP-ALL with a missense NRAS mutation in codon 61 (c.182A>G, p.Q61R). The patient also had a minor population of non-ETP T-ALL blasts and clinical features typically associated with juvenile myelomonocytic leukemia (JMML), namely, absolute monocytosis, splenomegaly, and elevated hemoglobin F. The treatment was initiated with chemotherapy, followed by cord blood transplantation. The patient achieved remission, but unfortunately died from transplant-related complications. This case highlights an NRAS mutation in ETP-ALL with JMML-like phenotype.

10512 Background: PI3K/mTOR signaling, a critical pathway in cell proliferation, metabolism, and apoptosis, is often dysregulated in acute lymphoblastic leukemia (ALL). A phase 1 trial of the mTOR inhibitor temsirolimus combined with etoposide and cyclophosphamide was performed in children with relapsed/refractory (r/r) ALL. Methods: Temsirolimus was administered intravenously (IV) on days 1 and 8 with cyclophosphamide 440 mg/m2 and etoposide 100 mg/m2 IV daily days 1-5. The starting dose level (DL) of temsirolimus was 7.5 mg/m2 (DL1) with escalation to 10 mg/m2 (DL2), 15 mg/m2 (DL3), and 25 mg/m2 (DL4). MRD was performed centrally. PI3K pathway inhibition was measured by phosphoflow cytometry (PFC) analysis of peripheral blood (PB) from treated patients (pts). Results: Sixteen heavily pretreated r/r ALL pts ages 2-19 years with marrow blasts > 25% were enrolled; 15 were evaluable [10 B-ALL/5 T-ALL]. One dose-limiting toxicity (DLT) of grade (Gr) 4 pleural and pericardial effusions with pneumonitis/lung infection leading to Gr 5 cardiorespiratory arrest occurred in a pt treated at DL3. No further DLTs were seen in the DL3 expansion and DL4 cohorts. Gr 3/4 non-hematologic toxicities occurring in ≥ 3 pts included febrile neutropenia, elevated ALT, hypokalemia, mucositis, and tumor lysis syndrome and were independent of dose. Of 15 evaluable pts, 4 (27%; 2 B-ALL/2 T-ALL) had a complete response (CR) after cycle 1, comprised of 1 pt at each DL. Three had MRD < 0.01%. Three pts (20%; 2 B-ALL/1 T-ALL) had partial response (PR). Overall response rate (CR+PR = ORR) was 47%. Pharmacodynamic PFC studies compared phosphoprotein levels pre (day 0) and post treatment (days 3-5) in 9 consenting pts with available PB. All tested pts showed basal activation of PI3K pathway signaling. Dose-dependent inhibition of mTOR targets phosphorylated (p) S6 and/or p4EBP1 was observed in 9/9 and 6/9 pts, respectively, following temsirolimus and chemotherapy treatment. Various patterns of compensatory upregulation of pPI3K, pmTOR, pAkt, and/or pERK was observed. Conclusions: Temsirolimus at 25 mg/m2 combined with salvage etoposide and cyclophosphamide has an acceptable safety profile in high-risk pediatric patients with r/r ALL. Responses were observed at all DLs. mTOR target inhibition was achieved and appeared to correlate with dose level. Future testing of other PI3K/mTOR pathway inhibitors in combination with chemotherapy may be warranted with a goal of further increasing response in r/r ALL. Clinical trial information: NCT01614197.

10532 Background: Anthracyclines (AC) are highly effective in treating acute myeloid leukemia (AML), but limited by cardiotoxicity (CTX). CPX-351, a liposomal preparation of daunorubicin (DNR) and cytarabine, may provide therapeutic benefit with less CTX. We evaluated acute changes in cardiovascular (CV) function and biomarkers after 1 cycle of CPX-351 in children with relapsed AML within the phase 1/2 study, AAML1421. Methods: Patients (pts) received 135 units/m2/dose of CPX-351 on days 1, 3, and 5. Echocardiograms were centrally quantified at baseline (BL) and day 29 (end of cycle (EOC)). High sensitivity troponin (cTnT) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) were measured at BL and days 5, 8, 15, 22, and 29. Differences between BL and post-CPX-351 echo/biomarker measurements were analyzed using pre-specified Wilcoxon signed rank tests. The relationship between EOC ejection fraction (EF) and clinical variables was assessed using repeated measures linear regression. Results: In 32 included pts, the median AC exposure prior to study entry was 337 mg/m2 DNR equivalents. At baseline, markers of CV function and stress were abnormal (Table). Over 1 cycle, there was a statistically significant decrease in EF and circumferential strain (Table). NT-proBNP and cTnT did not increase significantly over time. In multivariable analysis, only increasing body surface area was significantly associated with lower EOC EF (b:-5.9, 95% CI -10.8,-0.9). Cancer therapy–related cardiac dysfunction, defined as ≥10% decline in EF to < 50%, occurred in 6/32 pts at EOC. Conclusions: In this single arm study of AC pre-treated children, baseline abnormalities in CV function were common. CPX-351 was associated with a statistically significant decline in CV function without a rise in cardiac biomarkers. Absent a comparator population, it is not known how these cardiac trends compare to non-liposomal AC or non-AC salvage regimens. The COG AAML1831 trial will determine if CPX-351 offers cardioprotection compared to standard AC in pts with de novo AML. Clinical trial information: NCT02642965. [Table: see text]

10524 Background: Dependence on the prosurvival protein B-cell lymphoma 2 (BCL-2) occurs in certain pediatric solid tumors, resulting in tumorigenesis and resistance to therapies. Venetoclax (VEN), an orally administered BCL-2-selective inhibitor, has preclinical anticancer activity in human-derived neuroblastoma models. Reported here are preliminary results from pediatric patients (pts) with recurrent or refractory (R/R) solid tumors treated with VEN monotherapy or VEN with cyclophosphamide and topotecan (Cy-Topo). Methods: This phase 1 open-label, 2-part study (NCT03236857) enrolled pts < 25 yr old with R/R malignancies; we report only on pts with solid tumors. Following a dose ramp-up, pts received 800 mg VEN (age/weight-adjusted adult equivalent) once daily for the first 8 wk; Cy-Topo was added optionally after wk 8. Dose-limiting toxicities (DLTs) were assessed during the first 21 days of VEN therapy or cycle 1 of VEN-Cy-Topo. Objectives included safety, toxicity, and preliminary efficacy. Results: As of Dec 17, 2019, 11 solid tumor pts were enrolled: neuroblastoma (n = 6), rhabdomyosarcoma (n = 2), Wilms’ tumor, Carney-Stratakis syndrome, and low-grade fibromyxoid sarcoma (n = 1 each). Median age was 11 yr (range 3–22); median time on study was 6.9 mo (range 1.2–17.8). All pts experienced ≥1 treatment-emergent adverse event (TEAE); vomiting (72%; all grades) was most common. Grade ≥3 TEAEs were reported in 82% of pts; febrile neutropenia (64%), decreased blood cell count, and neutropenia (36% each) were the most common. Seven pts received 800-mg monotherapy for 8 wk; 3 of these pts did not receive Cy-Topo after monotherapy. Of the 7 pts who received VEN-Cy-Topo, 3 pts received 400 mg VEN with Cy-Topo. DLTs of grade 4 neutropenia/thrombocytopenia with delayed count recovery occurred in 2 pts on 800 mg VEN-Cy-Topo, necessitating a dose de-escalation (to 400 mg VEN). Grade 4 neutropenia occurred in 2 pts on 400 mg VEN with Cy-Topo, leading to the addition of myeloid growth factor to the therapy regimen. The best response after 8 wk of VEN monotherapy was stable disease (SD). Six pts were evaluable for tumor response with VEN-Cy-Topo; 1 neuroblastoma pt had a complete response after 5 cycles of 400 mg VEN, 4 pts had SD (3 on 800 mg and 1 on 400 mg VEN) and 1 (800 mg VEN) had progressive disease as best response. Conclusions: Continuous dosing of VEN with Cy-Topo was not tolerated due to cytopenias in 4/7 pts with solid tumors. Discontinuous dosing of VEN with Cy-Topo is being explored. Clinical trial information: NCT03236857.

Background Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations occur in 10%-15% of pediatric/adolescent patients with AML and are associated with treatment resistance, risk of relapse, and poor survival. Nearly 50% of children with newly diagnosed FLT3-ITD AML fail treatment or relapse as their first event (Levis, et al, Intl J Hematol 2005; Gilliland, Blood 2002). Therefore, targeting FLT3-ITD may improve clinical responses or prevent relapse in younger patients with FLT3-ITD mutations. No FLT3 inhibitors are approved for use in children. Q is an oral, highly potent and selective, type II FLT3 inhibitor. In the phase 3 QuANTUM-R trial, Q monotherapy prolonged overall survival vs salvage chemotherapy in adult patients with R/R FLT3-ITD AML (Cortes, et al, Lancet Oncol 2019). In another study, Q combined with chemotherapy in pediatric patients (age ≤ 21 years) with R/R AML or acute lymphoblastic leukemia, regardless of FLT3-ITD status, resulted in a marked reduction in bone marrow blasts for patients with FLT3-ITD vs other patients (Cooper, et al, Clin Cancer Res 2016). Thus, there is a strong rationale to investigate Q for treatment of R/R FLT3-ITD AML in pediatric/adolescent patients. Study Design and Methods This open-label, multicenter, single-arm, phase 1/2 study (NCT03793478) consists of dose-escalation and -expansion phases. Patients will be stratified into 2 age groups: 1 to < 12 months (younger age group) and 1 to ≤ 21 years (older age group). Key inclusion criteria are diagnosis of FLT3-ITD AML, first relapse or refractory to first-line high-dose chemotherapy with adequate organ function. Patients who received prior therapy with other FLT3 inhibitors or prior stem cell transplant (SCT) are eligible. Key exclusion criteria include acute promyelocytic leukemia or juvenile myelomonocytic leukemia, significant cardiovascular disease (including QTc > 450 ms), active systemic infections, liver disease, or HIV infection. Phases 1 and 2 include ≤ 2 reinduction cycles, optional consolidation therapy, and continuation therapy for those having a partial response (PR) or remission. Reinduction therapy will consist of fludarabine and cytarabine (FLA; 30 mg/m2/day and 2000 mg/m2/day, respectively) followed by Q in 28-day cycles. Patients will be treated with intrathecal (IT) triple chemotherapy prophylaxis before reinduction cycles 1 and 2 and after cycle 2. Additional IT triple chemotherapy prophylaxis may be given at investigator discretion for patients with CNS2 disease. Patients achieving at least a PR after reinduction may receive optional consolidation chemotherapy as a bridge to allogeneic hematopoietic SCT (HSCT). Optional consolidation regimens are high-intensity chemotherapy (cytarabine 500 mg/m2/day + etoposide 100 mg/m2/day) followed by Q, or low-intensity consolidation with either Q monotherapy or cytarabine (75 mg/m2/day). Patients who achieve at least a PR after reinduction or continue to be in remission after HSCT may receive continuation therapy with Q monotherapy (for up to twelve 28-day cycles). In phase 1, the starting dose of Q for the reinduction cycle for patients 1 to ≤ 21 years will be 40 mg/m2 once daily with body surface area (BSA) < 1.5 m2 or 60 mg once daily for patients with BSA ≥ 1.5 m2. The starting dose of Q for younger patients in phase 1 will be determined from the recommended phase 2 dose (RP2D) from the older age group and pharmacokinetic (PK) modeling. Up to 9 patients per dose level will be enrolled in the older age group, and younger patients will be enrolled using a rolling 6 design. Patients in phase 2 will receive the RP2D for their respective age group. The planned sample size is 41-65 patients to meet the target of 41 response-evaluable patients at the RP2D. QTc based dose modifications will be instituted for Q. The primary endpoints are safety, composite complete remission rate (CRc; defined as complete remission + complete remission with incomplete recovery), and PK parameters. Secondary endpoints include duration of responses, relapse rates, overall survival, and event-free survival at 1, 2, and 3 years, number of patients proceeding to HSCT, pharmacodynamics, gene mutations in bone marrow blasts, minimal residual disease, and FLT3 allelic ratio. This study is recruiting and being conducted in collaboration with the Children's Oncology Group in North America and the Innovative Therapies for Children with Cancer Consortium in Europe. Disclosures Zwaan: Servier: Consultancy; Pfizer: Research Funding; BMS: Research Funding; Janssen: Consultancy; Roche: Consultancy; Celgene: Consultancy, Research Funding; Sanofi: Consultancy; Jazz Pharmaceuticals: Other: Travel support; Incyte: Consultancy; Novartis: Consultancy. Mires:Daiichi Sankyo: Employment. Vigliotti:Daiichi Sankyo, Inc.: Employment. Xie:Daiichi Sankyo, Inc.: Employment. Downs:Daiichi Sankyo, Inc.: Employment. DeCillis:Pyramid: Consultancy; Codiak: Consultancy; Eleven: Consultancy; Arvinas: Consultancy; Exelixis: Consultancy; Novartis: Consultancy; Genocea: Consultancy; Daiichi Sankyo, Inc.: Consultancy; CytomX: Consultancy.

Introduction: Venetoclax (VEN) is a highly selective B-cell lymphoma 2 (BCL2) inhibitor approved for the treatment of adults with hematologic malignancies. VEN has antileukemic activity in pediatric patient (pt)-derived xenograft models and cell lines. Data from these and ongoing adult studies of acute myeloid leukemia (AML) provide rationale for trials in a pediatric population. This study evaluated VEN as monotherapy (monoTx) and in combination with disease-specific chemotherapy (CTx) regimens in pediatric and young adult pts with relapsed/refractory (R/R) malignancies. Preliminary results from acute lymphoblastic leukemia (ALL) and AML pts are reported. Methods: This phase 1 open-label, 2-part, multicenter study (NCT03236857) enrolled pts newborn to Results: As of May 30, 2019, 29 pts were enrolled with AML (n=18; 10 DD plus 8 expansion) or ALL (n=11; 5 DD plus 6 expansion). Median age was 6 years (range 1-25); 59% were male (Table). Pts had a median of ≥6 prior therapies; 52% had prior hematopoietic stem cell transplant (HSCT). Median time on study was 2.5 months (range 3-13.5 months); 76% of pts discontinued VEN, due to progressive disease (38%), physician decision (14%), to pursue HSCT (14%), or adverse events (AEs; 10%). Five pts (17%) had fatal AEs related to disease progression considered unrelated to VEN. Preliminary PK data for pts in the DD cohort demonstrated that the weight-based dosing scheme resulted in similar VEN plasma concentrations across the wide weight and age range of enrolled pts. The most common treatment-emergent AEs (TEAEs) in all 29 pts receiving VEN plus CTx were vomiting (52%), diarrhea (52%), hypokalemia (48%), increased ALT (48%), febrile neutropenia (45%), increased AST (45%), and anemia (41%). On VEN monoTx or combination Tx, grade ≥3 TEAEs related to VEN Tx occurred in 65% of pts; serious AEs related to VEN occurred in 24% of pts. Grade 5 AEs unrelated to VEN were observed in 2 pts with AML (respiratory failure and hypoxia) and 3 pts with ALL (n=1: lung infection; n=2: multiorgan failure), all in context of progressive disease. No DLTs occurred in the 21-day monoTx period. One AML and 1 ALL pt experienced laboratory TLS (grade 3) on VEN plus CTx; this resolved with standard care. For AML pts, the best response after 21 days of monoTx was stable disease. In Part 1, 1 pt had complete remission with incomplete marrow recovery (CRi) after 2 cycles of azacitidine plus VEN. In Part 2, 1 pt achieved a CR after 1 cycle of VEN plus azacitidine. Overall objective response rate (ORR) for AML pts was 11%. For ALL pts, ORR was 27% in Parts 1 and 2 with VEN combination Tx. The best response after 21 days of monoTx was partial remission, with 2 pts achieving CRi after addition of 1 or 2 cycles of VEN plus dexamethasone-vincristine-peg-asparaginase. In Part 2, 1 pt achieved CRi after 1 cycle of VEN plus vincristine-peg-asparaginase. MRD negativity ( Conclusions: VEN plus CTx was well tolerated, with no unexpected toxicities in pediatric pts with heavily treated/refractory ALL or AML. VEN monoTx led to few discontinuations or dose reductions due to AEs, and few serious AEs attributable to VEN. There was preliminary evidence of efficacy of VEN combined with CTx in this heavily treated pt population, including those with prior HSCT, which is notable. Further enrollment, follow-up, and correlative biomarker analyses are ongoing. Disclosures Karol: Abbvie: Other: Unrelated to this study, St. Jude has received a charitable contribution from AbbVie, Inc. The charitable contribution is not being used for clinical or research activities, including any activities related to this study.. Bittencourt:Jazz Pharmaceuticals: Consultancy, Other: Travel, accommodations expenses; Novartis: Consultancy. Gore:Amgen: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel expenses; Novartis: Consultancy, Other: Service on Data Safety Monitoring Committee; travel, accommodations, expenses; Roche/Genentech: Consultancy, Honoraria, Other: travel expenses; Anchiano: Equity Ownership, Other: spouse employment and company leadership; Blueprint Medicines: Equity Ownership; Celgene: Equity Ownership, Other: DSMC member; Clovis: Equity Ownership; Mirati: Equity Ownership; Sanofi Paris: Equity Ownership. O'Brien:Pfizer: Research Funding; Celgene: Research Funding; BMS: Research Funding; AbbVie: Research Funding; Amgen: Research Funding; BTG: Research Funding. Fraser:Novartis: Consultancy; Amgen: Consultancy. Gambart:Jazz Pharmaceuticals: Other: Travel, accommodations, expenses. Zwaan:Sanofi: Consultancy; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; Novartis: Consultancy; BMS: Research Funding; Incyte: Consultancy; Celgene: Consultancy, Research Funding; Servier: Consultancy; Jazz pharmaceuticals: Other: Travel support; Janssen: Consultancy; Roche: Consultancy. Bourquin:Servier: Other: Travel support. Loh:Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees. Caron:F. Hoffmann-La Roche Ltd: Employment, Other: may own stock. Prine:AbbVie: Employment, Other: Stock/stock options. Salem:AbbVie: Employment, Other: Stock/stock options. Unnebrink:AbbVie Germany: Employment, Other: Stock/stock options. Tong:AbbVie: Employment, Other: stock or options. Palenski:Abbvie: Employment, Other: Stock/ stock options. Place:Novartis: Consultancy, Research Funding.

Background: Children treated for acute myeloid leukemia (AML) receive high doses of anthracyclines. Anthracycline cardiotoxicity causes significant short and long term morbidity and mortality, thus it is crucial to understand its risk factors, natural history, and impact on treatment outcome. Methods: Between June 2011 and September 2018, AAML1031 enrolled patients aged 30% and EF returning to baseline or >60%. Resource utilization (RU) data for patients enrolled at hospitals contributing to the Pediatric Health Information Systems database (PHIS) were merged with AAML1031 study data. Cox regressions were used to assess risk factors for incident cardiotoxicity and recovery among cases, as well as to compare event-free (EFS) and overall survival (OS) in patients with and without cardiotoxicity. Poisson regressions were used to compare on-protocol RU. Cardiotoxicity was treated as a time-varying exposure introduced at time of documented onset in comparisons of EFS, OS and RU. Patients with high allelic ratio FLT3/ITD+ AML were excluded from analyses. Results: 1092 patients were treated on AAML1031 and included in the primary analyses. Echo compliance was high overall (mean ±sd: 74.5% ±19.6), but lower and more variable during off-protocol (mean ±sd: 58.1%, ±31.0) than on-protocol (mean ±sd: 90.6% ±19.5) reporting periods. Over a median follow-up of 3.5 years, 39% (n=428) of patients experienced protocol-defined cardiotoxicity, of whom 54% (n=232) suffered grade 2+ LVSD. Cumulative incidence was higher among patients randomized to receive bortezomib, non-infants, females, and patients with low risk disease who receive more cumulative anthracycline during frontline therapy (Table 1). Patients treated with dexrazoxane were less likely to develop cardiotoxicity. Among patients with grade 2+ LVSD, 49% experienced recovery of cardiac function within a median of 14 months (IQR: 9-20) from initial detection. High risk disease classification and dexrazoxane exposure were associated with LVSD recovery (Table 1). Both EFS (HR 1.23, 95% CI: 1.01-1.50) and OS (HR 1.45, 95% CI 1.15-1.84) were significantly worse in patients with cardiotoxicity meeting per-protocol thresholds for treatment modification. The magnitude of the association with EFS (HR 1.24, 95% CI: 0.98, 1.58) was similar when restricting to grade 2+ LVSD, but more pronounced for OS (HR 1.80, 95% CI 1.38, 2.34). A subset of 389 patients (23% suffered grade 2+ LVSD) were treated at PHIS hospitals and contributed to RU analyses. Cardiotoxicity was associated with greater cardiac ICU requirements, increased use of cardiac-directed interventions including antihypertensives, vasopressors, and heart failure medications, and reduced cardioprotection (Table 2). Conclusions: Echo compliance and cardiotoxicity rates were higher than previously described due in part to more frequent echoes and required reporting of raw echo results. Approximately 20% of pediatric AML patients on AAML1031 developed grade 2+ LVSD, only half of whom had documented resolution of LVSD. Dexrazoxane was strongly associated with reduced LVSD risk and potentially superior recovery. Our results offer further evidence that early cardiotoxicity decreases EFS/OS and that cumulative anthracycline dose is the principal predictor of LVSD. These data argue for comparative effectiveness studies of cardiac-directed interventions after LVSD, and identification of clinical and genetic risk factors for anthracycline cardiotoxicity and its recovery. Such studies may allow for anthracycline de-escalation in select patient populations. Work is ongoing to inform optimal supportive care practices for patients with LVSD using clinical data merged with PHIS. Disclosures Leger: Abbott: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; BTG Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

The MLLT10 gene, a known fusion partner for KMT2A, encodes AF10 protein, a transcription factor that binds unmodified histone H3 and regulates DOT1L expression. KMT2A-MLLT10 fusion portends adverse outcome, but MLLT10 function and prognostic implications in partnership with other genes has not been defined. In comprehensive transcriptome and karyotype evaluation of 2226 children and young adults (0-30 years), we defined the full spectrum of MLLT10 fusions, identified new fusion partners, and correlated MLLT10 structural variants with clinical outcome. We also evaluated transcription and methylation profiles to identify genes dysregulated in MLLT10 fusions with and without KMT2A. 2226 patients treated on Children's Oncology Group (COG) trials AAML0531 and AAML1031 were evaluated by transcriptome profiling and/or karyotyping to identify leukemia associated fusions and copy number changes associated with prognosis. Collectively, 127 patients (5.7%) had primary fusions involving MLLT10: 104 (82%) involving KMT2A (KMT2A-MLLT10), and 23 patients (18%) revealed other fusion partners (MLLT10-X). Alternate, recurrent fusion partners included PICALM (n=13), DDX3X (n=2), and TEC (n=2), while fusions with 6 other partner genes (DDX3Y, CEP164, NAP1L1, SCN2B, TREH, and XPO1) were each identified in single patients. Given the known association of KMT2A-MLLT10 fusions with adverse outcome, we sought to determine whether MLLT10-X had distinct characteristics and comparable outcomes. Initial comparison of disease characteristics in patients with and without KMT2A as fusion partner showed significant differences in age at diagnosis. Those with KMT2A-MLLT10 had a median age of 1.7 years (range 0-21.3), compared to 12.7 years (range 1.4-18.9) in those with MLLT10-X (p ≤ 0.001). There was no significant difference in gender, race, mutational status, or white blood cell count between these two cohorts. MLLT10 rearranged patients (n=127) demonstrated adverse outcomes, with 5-year event-free survival (EFS) of 18.6% vs. 49% in non-MLLT10 rearranged patients (N=1953, p Next we explored the transcriptome profile of patients with MLLT10 fusions to determine the impact of fusion partners, using ribodepleted RNA-seq data from 1049 patients treated on COG AAML1031. MLLT10-fusion-positive cases (n=66) were compared to other AML cases (n=983) in a differential expression (DE) analysis (limma/voom) (Fig 1C). Of 1,910 genes significantly differentially expressed, HOXA family genes were among the top 30 upregulated genes, with HOXA11 identified as >6 logFC, or over 400x higher on average in MLLT10 rearranged patients. To determine if patients with MLLT10 fusions had distinct epigenetic profiles, we performed differential methylation analyses on samples from normal bone marrow and patients with 4 high-risk molecular features: MLLT10 rearranged, KMT2A rearranged, NUP98-NSD1 fused, and FLT3-ITD, across nearly 1 million CpG sites on the Infinium EPIC array (Illumina, CA). After fitting a multivariate model with all of the interacting molecular features, the 250 most discriminative regions were extracted and plotted (ComplexHeatmap) (Fig 1D). Strikingly, patients with MLLT10-X fusions cluster discretely with ultra-high-risk NUP98-NSD1 fusion patients, showing a broadly hypermethylated profile, while KMT2A-MLLT10 patients cluster within the larger KMT2A category and show far fewer hypermethylated regions. We identified patients with MLLT10 fusion partners not previously described, and compared them to other AML patients, as well as patients with known MLLT10 partners KMT2A and PICALM. All MLLT10-aberrant cases had poor EFS and OS, high RR, overexpressed HOXA genes, and distinct DNA methylation profiles, while patients with MLLT10-X fusions tend to be older children. Regardless of fusion partner, patients with MLLT10 fusions exhibit very high risk, and should be prioritized for alternative therapeutic intervention. Disclosures Farrar: Novartis: Research Funding. Deshpande:A2A Pharmaceuticals: Consultancy; Salgomed Therapeutics: Consultancy.

Somatic mutations of CEBPA in AML are present at similar rates in all age groups and have been shown to be associated with favorable outcome. The most common cooperating variant in CEBPA mutations is a second CEBPA mutation, where the cooperation of TAD and bZip mutations lead to a highly penetrant variant which is sufficient for malignant transformation. Data suggested that only double CEBPA mutations (CEBPA-dm) were associated with favorable outcome. Currently, WHO classification of AML includes CEBPA-dm as a diagnostic entity in AML. We previously reported that in children and young adults, somatic single CEBPA mutations, which almost exclusively occurred in the bZip domain, had similar outcomes to CEBPA-dm patients. We have also shown that CSF3R and GATA2 mutations occur in childhood AML and are highly associated with CEBPA mutations. In this large analysis, we sought to define the prognostic impact of CEBPA-dm vs single bZip mutations and evaluate the impact of common co-occurring mutations. This study included a total of 2,958 patients (age 0-29.94 years) treated on the 4 large Children's Oncology Group trials CCG2961 (n=562), AAML03P1 (n=266), AAML0531 (n=917), and AAML1031(n=1233). DNA binding domain of CEBPA (bZip domain) was analyzed by fragment length analysis and 160 patients with mutations were identified and verified with sanger sequencing (n=23 2961; n=15 03P1; n=51 0531; n=71 1031). In all cases where bZip mutations were detected, N terminal region was sequenced for identification of TAD mutations. Of the 160 cases with bZip mutation, 132 patients had a secondary TAD mutation (CEBPA-dm; 82.5%) and in the remaining 28 cases (17.5%) no TAD mutation was detected (CEBPA-sm). Comprehensive NGS data was available in a cohort of patients (n=106) and demonstrated dm vs. sm prevalence of 81% and 19% respectively, similar to the cohort overall. We compared clinical and biologic characteristics among CEBPA-dm vs. CEBPA-sm patients. The two groups showed no significant differences across karyotype, complete remission rates, FLT3/ITD or NPM1 status, cyto-molecular risk group, age, or diagnostic WBC. CEBPA mutations were more prevalent in Asian patients (10.5%) compared to other races (p=0.019). Evaluation of outcomes for those with CEBPA-dm vs. sm in the entire cohort demonstrated that those with CEBPA bZip mutations regardless of dm vs. sm status had identical event free survival (EFS) of 64% (Fig 1A; p=0.777). Similarly, the overall survival (OS) for CEBPA-sm was 89% vs. 81% for CEBPA-dm (p=0.259; Figure 1A). Evaluation by study cohort demonstrated that presence of bZip mutations whether as sm or dm had similar favorable clinical impact (Figure 1B-D; p=NS in each study for EFS and OS). Analysis of CEBPA+ patients with comprehensive NGS data identified somatic mutations in GATA2 (n=20; 19%) and CSF3R (n=12; 11%)(Figure 1E). Co-occurrence of a GATA2 mutation did not impact the favorable outcome conferred by CEBPA+ on EFS or OS (p=0.356 and p=0.749 respectively; Figure 1F). However, presence of CSF3R mutation significantly modulated the clinical implications of CEBPA mutations. We previously showed an overlap with CEBPA and CSF3R mutations, which occur as SNVs in the extracellular domain or truncating mutations in the cytoplasmic domain. CSF3R mutations result in aberrant CSF3R activation and expression and have been shown to be amenable to targeted therapy with either JAK or SRC family inhibitors. Given the large number of CEBPA+ patients available for analysis, we evaluated the outcomes of patients with CEBPA+/CSF3R+ (n=12) and without CSF3R mutations (n=94). Dual mutant CEBPA+/CSF3R+ patients had a significantly inferior EFS of 17% vs. 63% (p In a large study of CEBPA mutations in pediatric AML, we show that patients with a bZip mutation, regardless of sm vs. dm status have a favorable prognosis. Further, we confirm the significant overlap of CEBPA and CSF3R mutations previously reported, and demonstrate that CEBPA+/CSF3R+ patients are at high risk for relapse and thus should not be considered a low risk cohort. Given the poor outcomes with current regimens, patients with CEBPA+/CSF3R+ mutations could be considered for addition of tyrosine kinase inhibitors with upfront therapy. Disclosures No relevant conflicts of interest to declare.

Introduction: Despite maximally intensified chemotherapy, cure rates remain suboptimal for children and young adults with acute myeloid leukemia (AML). As CD123 (IL3RA) is expressed on the majority of AML cells, it is a promising immunotherapeutic target. Increased CD123 expression has been linked to high-risk disease characteristics in adult AML, but clinical implications for childhood AML are less well-defined. Methods: The Children's Oncology Group AAML1031 phase 3 trial (NCT01371981) tested the efficacy of the addition of bortezomib to standard chemotherapy in a randomized fashion. A total of 1400 children and young adults were enrolled on study. All diagnostic specimens were centrally and prospectively evaluated for the expression of CD123 by multi-dimensional flow cytometry (MDF) at Hematologics, Inc. Patients were stratified to either a low-risk (LR) or high-risk (HR) arm of the therapy based on cytogenetic and molecular alterations and end-of-induction (EOI) minimal residual disease (MRD) measured by MDF. LR patients received four cycles of chemotherapy, while HR patients received 3 courses of chemotherapy followed by best allogeneic hematopoietic stem cell transplantation (HSCT). Patients with high allelic ratio FLT3-ITD enrolled on arm C received sorafenib in combination with standard chemotherapy followed by HSCT. Here we provide data on expression of CD123 across all patients and correlate this expression with disease characteristics and clinical outcomes. Results: Surface CD123 expression on AML cells was available for 1040 patients, and expression level varied significantly across the study population with a median CD123 molecules per cell of 1300 (range 120-13,100 molecules per cell). For analysis, the study population was divided into quartiles (n=260 each) based on CD123 expression levels. Significant variation in cytogenetic/molecular characteristics was observed across the four quartiles, where those with highest CD123 expression had a lower prevalence of t(8;21), inv(16), and CEBPA mutations (p Conclusions: CD123 expression is strongly associated with disease-relevant cytogenetic and molecular alterations in childhood AML. Patients with highest CD123 surface expression are more likely to have HR genetic alterations and a paucity of LR features. Further, despite similar induction remission rates, those with high CD123 expression had inferior clinical outcomes compared to patients with lower CD123 expression. However, despite an association with HR features, expression of CD123 appears to be independently associated with therapeutic response given that outcome differences were maintained in multivariate regression analysis. This suggests that CD123 expression may provide additional prognostic information, as highlighted by the inferior outcomes in LR patients that had high CD123 expression. In pediatric and young adult patients with the highest risk disease, the higher CD123 expression represents a valuable therapeutic target in the development of immunotherapies for childhood AML. Disclosures Eidenschink Brodersen: Hematologics, Inc: Employment. Pardo:Hematologics, Inc: Employment. Tasian:Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; Incyte Corportation: Research Funding. Loken:Hematologics, Inc: Employment, Equity Ownership.

Background FLT3/ITD is associated with poor outcomes in adult and pediatric acute myeloid leukemia (AML). Allogeneic hematopoietic stem cell transplantation (HSCT) can improve cure rates, however relapse is still common. Recent studies demonstrate the activity of FLT3 inhibitors, including sorafenib, in targeting the underlying mutation. Procedure We conducted a retrospective study of 15 pediatric patients with FLT3/ITD+ AML treated with sorafenib within 18 months after receiving HSCT. Sorafenib was administered either as prophylaxis in patients considered at very high risk for relapse (n = 6) or at the time of disease recurrence (n = 9). Results Sorafenib was initiated at a median of 100 days post HSCT. Overall, 11/15 (73%) of patients experienced medically significant toxicities. Among patients who experienced toxicity, 6/11 (55%) received treatment at doses above what was later determined to be the maximum tolerated dose of sorafenib for pediatric leukemia. Importantly, sorafenib did not appear to exacerbate graft versus host disease. Our findings suggest that sorafenib may be of particular efficacy in patients with minimal residual disease (MRD); all patients who received sorafenib for MRD immediately prior to transplant or with emergence post-HSCT are alive and remain in complete remission at a median of 48 months post HSCT. Conclusions Our case series suggests that sorafenib administration is feasible and tolerable in pediatric FLT3/ITD+ AML patients early post HSCT. Ongoing prospective controlled studies are needed to further define the dosing of sorafenib in the post-HSCT period and to determine the optimal context for this treatment approach. Pediatr Blood Cancer 2015;62:1048–1054. © 2015 Wiley Periodicals, Inc.

Juvenile myelomonocytic leukemia is a rare myeloproliferative disease in young children. While hematopoietic stem cell transplantation remains the only curative therapeutic option for most patients, children with juvenile myelomonocytic leukemia increasingly receive novel agents in phase I-II clinical trials as pre-transplant therapy or therapy for relapse after transplantation. However, response criteria or definitions of outcome for standardized evaluation of treatment effect in patients with juvenile myelomonocytic leukemia are currently lacking. Here we propose criteria to evaluate the response to the non-transplant therapy and definitions of remission status after hematopoietic stem cell transplantation. For the evaluation of non-transplant therapy, we defined 6 clinical variables (white blood cell count, platelet count, hematopoietic precursors and blasts in peripheral blood, bone marrow blast percentage, spleen size and extramedullary disease) and 3 genetic variables (cytogenetic, molecular and chimerism response) which serve to describe the heterogeneous picture of response to therapy in each individual case. It is hoped that these criteria will facilitate the comparison of results between clinical trials in juvenile myelomonocytic leukemia.

Introduction: Pediatric and young adult patients (pts) with relapsed/refractory (R/R) acute leukemia have a poor prognosis. Selinexor is a first-in-class antagonist of exportin (XPO1) with single agent activity in adults with acute myelogenous leukemia (AML). Dose-defining non-hematologic toxicities in adult studies included nausea, weight loss and anorexia. Methods: DFCI Protocol 13-563 was a Phase I dose-escalation trial conducted at nine institutions to test the safety, tolerability and pharmacokinetic (PK) characteristics of selinexor administered to pediatric and young adult pts with R/R acute leukemias. Selinexor was orally administered twice weekly during 4-week cycles. Three dose levels (DLs) were tested (30 mg/m2, 40 mg/m2 and 56 mg/m2) with dose-escalation decisions based on standard 3+3 Phase I design rules. Required supportive care medications included antiemetics and bacterial prophylaxis. All grade ≥2 non-hematologic adverse events (AEs) were recorded with the exception of non-clinically significant grade 2 laboratory abnormalities. Grades 1-4 hematologic AEs were expected in a relapsed leukemia patient population and were not considered AEs. Results: Sixteen pts enrolled on study, including 5 with primary refractory disease and 7 with second or greater relapse; 6 pts had received prior hematopoietic stem cell transplant. Median age was 9.5 years (range 2.3-21.8). Twelve pts had AML and 4 had acute lymphoblastic leukemia (ALL). Median number of completed cycles was 1 (range 0-3). Thirteen pts (81%) were evaluable for dose limiting toxicity (DLT) assessment, having received at least 7 doses of selinexor during the first cycle. Of 9 pts enrolled at DL1 (30 mg/m2), 6 were DLT-evaluable; 1 pt experienced a DLT of grade 5 pancreatitis (possibly related to study treatment). Three pts enrolled at DL1 were not DLT-evaluable; 2 pts were removed from study due to disease progression and 1 pt died from complications of a pulmonary infection. Six pts enrolled at DL2 (40 mg/m2) and none experienced a DLT. One pt was treated at DL3 (56 mg/m2) and developed a DLT of cognitive impairment (grade 3). Further enrollment onto DL3 was suspended secondary to an amendment capping dose escalation at 40 mg/m2 (equivalent to recommended single-agent dose in adult AML). During Cycle 1, the most common selinexor treatment-related AE's were hyponatremia (31%), elevated alanine aminotransferase (25%), nausea (25%), hyperkalemia (25%), elevated aspartate aminotransferase (19%) and diarrhea (19%). The most common grade ≥ 3 selinexor treatment-related AE's were hyponatremia (31%), elevated alanine aminotransferase (19%), hyperglycemia (13%) and hyperkalemia (13%). In contrast to adults treated with selinexor, there were no cases of grade ≥ 3 weight loss or nausea and only one case of grade 3 anorexia. One AML pt, after 2 cycles, achieved a complete remission with incomplete recovery of platelet count and one ALL pt, after 1 cycle, achieved a partial response, resulting in a protocol-defined overall response rate of 12.5%. Two of 4 pts with ALL (one each in DLs 1 and 2) developed clinically significant (grade 4) TLS. These included the pt who achieved a PR and another pt with early T-cell precursor ALL. Overall, 9 pts (56%), including 6 AML and 3 ALL, experienced an objective response and/or an investigator-defined clinical benefit (e.g., reduction in transfusions, clearance of peripheral blasts, decreased pain) from therapy. Conclusions: Selinexor, at a doses of 30 or 40 mg/m2 given twice weekly, was well tolerated in pediatric and young adult pts with R/R acute leukemia, demonstrating single agent clinical activity and less GI toxicity than had been seen in adults. The high frequency of TLS in ALL pts is particularly notable as treatment of ALL with selinexor has not been previously reported. Analysis of pharmacokinetic and correlative biology studies are underway. Based on the results of this Phase I study, the recommended Phase II dose of single-agent selinexor in children with R/R acute leukemia is 40 mg/m2 administered twice weekly. These results also support the continued investigation of selinexor in combination with multiagent chemotherapy. Disclosures Burke: AMGEN: Speakers Bureau; JAZZ: Speakers Bureau; Shire: Speakers Bureau.

Despite the remarkable pace of characterizing the genomics of pediatric acute leukemias, the integration of real-time sequencing results into clinical practice has lagged. With increased availability of molecularly-targeted therapies, the promise of matching genetic lesions in patients' leukemia cells to treatment has not yet been fully realized. We established the first pediatric leukemia clinical genomics consortium in the United States, known as the Leukemia Precision-based Therapy (LEAP) Consortium, which includes 13 major pediatric cancer institutions. We hypothesized that it is feasible to identify and match, in real-time, actionable alterations with a targeted therapy for pediatric patients with relapsed, refractory or high-risk leukemias or myelodysplastic syndrome (MDS). Using a combination of a DNA-based next-generation sequencing panel and RNA-based gene fusion testing, followed by data review by our multidisciplinary molecular tumor board, we are conducting a clinical trial to test this hypothesis. To date, we have enrolled and reviewed data from 143 patients stratified by disease status: Cohort 1, patients with relapsed or refractory leukemias (n=93), and Cohort 2, patients with de novo high-risk leukemias or MDS (n=50). A matched targeted therapy (MTT) recommendation has been made for 72% (n=103) of patients, tiered based on the level of evidence linking the mutation to potential activity of targeted therapy in the context of each patient's disease (Tier 1: 11%, Tier 2: 4%, Tier 3: 41%, Tier 4: 6%, Tier 5: 10%). Of the 44 patients in Cohort 1 with clinical follow-up data, 5 (11%) had alterations in therapy made based upon sequencing results and MTT recommendation. These include the use of the MEK inhibitor trametinib for RAS mutant acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) (n=2), dasatinib for B-ALL and T-ALL with NUP214-ABL1 translocations (n=2) and ponatinib for B-ALL with an ABL1 T315I mutation (n=1). In addition, this genomic data refined diagnosis and supported additional germline assessment in a subset of patients. In parallel to the genomic sequencing, we are conducting high-throughput drug sensitivity (HTS) assays to study in vitro anti-leukemia effects of a panel of up to 120 targeted inhibitors in the context of leukemia-associated genetic alterations. Of the initial 106 accrued patients, 40 (38%) had adequate amounts of blood or bone marrow for in vitro drug testing. All samples were tested in the inhibitor panel assay, and 65% of the samples yielded interpretable results. Inhibitor screening data was compiled for all 12 patients with genetic alterations resulting in Tier 1, 2 or 3 MTT recommendations. This subset of patients had leukemias with two distinct molecular profiles: 1) oncogenic RAS signaling pathway mutations or 2) oncogenic tyrosine kinase alterations. Our molecular tumor board recommended trametinib for the first group of patients and tyrosine kinase inhibitors (TKIs), specific to the mutation, for the second group of patients. In vitro HTS data analysis demonstrated dose-response sensitivity of leukemia cells with RAS pathway mutations to trametinib, many of which had half-maximal inhibitory concentration (IC50) less than 50 nM. Similarly, leukemia cells from patients with FLT3, ABL1 or KIT mutations generally showed dose-responses to relevant TKIs with low IC50s. Overall, HTS data were concordant with MTT recommendations informed by the sequencing results. This first in the US multi-institutional prospective leukemia genomics trial brings state-of-the-art clinical genetic testing, assessment of prognostic biomarkers, selection of patients for germline testing, and targeted therapeutic treatment recommendations to children and young adults with high-risk, relapsed or refractory leukemias or MDS. Our collaboration provides a unique opportunity to perform sophisticated patient-specific in vitro drug testing assays to impact MTT discovery efforts, which we plan to validate in vivo using patient-derived xenograft models where feasible. We believe that a model like the LEAP Consortium has the potential to transform precision medicine approaches for children with high-risk leukemias and to inform future genomics-guided therapeutic trials and drug discovery efforts. Disclosures Tasian: Aleta Biopharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding. Burke:JAZZ: Speakers Bureau; Shire: Speakers Bureau; AMGEN: Speakers Bureau. Tyner:AstraZeneca: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Janssen: Research Funding; Array: Research Funding; Constellation: Research Funding; Gilead: Research Funding; Aptose: Research Funding; Incyte: Research Funding; Genentech: Research Funding.

INTRODUCTION: Gemtuzumab ozogamicin (GO, MylotargTM) was re-approved by the FDA and has been commercially available since September 2017. Prior to re-approval, adults and children with relapsed/refractory AML or APL were treated with GO obtained through an expanded access program under an institutional IND at our Cancer Consortium: Fred Hutchinson Cancer Research Center (FHCRC), the University of Washington Medical Center (UWMC), Seattle Children's Hospital (SCH), and the Seattle Cancer Care Alliance (SCCA). METHODS: Between 2014-2017, we enrolled 44 patients (median age 40); patient characteristics are summarized in Table 1. Twelve were children treated at SCH and 32 adults at SCCA/UWMC. Forty had AML, 3 mixed phenotype acute leukemia and 1 APL. Forty-two had relapsed disease and 2 were primary refractory. Median duration of CR1 was 11.2 months (range 0-104). Median number of prior induction attempts was 2 (range 1-8). Eligibility initially required intermediate or "good" risk cytogenetics, though subsequently patients whose blasts were CD33 positive were included regardless of cytogenetics. Additional inclusion criteria were ECOG performance status (PS) ≤3, bilirubin ≤2.0 mg/dl, and ALT/AST ≤5 fold the institutional upper limit of normal. RESULTS: Two patients received single-agent GO (9mg/m2 day 1), 1 patient with APL received GO + ATRA + Aza, and the remainder typically received 3mg/m2 with chemotherapy for a single dose or on days 1, 4, and 7 (Table 2). Patients received a median of 2 cycles (range 1-6) of GO (the APL patient received 6 cycles). Timing of GO around hematopoietic stem cell transplant (HCT) is also noted in Table 2. Table 3 shows the grade 3-4 toxicities occurring within 60 days of receipt of GO per number of patients, rather than number of events. Two children developed sinusoidal obstructive syndrome (SOS) >60 days post GO. Both incidences were post an allogeneic hematopoietic cell transplant (HCT), one at 15 days post HCT and the other 28 days post HCT. The first patient had complete resolution of SOS and remains alive 2 years later, the second patient developed SOS day 28 post HCT then died of an invasive mucormycosis infection in the setting of immunosuppression on day 37 of the HCT. Six deaths occurred within 60 days of beginning GO: 1 from cardiogenic shock, 1 from multiorgan failure post aspiration event, 2 from pneumonia/sepsis, and 2 from disease progression. No deaths were attributed to GO. Thirteen of the 44 patients (29%; 95% CI:18-44%) achieved complete remission (CR), 3 with measurable residual disease (MRD 7%) (Table 4). The MRD was detected by flow cytometry in 2 patients, and PCR for inversion 16 in 1 patient. Sixteen had CR with incomplete hematologic recovery (CRi) and 15 were resistant or died before assessment. Eight remain alive in CR with a median event free survival (EFS) of 31 months (range 7-47). Thirty-six patients have died, with a median survival of 17.2 months (range 0.5-9.4). Table 4 outlines response by "more" or "less" intense regimens (intensity is defined in Table 2) and by adult vs. pediatric populations. We found higher response rates in patients who received GO combined with more intensive [78% CR+CRi] rather than less intensive therapy [44% CR+CRi] and in those with fewer prior regimens [100% CR+CRi in patients with 1 prior regimen compared to 48% with ≥2 prior regimens]. In addition, responses were obtained in patients with intermediate risk cytogenetics (11 out of 19 total intermediate = 58%) or unfavorable (3 out of 8 total unfavorable = 37%), although a higher fraction of responses were seen with favorable cytogenetics [t(8;21, inv16, t(15;17)] (15 out of 17 total favorable = 88%). CONCLUSION: GO was safe and well tolerated. Based on CR1 duration and number of salvage regimens, the observed 9/28 CR rate with GO + intense therapy compares with a rate of expected 7/28 had the same patients received prior intense salvage therapy without GO (Estey E, Blood [1996/88:756]). GO combinations are a reasonable option for relapsed/refractory AML, but might be of more value in patients with only measurable residual disease. ACKNOWLEDGEMENTS: The GO team wishes to acknowledge Pfizer Inc. for their commitment to patients by supplying drug, and our Investigational Pharmacy, the Institutional Review Board, and the FDA for supporting the efforts of this expanded access program in making gemtuzumab ozogamicin accessible to people with relapsed or refractory AML. Disclosures Walter: Boehringer Ingelheim Pharma GmbH & Co. KG: Consultancy; Seattle Genetics, Inc.: Consultancy, Other: Clinical trial support, Research Funding; Covagen AG: Consultancy, Other: Clinical trial support, Research Funding; Aptevo Therapeutic: Consultancy, Other: Clinical trial support, Research Funding; Amphivena Therapeutics: Consultancy, Equity Ownership, Other: Clinical trial support, Research Funding; Amgen Inc.: Other: Clinical trial support, Research Funding; Actinium Pharmaceuticals, Inc.: Other: Clinical trial support, Research Funding; Pfizer: Consultancy. Scott:Celgene: Consultancy, Research Funding; Agios: Consultancy; Alexion: Consultancy; Novartis: Research Funding. Cassaday:Adaptive Biotechnologies: Consultancy; Merck: Research Funding; Pfizer: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Seattle Genetics: Other: Spouse Employment, Research Funding; Jazz Pharmaceuticals: Consultancy; Kite Pharma: Research Funding; Incyte: Research Funding. Becker:GlycoMimetics: Research Funding.

Background: CD33 is variably expressed on leukemia blasts in most patients with acute myeloid leukemia (AML). Efforts to target CD33 therapeutically have focused on gemtuzumab ozogamicin (GO), an antibody-drug conjugate delivering a DNA-damaging calicheamicin derivative. Qualification for GO therapy has been determined by expression of CD33 by multidimensional flow cytometry (MDF); patients with positive CD33 expression receive GO. Previous studies from the AAML0531 protocol demonstrated that the cell surface intensity expression of CD33, determined by MDF, predicts the response to treatment with GO, and that in part this expression is regulated by a pair of CD33 single nucleotide polymorphisms (SNPs) in linkage disequilibrium that also, independently, predict response to therapy. Patients with CC genotype for rs12459419 have lower relapse risk and improved disease-free survival, compared to CT and TT genotypes. Because GO therapy is associated with treatment-related toxicity, it is important to identify biologic variables associated with benefits and risks. To date, there has been no report associating SNP status among CD33 positive versus CD33 negative patients that could assess how the combination of these biomarkers for determining administration of GO therapy could improve patient outcomes. Objective: In an effort to determine which children would benefit most from GO treatment in future prospective pediatric AML protocols, we aimed to elucidate if CD33 SNP genotype status should be combined with quantitative CD33 cell surface antigen expression on the diagnostic leukemia cells (CD33+ versus CD33-) to determine GO eligibility, with a retrospective analysis of children enrolled in Children's Oncology Group AAML0531. Methods: Of 1022 newly diagnosed pediatric patients with de novo AML enrolled on protocol AAML0531, 666 satisfied two criteria for this study: (1) submission of a blood or bone marrow sample for MDF at diagnosis with corresponding CD33 SNP genotype data available, and (2) proper consent for specimen testing. CD33 Expression Levels on Leukemic Blasts The diagnostic AML leukemia population was identified by gating on CD45 versus log-side scatter and CD33 expression levels were determined by measuring the mean fluorescence intensity (MFI) by flow cytometry. For a patient to be considered CD33+ two criteria were required: intensity of CD33 on blasts was at minimum four times the MFI of its correspondent autofluorescent control, and at least 80% of the total blasts were greater than this minimum. Genotyping CD33 SNPs CD33-coding SNP rs12459419-Ala14Val and linked promoter SNP rs3865444 were genotyped using the Sequenome (San Diego, CA) platform at the Biomedical Genomics Center, University of Minnesota. Both SNPs had a call rate of 0.98 and were in Hardy-Weinberg equilibrium (P=0.05). Results: Association of Genotype and cell surface expression of CD33 for AAML0531 patients Of 666 patients, 84% (560/666) were CD33+. CC genotype was observed in 54.5% (305/560) of CD33+ cases, 37.5% (210/560) had CT genotype and 8% (45/560) TT genotype. Of the 16% (106/666) of patients who were CD33 negative, 33% (35/106) had CC genotype, 47% (50/106) CT genotype, and 20% (21/106) had TT genotype. Comprehensively, 340/666 (51%) had CC genotype (51%) and 10% were CD33 negative (35/340). Conversely, out of a total of 66 patients with TT genotype, 45 (68%) were CD33+ and 32% (21/66) were CD33 negative. Conclusions: These results clarify the relationship between the amount of CD33 expressed on AML at diagnosis as measured by MDF and CD33 SNP genotype status. While correlation with clinical outcome analysis is ongoing, these data support inclusion of CD33 SNP genotyping for eligibility of GO therapy. Therefore, the current recommendation for future COG AML clinical protocols is that GO will be administered to patients with CD33 expression as determined by MDF and CC genotype patients regardless of CD33 expression. Disclosures Pardo: Hematologics, Inc: Employment. Eidenschink Brodersen:Hematologics, Inc: Employment. Paine:Hematologics, Inc: Employment. Loken:Hematologics, Inc: Employment, Equity Ownership.

Background We sought to determine the feasibility of co-administering everolimus with a four-drug reinduction in children and adolescents with acute lymphoblastic leukemia (ALL) experiencing a first marrow relapse. Procedure This phase I study tested everolimus with vincristine, prednisone, pegaspargase and doxorubicin in patients with marrow relapse occurring >18 months after first complete remission (CR). The primary aim was to identify the maximum tolerated dose of everolimus. Three dose levels (DLs) were tested during dose escalation (2, 3, and 5 mg/m2 /day). Additional patients were enrolled at the 3- and 5 mg/m2 /day DLs to further evaluate toxicity (dose expansion). Results Thirteen patients enrolled during dose escalation and nine during dose expansion. During dose escalation, one dose-limiting toxicity occurred (grade 4 hyperbilirubinemia) in six evaluable patients at DL3 (5 mg/m2 /day). The most common grade ≥3 adverse events were febrile neutropenia, infections, transaminitis, hyperbilirubinemia, and hypophosphatemia. Two of the 12 patients treated at DL3 developed Rothia mucilaginosa meningitis. Nineteen patients (86%) achieved a second CR (CR2). Of those, 13 (68%) had a low end-reinduction minimal residual disease (MRD) level (≤10-3 by polymerase chain reaction-based assay). The CR2 rate for patients with B-cell ALL treated at DL3 (n = 12) was 92%; 82% of these patients had low MRD. Conclusions Everolimus combined with four-drug reinduction chemotherapy was generally well tolerated and associated with favorable rates of CR2 and low end-reinduction MRD. The recommended phase 2 dose of everolimus given in combination with a four-drug reinduction is 5 mg/m2 /day. This promising combination should be further evaluated in a larger patient cohort.

Background Most patients with juvenile myelomonocytic leukemia (JMML) are curable only with allogeneic hematopoietic cell transplantation (HCT). However, the current standard conditioning regimen, busulfan-cyclophosphamide-melphalan (Bu-Cy-Mel), may be associated with higher risks of morbidity and mortality. ASCT1221 was designed to test whether the potentially less-toxic myeloablative conditioning regimen containing busulfan-fludarabine (Bu-Flu) would be associated with equivalent outcomes. Procedure Twenty-seven patients were enrolled on ASCT1221 from 2013 to 2015. Pre- and post-HCT (starting Day +30) mutant allele burden was measured in all and pre-HCT therapy was administered according to physician discretion. Results Fifteen patients were randomized (six to Bu-Cy-Mel and nine to Bu-Flu) after meeting diagnostic criteria for JMML. Pre-HCT low-dose chemotherapy did not appear to reduce pre-HCT disease burden. Two patients, however, received aggressive chemotherapy pre-HCT and achieved low disease-burden state; both are long-term survivors. All four patients with detectable mutant allele burden at Day +30 post-HCT eventually progressed compared to two of nine patients with unmeasurable allele burden (P = 0.04). The 18-month event-free survival of the entire cohort was 47% (95% CI, 21-69%), and was 83% (95% CI, 27-97%) and 22% (95% CI, 03-51%) for Bu-Cy-Mel and Bu-Flu, respectively (P = 0.04). ASCT1221 was terminated early due to concerns that the Bu-Flu arm had inferior outcomes. Conclusions The regimen of Bu-Flu is inadequate to provide disease control in patients with JMML who present to HCT with large burdens of disease. Advances in molecular testing may allow better characterization of biologic risk, pre-HCT responses to chemotherapy, and post-HCT management.

Purpose: To determine a safe and biologically active dose of quizartinib (AC220), a potent and selective class III receptor tyrosine kinase (RTK) FLT3 inhibitor, in combination with salvage chemotherapy in children with relapsed acute leukemia. Experimental Design: Quizartinib was administered orally to children with relapsed AML or MLL-rearranged ALL following 5 days of high-dose cytarabine and etoposide (AE). A 3+3 dose escalation design was used to identify a safe and biologically active dose. Plasma inhibitory assay (PIA) testing was performed weekly to determine biologic activity. Results: Toxicities were consistent with intensive relapsed leukemia regimens. One of 6 patients experienced a dose-limiting toxicity (DLT) at 40 mg/m2/day (elevated lipase) and 1 of 9 had a DLT (hyperbilirubinemia) at the highest tested dose of 60 mg/m2/day. Of 17 response evaluable patients, 2 had complete response (CR), 1 complete response without platelet recovery (CRp), 1 complete response with incomplete neutrophil and platelet recovery (CRi), 10 stable disease (SD), and 3 progressive disease (PD). Of 7 FLT3-ITD patients, 1 achieved CR, 1 CRp, 1 Cri, and 4 SD. FLT3-ITD patients, but not FLT3 wild-type (WT) patients, had significantly lower blast counts post-quizartinib. FLT3 phosphorylation was completely inhibited in all patients. Conclusions: Quizartinib plus intensive chemotherapy is well tolerated at 60 mg/m2/day with near complete inhibition of FLT3 phosphorylation in all patients. The favorable toxicity profile, pharmacodynamic activity, and encouraging response rates warrant further testing of quizartinib in children with FLT3-ITD AML. Clin Cancer Res; 22(16); 4014–22. ©2016 AACR.

Introduction: MLL-rearranged (MLL-r) acute leukemia in children is characterized by young age at presentation and a poor overall prognosis despite multi-agent chemotherapy. Aberrant fusion proteins involving the MLL histone methyltransferase (HMT) recruit another HMT, DOT1L, to a multi-protein complex leading to aberrant methylation of histone H3 lysine 79 (H3K79) at MLL target genes. This results in enhanced expression of critical genes for hematopoietic differentiation, including HOXA9 and MEIS1, and has been established as a key mechanism for leukemogenesis in MLL-r leukemias (Krivstov, 2007). Pinometostat is a small molecule inhibitor of DOT1L with sub-nanomolar affinity and >37,000 fold selectivity against non-MLL HMTs. Treatment of MLL-rearranged cells and xenograft models with pinometostat led to reduced histone 3 lysine 79 methylation (H3K79me2), decreased MLL target gene expression and selective leukemia cell kill (Daigle, 2013). Here we report the final results of the pinometostat phase 1 trial in children with relapsed/refractory (R/R) MLL-r acute leukemia. Methods: An open label dose escalation study of pinometostat was performed in patients (pts) aged 3 months to 18 years (yr) with R/R MLL-r leukemia (NCT02141828). Pinometostat was administered via continuous intravenous infusion (CIV) until disease progression or unacceptable toxicity. Pts were assigned to one of two aged-based dose escalation schemas developed from simulations of pediatric exposures using a previously reported physiologically-based PK (PBPK) model (Waters, 2014). All patients underwent serial collection of PK and peripheral blood mononuclear cells (PBMC). Leukemic blasts were isolated from PBMCs using flow cytometry and quantified for H3K79me2 levels by ChIP-Seq. Results: 18 pts were enrolled on study with 9 pts dosed at 70 mg/m2/day and 7 pts at 90 mg/m2/day in the older age cohort (1 to 18 yr) plus 2 pts dosed at 45 mg/m2/day in the younger age cohort ( Grade ≥3 treatment-emergent adverse events (TEAEs) regardless of attribution reported in >20% of pts were: febrile neutropenia; anemia; leukopenia; thrombocytopenia; hypokalemia; respiratory failure; lymphopenia; neutropenia. Drug-related TEAEs reported in >15% of pts were: anemia; thrombocytopenia; leukopenia; rash; lymphopenia; hypocalcemia; hypophosphatemia; neutropenia; ALT elevation; nausea; vomiting. Dose-limiting toxicities (DLT) were: apnea (70 mg/m2); elevated transaminases (2 at 90 mg/m2) in the >1 year of age cohort, thus defining 70 mg/m2 as the recommended phase 2 dose (RP2D) in older pts. A RP2D was not determined in pts < 1 year of age. Median duration of treatment was 23 days (range 7- 53 days). Pinometostat induced transient decreases in peripheral or marrow leukemic blasts in 7/18 pts, however, these reductions did not meet formal thresholds for objective response. Adjusted mean plasma pinometostat concentrations during the infusion in children >1 yr at 70 and 90 mg/m2/d doses (1151 ng/mL) was comparable to mean steady-state concentrations observed in adult patients at the 80 mg/m2/d and 90 mg/m2/d doses (1320 ng/mL and 1410 ng/mL, respectively) and was within the range of 1000-1600 ng/mL at 90 mg/m2/d in ≥ 1 yr predicted by earlier PBPK modeling results. CSF concentrations of pinometostat were below the lower limit of quantification of 1 ng/mL (n = 8) or very low (< 12 ng/mL) (n = 4), suggesting negligible CSF exposure. H3K79me2 ChIP-Seq on leukemic blasts demonstrated that pinometostat induced reductions in methylation at MLL-r target genes (e.g. HOXA9 and MEIS1) of ≥ 80 % at all post dose time points (15 and 28 days) and doses (70 & 90 mg/m2) tested consistent with DOT1L inhibition. Conclusions: In pediatric pts with R/R MLL-r pinometostat has an acceptable safety profile with a RP2D defined as 70 mg/m2 CIV in children > 1 yr. Pinometostat dose/exposure relationships were comparable between adults and children > 1 yr. Pharmacodynamic evidence of DOT1L inhibition was observed in leukemic blasts. Transient reductions in peripheral or bone marrow blasts were detected in ~40 % of pts, however no objective responses were observed. Based on the biological activity observed and evidence of combination benefit of pinometostat with standard of care and novel agents in preclinical MLL-r models (Daigle 2015 and Klaus 2015) further clinical investigation of pinometostat combinations is warranted. Figure Figure. Disclosures O'Brien: Seattle Genetics: Research Funding; Celgene: Other: travel expenses for required site investigator meeting, October 2015;; steering committee member for pediatric AML trial, Research Funding; Epizyme: Research Funding; Amgen: Other: participated in one pediatric advisory board for blinatumomab in May 2015, paid consultant fee and travel expenses, Research Funding. Blakemore:Epizyme: Employment. Daigle:Epizyme: Employment. Suttle:Epizyme: Employment. Armstrong:Epizyme, Inc: Consultancy; Vitae Pharmaceuticals: Consultancy; Imago Biosciences: Consultancy; Janssen Pharmaceutical: Consultancy. Ho:Epizyme: Employment, Equity Ownership.

The range of genomic drivers of leukemogenesis and clonal nature of the disease illustrate the heterogeneity of the mutational spectrum in AML. Genomic interrogation of the evolution of AML has begun to highlight the scope of somatic changes that occur between diagnosis and relapse. A total of 1,214 patients were treated on the Children's Oncology Group trials AAML03P1 and AAML0531, of which 398 had relapse after initial remission. Of this cohort, 201 patients had matching diagnostic and relapse specimens for molecular profiling for the most common somatic mutations in pediatric AML (FLT3/ITD, FLT3/ALM, NPM1, CEBPA, WT1, NRAS, and KIT). Sequencing techniques included PCR with Sanger sequencing for detection of point mutations and indels and fragment length analysis for FLT3/ITD. In the cohort, FLT3/ITD was detected in 31/201 (15%) cases at diagnosis. Of the cases with diagnostic ITD, 22 (71%) relapsed with FLT3/ITD. Conversely, of the 28 cases with FLT3/ITD detected at relapse, 6 (21%) did not have detectable ITD at diagnosis. Overall, there were 37 patients (18%) with FLT3/ITD mutations detected at either time point. Of the 37 patients, 22 (59%) demonstrated stability of the mutation from diagnosis to relapse. Discordant mutation status was observed in 15 patients (41%). Among the discordant patients, 9 had FLT3/ITD detected at diagnosis only. Conversely, 6 patients were ITD-positive at relapse only, demonstrating disease evolution with continued mutational acquisition (Table 1). In every discordant case, ultra sensitive PCR analysis confirmed absence of an ITD. The median ITD allelic ratio (AR) for patients with concordant status was 0.47 (range 0.03-2.67) vs. 0.24 (range 0.04-0.47) for those with disappearance of the ITD at relapse, suggesting an association of diagnostic AR with mutation stability. NPM1 mutations were detected in 8 patients at diagnosis and 100% concordance was observed in the cohort. CEBPA mutations were detected in 6 patients at diagnosis, and in 5 cases remained at relapse. One patient had a CEBPA mutation detected at diagnosis only. FLT3/ALM mutations were detected in 7 patients at either time point. Seven patients had an ALM at diagnosis, however concordance was observed in 2 cases, whereas 4 patients had detection at diagnosis only. There were 22 patients (11%) with NRAS mutations detected at either time point. Diagnostic NRAS mutations were detected in 18 patients, while only 3 (17%) had the identical mutation detected at relapse, as one patient had a distinct mutational sequence present at relapse. NRAS mutations were detected at diagnosis only in 13 patients (59%), where as 5 patients (23%) had a mutation detected at relapse only. NRAS was the most discordant mutations analyzed, with only 3/22 patients (14%) demonstrating stability of the mutation from diagnosis to relapse (Table 1). WT1 exon 17 indels were observed in 24 patients (12%) at either time point. Nineteen patients had diagnostic mutations, with 18 patients demonstrating stability at relapse. Five patients had mutations detected at relapse only. Overall, concordance was observed in 18 patients (75%). Only 1 alteration was detected at diagnosis in all patients, however 6 patients with concordant WT1 status had multiple indels detected at relapse, demonstrating continued mutational acquisition. KIT mutations (missense and indels) in exons 8 (n=11) and 17 (n=7) were detected in 17 patients. Mutational concordance was observed in 7 patients. Eight patients had mutations detected at diagnosis only, while 2 patients had mutations detected at relapse only (Table 1). We demonstrate the complexity of the evolving somatic landscape from diagnosis to relapse in pediatric AML. The stability of NPM1 mutations, considered an early leukemogenic event, is in contrast to the discordant NRAS and KIT mutations. There was evolution of FLT3/ITD status, and we observed overall higher ARs in the concordant cohort, perhaps suggesting mutations in this cohort served as stronger leukemic drivers. Further investigation on the biologic implications and clonal prevalence is critical to determine a mutation's significance in leukemogenesis, timing of acquisition, and if appropriate for therapeutic targeting and disease monitoring. Table 1 Mutational concordance from diagnosis to relapse. Legend: Discordant D+/R-: Discordant status with diagnostic only positive Discordant D-/R+: Discordant status with relapse only positive Table 1. Mutational concordance from diagnosis to relapse. / Legend:. / Discordant D+/R-: Discordant status with diagnostic only positive. / Discordant D-/R+: Discordant status with relapse only positive Disclosures No relevant conflicts of interest to declare.

Introduction: The Children's Oncology Group (COG) conducted a randomized, Phase III study evaluating Gemtuzumab Ozogamicin (GO), a humanized anti-CD33 antibody, for children with de novo AML. This analysis describes longer term outcomes for patients assigned to GO as well as relapse risk factors and subsequent outcome for patients experiencing relapse after AAML0531 therapy. Methods: AAML0531 enrolled 1,022 evaluable patients ages 0-29 to receive either standard five-course chemotherapy with or without 2 doses of GO. All high risk and those intermediate risk patients with family donors received stem cell transplant rather than the last 2 chemotherapy cycles and the 2nd GO dose. Analysis of characteristics impacting cumulative incidence to relapse and overall survival from relapse were performed (data cutoff 3/31/16). Results: Updated outcome analyses demonstrated GO improved 5 year (yr) event-free survival (EFS) of 51.4 ± 4.5% vs 48.5 ± 3.2% p=0.055, but no benefit in 5 yr overall survival (OS), 64.9 ± 4.4% v 64.1 ± 3.1% p=0.406. A 5-yr cumulative incidence to relapse (REL) from complete remission (CR) (N= 849) was seen in 37.8% of patients, with significantly less among those receiving GO vs no-GO (33.6% vs 42.2% p=0.01). In Univariate analysis, children with NPM and CEPBA mutations, low risk cytogenetics, and higher CD33 expression had lower relapse risk (RR). Those with extramedullary disease (EMD) at diagnosis, FLT3/ITD mutations, CNS3 disease, and minimal residual disease at end of induction 1 and 2 predicted higher RR (Table 1). Multivariate analysis determined that EMD at diagnosis (HR 1.68, 95% CI 1.10-2.56, p=0.016) and minimal residual disease (MRD) at end of induction 1 (EOI1) (HR 1.61, 95% CI 1.13-2.30, p= 0.008) predicted significantly higher RR, while low risk cytogenetics predicted lower RR (HR 0.48 95% CI 0.33-0.70, p 1 yr from first CR had significantly better 5-yr OS after REL (Table 2). Conclusions: AAML0531 previously reported GO significantly improves EFS by reducing RR when given to children with de novo AML. This cumulative analysis updates prognostic factors influencing RR and subsequent impact upon OS after REL. EMD at diagnosis and MRD at EOI1 are significantly predictive of relapse risk in multivariate analysis while those with low risk cytogenetics have significantly lower incidence of relapse. While GO significantly improves the 5 yr incidence of relapse from first remission, it does not impact 5-yr OS after first relapse. GO does improve 180-day OS after first relapse, but this impact loses significance at 1 yr post REL. Overall survival is significantly impacted by the length of 1st CR, with worst prognosis for those relapsing within 180 days. Work is ongoing to define other risk factors for altered OS. Disclosures No relevant conflicts of interest to declare.

Nat. Genet. 47, 1326–1333 (2015); published online 12 October 2015; corrected after print 7 December 2015 In the version of this article initially published, two patients were stated on page 5 to have been excluded owing to insufficient follow-up data. These patients were included in the final analysis, but two additional patients were excluded owing to the presence of Noonan syndrome.

Anita K. McElroy, Joseph A. Hilinski, Carlos R. Abramowsky, Ronald Jaffe, Sunita I. Park, Bahig M. Shehata, and ToddM. Cooper Divisions of Infectious Diseases and Hematology/Oncology, Department of Pediatrics, and Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia;, and Department of Pathology, University of Pittsburgh School of Medicine, Pennsylvania

Introduction: Treatment of relapsed childhood acute lymphoblastic leukemia (ALL) remains a significant clinical challenge with a need for novel, more effective therapies. Pre-clinical studies have demonstrated that mTOR inhibitors have single agent activity in ALL and are synergistic with other chemotherapy agents typically used to treat ALL, including vincristine, corticosteroids, doxorubicin and L-asparaginase. Methods: Dana-Farber Cancer Institute Protocol 11-237 tested the combination of everolimus (given orally on days 1-32) with a standard reinduction regimen of prednisone (40 mg/m2/daily orally on days 4-32), weekly vincristine (1.5 mg/m2/dose, max 2 mg, on days 4,11,18 and 25), PEG-asparaginase (2,500 U/m2/dose IV on days 5 and 18) and doxorubicin (30 mg/m2/dose on days 4 and 5) with dexrazoxane pretreatment (300 mg/m2/dose on days 4 and 5). Intrathecal cytarabine was administered on day 1 and intrathecal methotrexate, cytarabine and hydrocortisone (IT-MAH) was administered on days 18 and 32; patients (pts) with cerebrospinal fluid blasts at study entry received additional IT-MAH on days 11 and 25. Pts with first bone marrow relapse ALL occurring greater than 18 months after continuous remission were eligible for participation. The primary aim was to identify a maximum tolerated dose of everolimus in combination with this backbone utilizing a classic "3 + 3" dose escalation design. Three dose levels (DL) of everolimus were tested (2-, 3-, and 5-mg/m2/day). 5 mg/m2/day (DL3) was the highest dose tested because it was the recommended phase 2 dose (RP2D) of single-agent everolimus in children with solid tumors. Secondary aims included assessment of second complete remission (CR2) rate and levels of end-reinduction minimal residual disease (MRD) assessed by allele-specific oligonucleotide PCR. On-target activity of everolimus was assessed 6 hours after the first dose of everolimus by examining phosphorylated S6 (pS6) levels in peripheral blood blasts by flow cytometry. Results: Thirteen pts with first relapse B-ALL were enrolled. (Table 1) Median age at enrollment was 10 yrs (range: 2-22 yrs). Median duration of first CR was 3.7 yrs (range 1.5-7.7 yrs). One pt treated at DL 3 stopped study drug early due to Grade 4 infection (meningitis due to Rothia mucilaginosa ) and was inevaluable for dose limiting toxicity (DLT) assessment. Table 2 displays the most common Grade 2 or higher adverse events (AE's) attributable to everolimus. There were no Grade 5 events and no episodes of interstitial pneumonitis. No DLTs were observed at DL1 or DL2. One of 6 evaluable pts treated at DL3 experienced a DLT (Grade 4 hyperbilirubinemia). 12 of the 13 pts achieved CR (92%). Of the 12 pts who achieved CR, 9 (75%) had low end-reinduction MRD (²0.001). (Table 1) Of the 7 pts with evaluable samples (2 at DL1, 1 at DL2 and 4 at DL3), 6 (86%) had decrease in pS6 in peripheral blasts (>20% decrease in mean fluorescence intensity), consistent with on-target mTOR inhibition. Only 1 pt (treated at DL1) did not have evidence of pS6 inhibition; this pt achieved CR2 with low MRD. Discussion: Everolimus given in combination with a standard 4-drug reinduction was well tolerated at all dose levels tested in pediatric pts with first relapse ALL. CR2 rates were comparable to published re-induction regimens, with a promising proportion of pts with low end-reinduction MRD. An expansion cohort is currently enrolling to further assess toxicity of this combination, particularly infectious risks, in order to define the RP2D. | Pt # | Dose Level | Age (yrs) | Duration of CR1 (yrs) | DLT? | CR? | MRD | | ---- | ---------- | --------- | --------------------- | ---- | --- | ---- | | 001 | 1 | 10 | 7.7 | No | Yes | High | | 002 | 1 | 20 | 3.5 | No | Yes | Low | | 003 | 1 | 8 | 3.1 | No | Yes | Low | | 004 | 2 | 2 | 1.5 | No | Yes | High | | 005 | 2 | 22 | 5.1 | No | No | N/A | | 006 | 2 | 5 | 3.7 | No | Yes | Low | | 007 | 3 | 12 | 5.7 | No | Yes | Low | | 008 | 3 | 10 | 3.0 | N/A | Yes | Low | | 009 | 3 | 11 | 4.3 | No | Yes | Low | | 010 | 3 | 21 | 4.8 | Yes | Yes | High | | 011 | 3 | 7 | 3.4 | No | Yes | Low | | 012 | 3 | 13 | 5.0 | No | Yes | Low | | 013 | 3 | 17 | 3.0 | No | Yes | Low | Table 1. Patients and Response to Therapy | Toxicity | Grade 2 | Grade 3 | Grade 4 | Grade 5 | | ----------------------- | ------- | ------- | ------- | ------- | | Bacteremia | | 3 | | | | Bacterial meningitis | | | 1 | | | Invasive fungal disease | | 1 | | | | Febrile Neutropenia | | 7 | | | | Mucositis | 2 | 2 | | | | Anorexia | 3 | 1 | | | | AST increase | 1 | 5 | 2 | | | ALT increase | 1 | 5 | 2 | | | Bilirubin increase | | 2 | 1 | | | Hypophosphatemia | 2 | 5 | 1 | | Table 2. Grade 2 or higher Adverse Events Attributable to Everolimus (Possible, Probable, Definite) at all dose levels; N=13 patients Disclosures Pauly: Seattle Genetics, Inc.: Research Funding. Stegmaier: Novartis Pharmaceuticals: Consultancy.