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Acute myeloid leukemia (AML) remains a devastating disease in need of new therapies to improve patient survival. Targeted adoptive T-cell therapies have achieved impressive clinical outcomes in some B-cell leukemias and lymphomas but not in AML. Double-negative T cells (DNTs) effectively kill blast cells from the majority of AML patients and are now being tested in clinical trials. However, AML blasts obtained from ∼30% of patients show resistance to DNT-mediated cytotoxicity; the markers or mechanisms underlying this resistance have not been elucidated. Here, we used a targeted clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) screen to identify genes that cause susceptibility of AML cells to DNT therapy. Inactivation of the Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitinating complex components sensitized AML cells to DNT-mediated cytotoxicity. In contrast, CD64 inactivation resulted in resistance to DNT-mediated cytotoxicity. Importantly, the level of CD64 expression correlated strongly with the sensitivity of AML cells to DNT treatment. Furthermore, the ectopic expression of CD64 overcame AML resistance to DNTs in vitro and in vivo. Altogether, our data demonstrate the utility of CRISPR/Cas9 screens to uncover mechanisms underlying the sensitivity to DNT therapy and suggest CD64 as a predictive marker for response in AML patients.

The impact of adverse risk genetic profiles on outcomes in acute myeloid leukemia (AML) patients following allogeneic hematopoietic stem cell transplantation (HCT) has not been fully elucidated. Accordingly, we have profiled somatic mutations at diagnosis using next-generation sequencing (NGS) in 178 AML patients who received allogeneic HCT. NGS revealed 598 somatic mutations in 165/178 patients (92.7%). Frequently mutated genes include DNMT3A, TET2, NPM1, RUNX1, IDH2, and FLT3. Commonly detected cytogenetic profiles include normal karyotype, trisomy 8, monosomal karyotype (MK), deletion 5, complex karyotype (CK), and monosomy 7. In univariate analyses, TP53 mutation, MK, CK, and monosomy 7 were associated with decreased overall survival (OS), relapse-free survival (RFS), and a higher relapse incidence (RI). We defined adverse molecular-genetic profile as harboring at least one of the molecular/genetic abnormalities of TP53 mutation, MK, CK, monosomy 7, and deletion 5. The patients harboring adverse molecular-genetic profile (n = 30) showed a lower 2-year OS (24.9% vs. 57.9%; p = 0.003), RFS (23.7% vs. 57.9%; p = 0.002), and higher RI (47.2% and 17.2%; p = 0.001) after HCT when compared to patients without those lesions. Multivariate analysis confirmed adverse molecular-genetic profile as an independent prognostic factor, associated with decreased OS (HR 2.19), RFS (HR 2.23), and higher RI (HR 2.94).

Background: venous thromboembolism (VTE) is a well-known complication in adults with acute lymphoblastic leukemia (ALL), especially in patients treated with asparaginase (ASNase)-including regiments. However, VTE risk in adult Philadelphia-positive ALL (Ph+ve ALL) patients treated with non-hyperCVAD chemotherapy is unclear. In this study, we examined VTE incidence in adult Ph+ve ALL patients treated with imatinib plus a pediatric-inspired asparaginase (ASNase)-free regimen modified from the Dana Farber Cancer Institute (DFCI) ALL protocol. Methods: a single centre retrospective review of Ph+ve ALL patients treated at Princess Margaret Cancer Center (PMCC) from 2008&ndash, 2019 with imatinib plus modified DFCI protocol was conducted. Results: of the 123 patients included, 30 (24.3%) had at least 1 radiology confirmed VTE event from diagnosis to the end of maintenance therapy. 86.7% (26/30) of the VTE events occurred during active treatment. Of all VTE events, the majority (53.3%) were DVT and/or PE while another significant portion were catheter-related (40.0%). Major bleeding was observed in 1 patient on VTE treatment with low molecular weight heparin (LMWH). Conclusion: a high VTE incidence (24.3%) was observed in adults Ph+ve ALL patients treated with imatinib plus an ASNase-free modified DFCI pediatric ALL protocol, suggesting prophylactic anticoagulation should be considered for all adult Ph+ve ALL patients including those treated with ASNase-free regimens.

Purpose: Spleen tyrosine kinase (SYK) signaling is a proposed target in acute myeloid leukemia (AML). Sensitivity to SYK inhibition has been linked to HOXA9 and MEIS1 overexpression in preclinical studies. This trial evaluated the safety and efficacy of entospletinib, a selective inhibitor of SYK, in combination with chemotherapy in untreated AML. Patients and Methods: This was an international multicenter phase Ib/II study, entospletinib dose escalation (standard 3+3 design between 200 and 400 mg twice daily) + 7+3 (cytarabine + daunorubicin) in phase Ib and entospletinib dose expansion (400 mg twice daily) + 7+3 in phase II. Results: Fifty-three patients (n = 12, phase Ib and n = 41, phase II) with previously untreated de novo (n = 39) or secondary (n = 14) AML were enrolled (58% male; median age, 60 years) in this study. The composite complete response with entospletinib + 7+3 was 70%. Patients with baseline HOXA9 and MEIS1 expression higher than the median had improved overall survival compared with patients with below median HOXA9 and MEIS1 expression. Common adverse events were cytopenias, febrile neutropenia, and infection. There were no dose-limiting toxicities. Entospletinib-related skin rash and hyperbilirubinemia were also observed. Conclusions: Entospletinib with intensive chemotherapy was well-tolerated in patients with AML. Improved survival was observed in patients with HOXA9/MEIS1 overexpression, contrasting published data demonstrating poor survival in such patients. A randomized study will be necessary to determine whether entospletinib was a mediator this observation.

NPM1 mutation status and the allelic ratio (AR) of FLT3-internal tandem duplication (FLT3-ITD) are routinely tested for disease risk stratification in patients with normal karyotype (NK) acute myelogenous leukemia (AML); however, the predictive impact of immunophenotypic markers on different NPM1/FLT3 genotypes remains unclear. We performed a retrospective analysis of 423 patients with NK-AML subclassified into groups based on NPM1/FLT3 genotype. Allogeneic hematopoietic stem cell transplantation (HSCT) was performed in 124 of 423 patients (29%) and was significantly associated with longer event-free survival (EFS) and overall survival (OS), except for patients with the favorable genotype, defined as mutated NPM1 (NPM1mut) combined with normal FLT3 status (FLT3-ITDneg) or FLT3-ITD AR

Acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) generally confers poor prognosis; however, the clinical outcome remains heterogeneous. We sought to further stratify this subentity of AML by performing a retrospective analysis of 179 adult patients with AML-MRC diagnosed at our institution. Based on 2016 World Health Organization diagnostic criteria, 44 (25%) patients had multilineage dysplasia alone (AML-MRC-M), 74 (41%) had history of myelodysplastic syndrome (MDS) or myelodysplastic/myeloproliferative disease (AML-MRC-H), and 61 (34%) had MDS-related cytogenetics (AML-MRC-C). AML-MRC-M and hematopoietic stem cell transplantation (HSCT) were associated with prolonged event-free survival (EFS) (P = 0.0051 and P 0.0001, respectively) and overall survival (OS) (P = 0.0015 and P 0.0001, respectively), whereas AML-MRC-C and age ≥60 years were associated with shorter EFS (P = 0.028 and P = 0.015, respectively) and OS (P = 0.021 and P = 0.013, respectively). Of note, NPM1

Dipyridamole, an antiplatelet drug, synergizes with statins to induce tumor‐specific apoptosis. Here, we took a pharmacological approach to dissect the mechanism of action. By evaluating compounds that phenocopy the individual activities of dipyridamole, we uncovered that cAMP‐hydrolyzing phosphodiesterase inhibitors can potentiate statin‐induced apoptosis via a dual mechanism. This approach may be useful when interrogating novel functions of other repurposed drugs., Dipyridamole, an antiplatelet drug, has been shown to synergize with statins to induce cancer cell‐specific apoptosis. However, given the polypharmacology of dipyridamole, the mechanism by which it potentiates statin‐induced apoptosis remains unclear. Here, we applied a pharmacological approach to identify the activity of dipyridamole specific to its synergistic anticancer interaction with statins. We evaluated compounds that phenocopy the individual activities of dipyridamole and assessed whether they could potentiate statin‐induced cell death. Notably, we identified that a phosphodiesterase (PDE) inhibitor, cilostazol, and other compounds that increase intracellular cyclic adenosine monophosphate (cAMP) levels potentiate statin‐induced apoptosis in acute myeloid leukemia and multiple myeloma cells. Additionally, we demonstrated that both dipyridamole and cilostazol further inhibit statin‐induced activation of sterol regulatory element‐binding protein 2, a known modulator of statin sensitivity, in a cAMP‐independent manner. Taken together, our data support that PDE inhibitors such as dipyridamole and cilostazol can potentiate statin‐induced apoptosis via a dual mechanism. Given that several PDE inhibitors are clinically approved for various indications, they are immediately available for testing in combination with statins for the treatment of hematological malignancies.

Simple Summary Approximately 50–60% of patients with chronic myeloid leukemia (CML) achieve a stable deep molecular response (DMR) after tyrosine kinase inhibitor (TKI) therapy. The achievement of DMR is a prerequisite for treatment-free remission. Repurposing statins is a straightforward strategy for enhancing molecular response in CML treatment. Second-generation TKIs have been reported to exhibit cardiovascular toxicity. Thus, statins have been widely prescribed for patients with CML undergoing second-generation TKI therapy for modifying cardiovascular risk factors, such as hyperlipidemia. Furthermore, the results of this study support the therapeutic benefit of the concomitant use of statins in TKI therapy for patients with CML. Additionally, the potential additive effects of statins and TKIs enhance the DMR rate in patients with CML, rendering these effects clinically relevant in these patients. In particular, this combination is a strong candidate for the achievement of DMR in patients with CML who have not achieved DMR with TKI therapy alone. Abstract Previous studies have suggested that statins can be repurposed for cancer treatment. However, the therapeutic efficacy of statins in chronic myeloid leukemia (CML) has not yet been demonstrated. In this study, we retrospectively evaluated the outcomes of 408 CML patients who underwent imatinib therapy. The deep molecular response rates in patients treated with the statin/TKI combination were significantly higher than those in patients treated with TKI alone (p = 0.0016). The statin/TKI combination exerted potent cytotoxic effects against wild-type and ABL1 mutant CML, BaF3, and K562/T315I mutant cells. Furthermore, the statin/TKI combination additively inhibited the colony-forming capacity of murine CML-KLS+ cells in vitro. In addition, we examined the additive growth-inhibitory effects of the statin/tyrosine kinase inhibitor (TKI) combination against CML patient-derived CD34+ cells. The growth-inhibitory effects of the statin/imatinib combination against CD34+/CML primary cells were higher than those against CD34+/Norm cells (p = 0.005), suggesting that the combination of rosuvastatin and imatinib exerted growth-inhibitory effects against CML CD34+ cells, but not against normal CD34+ cells. Furthermore, results from RNA sequencing of control and statin-treated cells suggested that statins inhibited c-Myc-mediated and hematopoietic cell differentiation pathways. Thus, statins can be potentially repurposed to improve treatment outcomes in CML patients when combined with TKI therapy.

Background CD47 is an innate immune checkpoint that binds signal regulatory protein alpha (SIRPα) and delivers a "do not eat" signal to suppress macrophage phagocytosis. Overexpression of CD47 can allow tumor cells to escape immune surveillance. TTI-621 (SIRPαFc) is a fusion protein consisting of the CD47 binding domain of human SIRPα linked to the Fc region of human IgG1, designed to enhance phagocytosis and antitumor activity by blocking the CD47-SIRPα interaction between malignant cells and macrophages, and engaging Fcγ receptors. Here we report updates from the first-in-human study of TTI-621 (NCT02663518) in hematologic malignancies. Methods Study Part 1 tested increasing weekly intravenous doses of TTI-621 based on 3+3 escalation. The maximum tolerated dose was initially determined to be 0.2 mg/kg based on dose limiting toxicity (DLT) of thrombocytopenia [Grade (Gr) 4 of any duration]. Expanded testing followed in patients (pts) with hematologic malignancies, including leukemia, lymphoma, and multiple myeloma. In Part 2, most pts received 0.2 mg/kg. However, based on investigator discretion, a subset of pts received escalating doses up to 0.5 mg/kg. In Part 3, pts with T-cell lymphomas received stepwise dose escalations from 0.2 to 0.5 mg/kg over the first 5−8 weeks. In over 200 pts tested in Parts 1−3, thrombocytopenia did not increase with dose, typically recovered within 2−4 days, and was not associated with clinical sequelae. Part 4 was then undertaken to optimize TTI-621 dosing and is currently escalating doses in a 3+3 manner through pre-planned dose levels (0.5, 0.7, 1, and 1.4 mg/kg) in pts with cutaneous T-cell lymphoma (CTCL). The DLT criteria was modified to require Gr 4 thrombocytopenia lasting >72 hours. Safety monitoring includes weekly clinical laboratories and assessments of adverse events (AEs) per CTCAE v 4.03. Blood samples are obtained for pharmacokinetics (PK) and for pharmacodynamic (PD) assessments of receptor occupancy (RO) on normal peripheral T cells. Disease assessments are performed per Olsen's criteria. Results In Parts 1−3 (n=214), the most common related AEs were infusion-related reaction (IRR, 43%; 3% Gr ≥3), thrombocytopenia (30%; 22% Gr ≥3), chills (21%; 0% Gr ≥3), and fatigue (15%; 1% Gr ≥3). Objective responses to single agent TTI-621 were achieved in 14/71 (20%) NHL pts including CTCL (n=42, 1 CR, 7 PRs), PTCL (n=22, 2 CRs, 2 PRs) and DLBCL (n=7, 1 CR, 1 PR). In Part 4, as of July 10, 2020, 15 pts (9M/6F, median age 67 years) have enrolled into 4 dose cohorts (0.5−1.4 mg/kg). CTCL subtypes include mycosis fungoides (MF, n=10) and Sézary syndrome (n=5) with advanced (≥IIB) disease in 9 (60%) pts who received a median of 3 (range 1−12) prior systemic therapies. Related AEs have occurred in 11 (73%) pts including IRR (n=10) and thrombocytopenia (n=3); Gr ≥3 AEs have occurred in 4 (27%) pts including thrombocytopenia (n=3), IRRs (n=2), and exfoliative dermatitis (n=1). Thrombocytopenia generally occurred on dosing days, recovered in 2-4 days, and has not worsened with increasing doses. IRRs typically occurred during initial infusions. The Gr 3 IRR events occurred in 2 pts in the 1 and 1.4 mg/kg cohorts; low Gr IRRs have occurred across doses in 8 pts. IRRs typically resolved without recurrence and low Gr events often resolved allowing for completion of infusions. For initial infusions, the Gr 3 IRRs prompted increasing infusion times from 1 hour up to 4 hours and discretional use of steroid pre-medication. The exfoliative dermatitis occurred Day 80 and led to treatment discontinuation in 1 pt with MF whose underlying disease confounded the etiology. PK results reveal dose dependent increases in exposure; PD studies indicate ~60% RO at end of infusion up to 1 mg/kg. Antitumor activity to date includes 1 PR and 1 skin CR in 6 evaluable pts in the 1 mg/kg cohort; 2 responding pts bridged to allogeneic transplantation. The mean % change in mSWAT scores were -0.4%, -27%, and -37% for 0.5, 0.7 and 1 mg/kg cohorts, respectively. 1.4 mg/kg cohort results will be presented at the meeting. Conclusions In Parts 1−3, TTI-621 doses of 0.05 to 0.5 mg/kg were well-tolerated and demonstrated single agent activity in multiple hematologic malignancies. Preliminary data from Part 4 dose optimization indicate that weekly infusions of TTI-621 up to 1.4 mg/kg are well-tolerated without dose limiting or cumulative thrombocytopenia. Antitumor activity was seen at 1 mg/kg; dose escalation is continuing at 2 mg/kg. Disclosures Horwitz: Janssen: Consultancy; Verastem: Consultancy, Research Funding; Daiichi Sankyo: Research Funding; GlaxoSmithKline: Consultancy; ASTEX: Consultancy; Portola: Consultancy, Research Funding; C4 Therapeutics: Consultancy; Beigene: Consultancy; Myeloid Therapeutics: Consultancy; Vividion Therapeutics: Consultancy; Infinity/Verastem: Research Funding; Aileron: Consultancy, Research Funding; ADCT Therapeutics: Consultancy, Research Funding; Affirmed: Consultancy; Trillium: Consultancy, Research Funding; Corvus: Consultancy; Millenium/Takeda: Consultancy, Research Funding; Kyowa Hakka Kirin: Consultancy, Research Funding; Innate Pharma: Consultancy; Forty Seven: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Kura Oncology: Consultancy; Miragen: Consultancy; Mundipharma: Consultancy. Foran:Agios: Honoraria, Research Funding; H3Biosciences: Research Funding; Xencor: Research Funding; Trillium: Research Funding; Takeda: Research Funding; Kura Oncology: Research Funding; Aptose: Research Funding; Aprea: Research Funding; Actinium: Research Funding; Boehringer Ingelheim: Research Funding; Abbvie: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Revolution Medicine: Consultancy. Sawas:Flatiron Health: Current Employment; Gilead: Speakers Bureau; Seattle Genetics: Speakers Bureau; Daiichi Sankyo: Speakers Bureau; Affimed: Research Funding; Roche: Current equity holder in publicly-traded company. Feldman:AstraZeneca: Consultancy; Viracta: Research Funding; Trillium: Research Funding; Pfizer: Research Funding; Janssen: Speakers Bureau; Bayer: Consultancy, Honoraria; Kyowa Kirin: Consultancy, Research Funding; Eisai: Research Funding; Cell Medica: Research Funding; Amgen: Research Funding; Pharmacyclics: Honoraria, Other, Speakers Bureau; Abbvie: Honoraria; Takeda: Honoraria, Other: Travel expenses; Celgene: Honoraria, Research Funding; Kite: Honoraria, Other: Travel expenses, Speakers Bureau; BMS: Consultancy, Honoraria, Research Funding; Corvus: Research Funding; Rhizen: Research Funding; Seattle Genetics, Inc.: Consultancy, Honoraria, Other: Travel expenses, Research Funding, Speakers Bureau; Portola: Research Funding. Sokol:EUSA Pharma: Consultancy, Honoraria, Speakers Bureau; Kyowa/Kirin Inc.: Membership on an entity's Board of Directors or advisory committees; Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees. Mei:Sanofi: Consultancy; Morphosys: Membership on an entity's Board of Directors or advisory committees. Flinn:Gilead Sciences: Consultancy, Research Funding; F. Hoffmann-La Roche: Research Funding; Roche: Consultancy, Research Funding; Vincera Pharma: Consultancy; Agios: Research Funding; Iksuda Therapeutics: Consultancy; Great Point Partners: Consultancy; Genentech, Inc.: Research Funding; Forty Seven: Research Funding; Forma Therapeutics: Research Funding; ArQule: Research Funding; Kite Pharma: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Trillium Therapeutics: Research Funding; Triphase Research & Development Corp.: Research Funding; Curio Science: Consultancy; Constellation Pharmaceuticals: Research Funding; MorphoSys: Consultancy, Research Funding; Curis: Research Funding; Nurix Therapeutics: Consultancy; Novartis: Research Funding; Pfizer: Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Portola Pharmaceuticals: Research Funding; Seattle Genetics: Consultancy, Research Funding; Teva: Research Funding; Loxo: Research Funding; BeiGene: Consultancy, Research Funding; Calithera Biosciences: Research Funding; Johnson & Johnson: Other; Rhizen Pharmaceuticals: Research Funding; Yingli Pharmaceuticals ≠: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Karyopharm Therapeutics: Research Funding; AstraZeneca: Consultancy, Research Funding; Incyte: Research Funding; Takeda: Consultancy, Research Funding; IGM Biosciences: Research Funding; Infinity Pharmaceuticals: Research Funding; Unum Therapeutics: Consultancy, Research Funding; Juno Therapeutics: Consultancy, Research Funding; Acerta Pharma: Research Funding; Celgene: Research Funding; Merck: Research Funding. Villa:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; AZ: Consultancy, Honoraria, Research Funding; Kite/Gilead: Consultancy, Honoraria; Nano String: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Sandoz Canada: Consultancy, Honoraria; Immunovaccine: Consultancy, Honoraria; Purdue Pharma: Consultancy, Honoraria. Percival:Pfizer: Research Funding; Trillium: Research Funding; Nohla Therapeutics: Research Funding; Biosight: Research Funding; Oscotec: Research Funding; Cardiff Oncology: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Glycomimetics: Research Funding. Jagadeesh:Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Regeneron: Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Research Funding; Debiopharm Group: Research Funding. Savage:Merck, BMS, Seattle Genetics, Gilead, AstraZeneca, AbbVie, Servier: Consultancy; Roche (institutional): Research Funding; Merck, BMS, Seattle Genetics, Gilead, AstraZeneca, AbbVie: Honoraria; BeiGene: Other: Steering Committee. Akilov:Mallinckrodt: Consultancy; Medivir: Consultancy; Seattle Genetics, Inc.: Consultancy; Kyowa Hakko Kirin: Consultancy; Soligenix: Honoraria; Pfizer: Research Funding; Actelion: Consultancy, Research Funding; Trillium Therapeutics Inc.: Consultancy, Research Funding. Diefenbach:Trillium: Research Funding; Millenium/Takeda: Research Funding; Seattle Genetics: Consultancy, Research Funding; Merck: Consultancy, Research Funding; MEI: Research Funding; LAM Therapeutics: Research Funding; Incyte: Research Funding; Genentech, Inc.: Consultancy, Research Funding; Denovo: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding. Kim:Kyowa-Kirin Pharma: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Medivir: Membership on an entity's Board of Directors or advisory committees; Horizon Pharma: Consultancy, Research Funding; Merck: Research Funding; miRagen: Research Funding; Trillium: Research Funding; Galderma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Corvus: Research Funding; Elorac: Research Funding; Forty Seven Inc: Research Funding; Neumedicine: Consultancy, Research Funding; Innate Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Solingenix: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Portola: Research Funding. Lin:Trillium Therapeutics Inc.: Current Employment. Catalano:Trillium Therapeutics Inc.: Current Employment. Petrova:Trillium Therapeutics Inc.: Current Employment. Uger:Trillium Therapeutics Inc.: Current Employment. Molloy:Trillium Therapeutics Inc.: Current Employment. Large:Trillium Therapeutics Inc.: Current Employment. Shou:Trillium Therapeutics Inc.: Current Employment. Ansell:Affimed: Research Funding; Bristol Myers Squibb: Research Funding; Regeneron: Research Funding; Trillium: Research Funding; ADC Therapeutics: Research Funding; Seattle Genetics: Research Funding; Takeda: Research Funding; AI Therapeutics: Research Funding.

Multiagent combination chemotherapy can be curative in acute lymphoblastic leukemia (ALL). Still, patients with primary refractory disease or with relapsed leukemia have a very poor prognosis. Here we integrate an in-depth dissection of the mutational landscape across diagnostic and relapsed pediatric and adult ALL samples with genome-wide CRISPR screen analysis of gene–drug interactions across seven ALL chemotherapy drugs. By combining these analyses, we uncover diagnostic and relapse-specific mutational mechanisms as well as genetic drivers of chemoresistance. Functionally, our data identify common and drug-specific pathways modulating chemotherapy response and underscore the effect of drug combinations in restricting the selection of resistance-driving genetic lesions. In addition, by identifying actionable targets for the reversal of chemotherapy resistance, these analyses open therapeutic opportunities for the treatment of relapse and refractory disease. Ferrando and colleagues analyze matched diagnostic and relapsed acute lymphocytic leukemia by whole-genome sequencing, and perform in vitro genome-wide CRISPR screens, to examine alterations associated with chemotherapy resistance.

Key Points DNMT3A R882, TET2, ASXL1, and SRSF2 mutations identified at the time of diagnosis are associated with delayed count recovery. Persistence of preleukemic mutations in remission at high variant allele frequency is associated with delayed count recovery.

TO THE EDITOR: BCR-ABL -negative myeloproliferative neoplasms (MPNs) are a group of clonal stem cell disorders characterized by the overproduction of mature myeloid cells. However, MPN patients also have a 2.8- to 3.4-fold increased risk of developing a lymphoproliferative disorder (LPD) compared

Older patients with newly diagnosed acute myeloid leukemia (AML) in the phase 3 AZA-AML-001 study were evaluated at entry for cytogenetic abnormalities, and a subgroup of patients was assessed for gene mutations. Patients received azacitidine 75 mg/m2/day x7 days (n = 240) or conventional care regimens (CCR; n = 245): intensive chemotherapy, low-dose cytarabine, or best supportive care only. Overall survival (OS) was assessed for patients with common (occurring in ≥10% of patients) cytogenetic abnormalities and karyotypes, and for patients with recurring gene mutations. There was a significant OS improvement with azacitidine vs CCR for patients with European LeukemiaNet-defined Adverse karyotype (HR 0.71 [95%CI 0.51-0.99]; P = 0.046). Azacitidine-treated patients with -5/5q-, -7/7q-, or 17p abnormalities, or with monosomal or complex karyotypes, had a 31-46% reduced risk of death vs CCR. The most frequent gene mutations were DNMT3A (27%), TET2 (25%), IDH2 (23% [R140, 15%; R172, 8%]), and TP53 (21%). Compared with wild-type, OS was significantly reduced among CCR-treated patients with TP53 or NRAS mutations and azacitidine-treated patients with FLT3 or TET2 mutations. Azacitidine may be a preferred treatment for older patients with AML with Adverse-risk cytogenetics, particularly those with chromosome 5, 7, and/or 17 abnormalities and complex or monosomal karyotypes. The influence of gene mutations in azacitidine-treated patients warrants further study.

Despite advances that have improved the treatment of chronic myeloid leukemia (CML) patients in chronic phase, the mechanisms of the transition from chronic phase CML to blast crisis (BC) are not fully understood. Considering the key role of miR-15/16 loci in the pathogenesis of myeloid and lymphocytic leukemia, here we aimed to correlate the expression of miR-15a/16 and miR-15b/16 to progression of CML from chronic phase to BC. We analyzed the expression of the two miR-15/16 clusters in 17 CML patients in chronic phase and 22 patients in BC and in 11 paired chronic phase and BC CML patients. BC CMLs show a significant reduction of the expression of miR-15a/-15b/16 compared to CMLs in chronic phase. Moreover, BC CMLs showed an overexpression of miR-15/16 direct targets such as Bmi-1, ROR1, and Bcl-2 compared to CMLs in chronic phase. This study highlights the loss of both miR-15/16 clusters as a potential oncogenic driver in the transition from chronic phase to BC in CML patients.

TRAF1 is a pro-survival adaptor molecule in TNFR superfamily (TNFRSF) signaling. TRAF1 is overexpressed in many B cell cancers including refractory chronic lymphocytic leukemia (CLL). Little has been done to assess the role of TRAF1 in human cancer. Here we show that the protein kinase C related kinase Protein Kinase N1 (PKN1) is required to protect TRAF1 from cIAP-mediated degradation during constitutive CD40 signaling in lymphoma. We show that the active phospho-Thr774 form of PKN1 is constitutively expressed in CLL but minimally detected in unstimulated healthy donor B cells. Through a screen of 700 kinase inhibitors, we identified two inhibitors, OTSSP167, and XL-228, that inhibited PKN1 in the nanomolar range and induced dose-dependent loss of TRAF1 in RAJI cells. OTSSP167 or XL-228 treatment of primary patient CLL samples led to a reduction in TRAF1, pNF-κB p65, pS6, pERK, Mcl-1 and Bcl-2 proteins, and induction of activated caspase-3. OTSSP167 synergized with venetoclax in inducing CLL death, correlating with loss of TRAF1, Mcl-1, and Bcl-2. Although correlative, these findings suggest the PKN1-TRAF1 signaling axis as a potential new target for CLL. These findings also suggest the use of the orally available inhibitor OTSSP167 in combination treatment with venetoclax for TRAF1 overexpressing CLL.

Novel targeted therapies improve the survival of specific subgroups (defined by genetic variants) of patients with acute myeloid leukemia (AML), validating the paradigm of molecularly targeted therapy. However, identifying correlations between AML molecular attributes and effective therapies is challenging. Recent advances in highthroughput, in vitro drug sensitivity screening applied to primary AML blasts were used to uncover such correlations; however, these methods cannot predict the response of leukemic stem cells. Our study aimed to predict in vitro response to targeted therapies, based on molecular markers, with subsequent validation in leukemic stem cells. We performed ex vivo screening of sensitivity to 46 drugs on 29 primary AML samples at diagnosis or relapse. Using unsupervised hierarchical clustering analysis we identified a group with sensitivity to several tyrosine kinase inhibitors, including the multi-tyrosine kinase inhibitor, dasatinib, and searched for correlations between the response to dasatinib, exome sequencing and gene expression in our dataset and in the Beat AML dataset. Unsupervised hierarchical clustering analysis of gene expression resulted in clustering of dasatinib responders and non-responders. In vitro response to dasatinib could be predicted based on gene expression (area under the curve=0.78). Furthermore, mutations in FLT3/ITD and PTPN11 were enriched in the dasatinib-sensitive samples as opposed to mutations in TP53 which were enriched in resistant samples. Based on these results, we selected FLT3/ITD AML samples and injected them into NSG-SGM3 mice. Our results demonstrate that in a subgroup of FLT3/ITD AML (4 out of 9) dasatinib significantly inhibited leukemic stem cell engraftment. In summary we show that dasatinib has an anti-leukemic effect both on bulk blasts and, more importantly, on leukemic stem cells from a subset of AML patients that can be identified based on mutational and expression profiles. Our data provide a rational basis for clinical trials of dasatinib in a molecularly selected subset of AML patients.

Purpose: TTI-621 (SIRPα-IgG1 Fc) is a novel checkpoint inhibitor that activates antitumor activity by blocking the CD47 “don't eat me” signal. This first-in-human phase I study (NCT02663518) evaluated the safety and activity of TTI-621 in relapsed/refractory (R/R) hematologic malignancies. Patients and Methods: Patients with R/R lymphoma received escalating weekly intravenous TTI-621 to determine the maximum tolerated dose (MTD). During expansion, patients with various malignancies received weekly single-agent TTI-621 at the MTD; TTI-621 was combined with rituximab in patients with B-cell non-Hodgkin lymphoma (B-NHL) or with nivolumab in patients with Hodgkin lymphoma. The primary endpoint was the incidence/severity of adverse events (AEs). Secondary endpoint included overall response rate (ORR). Results: Overall, 164 patients received TTI-621: 18 in escalation and 146 in expansion (rituximab combination, n = 35 and nivolumab combination, n = 4). On the basis of transient grade 4 thrombocytopenia, the MTD was determined as 0.2 mg/kg; 0.1 mg/kg was evaluated in combination cohorts. AEs included infusion-related reactions, thrombocytopenia, chills, and fatigue. Thrombocytopenia (20%, grade ≥3) was reversible between doses and not associated with bleeding. Transient thrombocytopenia that determined the initial MTD may not have been dose limiting. The ORR for all patients was 13%. The ORR was 29% (2/7) for diffuse large B-cell lymphoma (DLBCL) and 25% (8/32) for T-cell NHL (T-NHL) with TTI-621 monotherapy and was 21% (5/24) for DLBCL with TTI-621 plus rituximab. Further dose optimization is ongoing. Conclusions: TTI-621 was well-tolerated and demonstrated activity as monotherapy in patients with R/R B-NHL and T-NHL and combined with rituximab in patients with R/R B-NHL.

Although most children survive B cell acute lymphoblastic leukemia (B-ALL), they frequently experience long-term, treatment-related health problems, including osteopenia and osteonecrosis. Because some children present with fractures at ALL diagnosis, we considered the possibility that leukemic B cells contribute directly to bone pathology. To identify potential mechanisms of B-ALL–driven bone destruction, we examined the p53−/−; Rag2−/−; Prkdcscid/scid triple mutant (TM) mice and p53−/−; Prkdcscid/scid double mutant (DM) mouse models of spontaneous B-ALL. In contrast to DM animals, leukemic TM mice displayed brittle bones, and the TM leukemic cells overexpressed Rankl, encoding receptor activator of nuclear factor κB ligand. RANKL is a key regulator of osteoclast differentiation and bone loss. Transfer of TM leukemic cells into immunodeficient recipient mice caused trabecular bone loss. To determine whether human B-ALL can exert similar effects, we evaluated primary human B-ALL blasts isolated at diagnosis for RANKL expression and their impact on bone pathology after their transplantation into NOD.Prkdcscid/scidIl2rgtm1Wjl/SzJ (NSG) recipient mice. Primary B-ALL cells conferred bone destruction evident in increased multinucleated osteoclasts, trabecular bone loss, destruction of the metaphyseal growth plate, and reduction in adipocyte mass in these patient-derived xenografts (PDXs). Treating PDX mice with the RANKL antagonist recombinant osteoprotegerin–Fc (rOPG-Fc) protected the bone from B-ALL–induced destruction even under conditions of heavy tumor burden. Our data demonstrate a critical role of the RANK-RANKL axis in causing B-ALL–mediated bone pathology and provide preclinical support for RANKL-targeted therapy trials to reduce acute and long-term bone destruction in these patients.

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous hematological malignancy. Although immunotherapy may be an attractive modality to exploit in patients with AML, the ability to predict the groups of patients and the types of cancer that will respond to immune targeting remains limited. This study dissected the complexity of the immune architecture of AML at high resolution and assessed its influence on therapeutic response. Using 442 primary bone marrow samples from three independent cohorts of children and adults with AML, we defined immune-infiltrated and immune-depleted disease classes and revealed critical differences in immune gene expression across age groups and molecular disease subtypes. Importantly, interferon (IFN)-γ-related mRNA profiles were predictive for both chemotherapy resistance and response of primary refractory/relapsed AML to flotetuzumab immunotherapy. Our compendium of microenvironmental gene and protein profiles provides insights into the immuno-biology of AML and could inform the delivery of personalized immunotherapies to IFN-γ-dominant AML subtypes.

Background Fatigue and insomnia are common symptoms experienced by breast cancer patients undergoing adjuvant radiation therapy (RT), yet the underlying mechanisms of these symptoms are unclear. In particular, the roles of hematopoietic stem cells (HSCs) and inflammatory cytokines remain to be elucidated. Methods Breast cancer patients (n = 147) completed questionnaires to longitudinally assess symptoms before, during, and after adjuvant RT. Phlebotomies were performed prior to RT, at the second and fifth treatment fractions, end of treatment (EOT), and 1 month after completing RT, assessing for CD34+, CD45+, full hematology, and 17 inflammatory cytokines. The associations between symptoms and all biomarkers were evaluated. All statistical tests were 2-sided. Results General fatigue and insomnia worsened with RT, with peak levels observed at EOT, which remained statistically significant even after controlling for anxiety and depression (P Conclusions This is the first longitudinal study to examine linkages between symptoms, HSCs, and cytokines, demonstrating that fatigue and insomnia shared associations with increasing serum levels of IP-10 and TNF-RII, and mental fatigue was associated with increasing serum levels of MMP-2. Our findings highlight opportunities for further research into mechanisms and potential interventions for these symptoms.

Core-binding factor acute myeloid leukemia is characterized by t(8;21) or inv(16) and the fusion proteins RUNX1-RUNX1T1 and CBFB-MYH11. International guidelines recommend monitoring for measurable residual disease every 3 months for 2 years after treatment. However, it is not known whether serial molecular monitoring can predict and prevent morphological relapse. We conducted a retrospective singlecenter study of 114 patients in complete remission who underwent molecular monitoring with real-time quantitative polymerase chain reaction analysis of RUNX1-RUNX1T1 or CBFB-MYH11 transcripts every 3 months. Morphological relapse was defined as re-emergence of >5% blasts and molecular relapse as ≥1 log increase in transcript level between two samples. Over a median follow-up time of 3.7 years (range, 0.2-14.3), remission persisted in 71 (62.3%) patients but 43 (37.7%) developed molecular or morphological relapse. Patients who achieved

Patients with acute myeloid leukemia (AML) often relapse after initial therapy because of persistence of leukemic stem cells that frequently express the IL-3 receptor alpha chain CD123. Natural killer (NK) cell-based therapeutic strategies for AML show promise and we explore the NK cell lines, NK-92 and CD16+ NK-92, as a treatment for AML. NK-92 has been tested in phase I clinical trials with minimal toxicity; irradiation prior to infusion prevents risk of engraftment. The CD16 negative NK-92 parental line was genetically modified to express the high affinity Fc gamma receptor, enabling antibody-dependent cell-mediated cytotoxicity, which we utilized in combination with an anti-CD123 antibody to target leukemic stem cells. NK-92 was preferentially cytotoxic against leukemic stem and progenitor cells compared with bulk leukemia in in vitro assays, while CD16+ NK-92 in combination with an anti-CD123 mAb mediated antibody-dependent cell-mediated cytotoxicity against CD123+ leukemic targets. Furthermore, NK-92 infusions (with or without prior irradiation) improved survival in a primary AML xenograft model. Mice xenografted with primary human AML cells had a superior survival when treated with irradiated CD16+NK-92 cells and an anti-CD123 monoclonal antibody (7G3) versus treatment with irradiated CD16+NK-92 cells combined with an isotype control antibody. In this proof-of-principle study, we show for the first time that a CD16+NK-92 cell line combined with an antibody that targets a leukemic stem cell antigen can lead to improved survival in a relevant pre-clinical model of AML.

Therapy for acute myeloid leukemia (AML) involves intense cytotoxic treatment and yet approximately 70% of AML are refractory to initial therapy or eventually relapse. This is at least partially driven by the chemo-resistant nature of the leukemic stem cells (LSCs) that sustain the disease, and therefore novel anti-LSC therapies could decrease relapses and improve survival. We performed in silico analysis of highly prognostic human AML LSC gene expression signatures using existing datasets of drug–gene interactions to identify compounds predicted to target LSC gene programs. Filtering against compounds that would inhibit a hematopoietic stem cell (HSC) gene signature resulted in a list of 151 anti-LSC candidates. Using a novel in vitro LSC assay, we screened 84 candidate compounds at multiple doses and confirmed 14 drugs that effectively eliminate human AML LSCs. Three drug families presenting with multiple hits, namely antihistamines (astemizole and terfenadine), cardiac glycosides (strophanthidin, digoxin and ouabain) and glucocorticoids (budesonide, halcinonide and mometasone), were validated for their activity against human primary AML samples. Our study demonstrates the efficacy of combining computational analysis of stem cell gene expression signatures with in vitro screening to identify novel compounds that target the therapy-resistant LSC at the root of relapse in AML.

Purpose: To explore the potential of ex vivo expanded healthy donor–derived allogeneic CD4 and CD8 double-negative cells (DNT) as a novel cellular immunotherapy for leukemia patients. Experimental Design: Clinical-grade DNTs from peripheral blood of healthy donors were expanded and their antileukemic activity and safety were examined using flow cytometry–based in vitro killing assays and xenograft models against AML patient blasts and healthy donor–derived hematopoietic cells. Mechanism of action was investigated using antibody-mediated blocking assays and recombinant protein treatment assays. Results: Expanded DNTs from healthy donors target a majority (36/46) of primary AML cells, including 9 chemotherapy-resistant patient samples in vitro, and significantly reduce the leukemia load in patient-derived xenograft models in a DNT donor–unrestricted manner. Importantly, allogeneic DNTs do not attack normal hematopoietic cells or affect hematopoietic stem/progenitor cell engraftment and differentiation, or cause xenogeneic GVHD in recipients. Mechanistically, DNTs express high levels of NKG2D and DNAM-1 that bind to cognate ligands preferentially expressed on AML cells. Upon recognition of AML cells, DNTs rapidly release IFNγ, which further increases NKG2D and DNAM-1 ligands’ expression on AML cells. IFNγ pretreatment enhances the susceptibility of AML cells to DNT-mediated cytotoxicity, including primary AML samples that are otherwise resistant to DNTs, and the effect of IFNγ treatment is abrogated by NKG2D and DNAM-1–blocking antibodies. Conclusions: This study supports healthy donor–derived allogeneic DNTs as a therapy to treat patients with chemotherapy-resistant AML and also reveals interrelated roles of NKG2D, DNAM-1, and IFNγ in selective targeting of AML by DNTs. Clin Cancer Res; 24(2); 370–82. ©2017 AACR.

Acute myeloid leukemia (AML) continues to be a deadly disease, with only 50–70% of patients achieving complete remission and less than 30% of adults having sustained long-term remissions. In order to address these unmet medical needs, we carried out a high-throughput screen of an in-house library of on- and off-patent drugs with the OCI/AML-2 cell line. Through this screen, we discovered adefovir dipi­voxil (adefovir-DP) as being active against human AML. In addition to adefovir-DP, there are second-generation formulations of adefovir, including octadecyloxyethyl adefovir (ODE-adefovir) and hexadecyloxypropyl adefovir (HDP-adefovir), which were designed to overcome the pharmacokinetic problems of the parent compound adefovir. Given the known clinical benefit of nucleoside analogs for the treatment of AML, we undertook studies to evaluate the potential benefit of adefovir-based molecules. In AML cell lines and patient samples, adefovir-DP and ODE-adefovir were highly potent, whereas HDP-adefovir was significantly less active. Interestingly, ODE-adefovir was remarkably less toxic than adefovir-DP towards normal hematopoietic cells. In addition, ODE-adefovir at a dose of 15 mg/kg/day showed potent activity against human AML in a NOD/SCID mouse model, with a reduction of human leukemia in mouse bone marrow of > 40% in all mice tested within 20 days of treatment. Based on its chemical structure, we hypothesized that the cytotoxicity of ODE-adefovir toward AML was through cell cycle arrest and DNA damage. Indeed, ODE-adefovir treatment induced cell cycle arrest in the S phase and increased levels of pH2Ax, indicating the induction of DNA damage. Furthermore, there was an increase in phospho-p53, transactivation of proapoptotic genes and activation of the intrinsic apoptotic pathway. Subsequent investigation unveiled strong synergism between ODE-adefovir and ara-C, making their coadministration of potential clinical benefit. Expression of MRP4, a nucleoside transporter, appeared to influence the response of AML cells to ODE-adefovir, as its inhibition potentiated ODE-adefovir killing. Taken together, our findings indicate that clinical development of ODE-adefovir or related compounds for the treatment of AML is warranted.

The high frequency of TET2 mutations in myelodysplastic syndromes (MDS) and the sole function of TET-dioxygenases as 5-hydroxymethylcytosine (5-hmC) hydroxylases emphasize the key role of this gene in disease pathogenesis. However, the broad down-regulation of 5-hmC argues for a role of DNA demethylation in MDS beyond TET2 lesions, which albeit the high frequency, do not convey any impact on survival outcomes. In fact, decrease in 5-hmC levels is by far more widely spread than TET2 lesions pointing towards other pathways affecting TET2 activity, thereby obscuring a precise determination of its mutational and clinical consequences. Herein, we investigated TETs expression to identify factors explaining the widespread deficiency of 5-hmC in MDS possibly determining clinical phenotypes and prognosis. An integrative data analysis of genomic studies (whole genome and deep targeted NGS), RNA-sequencing and 5-hmC quantification was performed on 1,665 patients with MDS and 91 healthy controls (HC). Meta-analytic studies of 5-hmC levels in myeloid neoplasia (n=598) and data of RNA-sequencing of fractionated CD34 (GSE63569) were also included as confirmatory cohorts. We started by analyzing the clinical impact of TET2 mutations carried by 23% of our study population. No impact on survival was found in carriers of TET2 lesions including those with biallelic, truncating or missense mutations compared to wild-type (WT) (Fig1A). By using 5-hmC levels as a functional readout of TET activity, we found a TET deficiency in about 70% of patients, a proportion higher than one would conclude by considering the mere presence of TET2 mutations (Fig1B). To explain the decrease in 5-hmC levels in WT cases, we next examined transcriptome modifications. Analysis of the expression of TET family of genes showed that MDS patients had lower TET2 mRNA levels in total and in CD34+ cells as compared to HC, irrespective of their TET2 status. Therefore, we reasoned that TET2 deficiency is more ubiquitously involved in MDS pathogenesis than what would be expected by the only estimation of mutant cases. Indeed, "low expressor" status (defined by TET2 expression < 25%ile of HC) was found in 74% of MDS. Along with variable 5-hmC levels, concomitant differences in TET1/TET3 expression were also investigated. While TET1 levels were too low to be evaluated, TET3 expression levels were markedly higher in all and in WT MDS compared to HC, possibly in an attempt to compensate TET2 dysfunction (Fig1C). In addition, TET3 expression did not correlate with TET2 mutational burden, confuting a compensatory feedback mechanism in TET2 mutant cases. Further uni- and multivariate analyses showed that elevated TET3 levels compensated TET2 deficiency in terms of clinical outcomes (Fig1D) and linear regression analyses confirmed that indeed lack of compensation by TET3 (low TET3 expression) was associated with high risk features. To explore whether other factors might be associated with low TET2 levels, we studied TET2 expression in WT cases as to the presence of other mutations. We found that TET2 expression was significantly lower in patients harboring DNMT3A (P< 0.0001), SF3B1 (P< 0.0001) and SRSF2 (P= 0.04) compared to HC. However, lack of correlation between levels of TET2 and mutational burden failed to prove a direct relationship of these mutations (Fig1E). Decreased hydroxylation of 5-mC may also be caused by endogenous L-2-hydroxyglutarate (L2HG) produced via malate shunt. Accordingly, L2HG dehydrogenase (L2HGDH) levels catabolizing L2HG and malate dehydrogenases (MDH1/2) supplying L2HG, would influence TET2 activity in a reciprocal fashion. Consistently we found that MDH1/2 levels were increased in MDS and that L2HGDH showed also a likely compensatory increase to handle elevated L2HG loads. Further, linear regression analyses revealed that L2HGDH levels were correlated inversely with TET2 and positively with TET3 expression in WT cases (Fig1F). In sum, MDS can be considered a wide-ranging 5-hmC deficiency disorder driven by direct or indirect loss of TET2functions by mutations or down-modulation due to a variety of mechanisms. Disease phenotypes and outcomes are both influenced by counteracting factors such as expression of TET3. Application of precision therapeutic approaches should be informed by the analyses of all these factors. Figure 1 Figure 1. Disclosures Carraway: Astex: Other: Independent review committee; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Other: Independent review committee; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Other: Independent review committee; Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kim: Paladin: Consultancy, Honoraria, Research Funding; Bristol-Meier Squibb: Research Funding; Pfizer: Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Minden: Astellas: Consultancy. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy; Regeneron: Consultancy; Alexion: Consultancy.

Background: Gilteritinib (GIL), a FLT3 inhibitor, shows efficacy/safety in patients (pts) with FLT3mut+ relapsed/refractory (R/R) AML. For pts with newly diagnosed (ND) AML with FLT3 mutations unable to receive intensive induction chemotherapy (IIC), survival is limited, and therapy options are few. In preclinical studies, GIL plus azacitidine (AZA) impeded tumor growth and induced apoptosis/differentiation of FLT3-ITD AML cell lines. Antitumor effects appeared synergistic in xenografted mouse models (data on file). We investigated GIL+AZA vs AZA in adults with ND FLT3mut+ AML ineligible for IIC (NCT02752035). Aim/Objective: To compare efficacy and safety/tolerability of GIL+AZA and AZA in pts with ND FLT3mut+ AML ineligible for IIC. Methods: Pts were initially randomized (1:1:1) to GIL (120 mg/day orally on Days 1-28) plus AZA (75 mg/m 2/day SC/IV on Days 1-7), AZA (same regimen), or GIL (same regimen) during 28-day cycles. The GIL arm was removed due to preferred therapy changes. Pts were then randomized (2:1) to GIL+AZA or AZA alone. The primary endpoint was overall survival (OS) and key secondary endpoint was event-free survival (EFS). Treatment failure date was randomization date if complete remission (CR) was not achieved after 6 cycles. Subgroup/sensitivity analyses were prespecified. Response rates, safety/tolerability, and pharmacokinetic endpoints were analyzed. Data from long-term follow-up ≤3 yrs were obtained, including subsequent AML therapy. These results are from an interim analysis at 70 deaths (~50% of total deaths). Results: As of August 26, 2020, 123 pts were randomized to GIL+AZA (n=74) and AZA (n=49); 39 (52.7%) and 31 (63.3%) deaths, respectively, occurred. Median age was 78 yrs with GIL+AZA and 76 yrs with AZA; ECOG PS ≥2 was 47.3% and 32.7%, and FLT3-ITD alone was in 78.4% and 81.6% of pts, respectively (TKD alone 18.9% vs 14.3%; ITD with TKD 2.7% vs 4.1%). Median follow-up was 9.76 mo for GIL+AZA and 17.97 mo for AZA. Median exposure duration was 112 days for GIL in the GIL+AZA arm (n=73); AZA exposure was 98 and 99 days in the GIL+AZA and AZA (n=47) arms, respectively. Subsequent AML therapy was received by 20.3% pts on GIL+AZA and 44.9% pts on AZA; median time to first subsequent therapy was 8.2 and 4.5 mo, respectively. In the AZA arm, 22 pts received subsequent AML therapy, including 10 pts on GIL and 4 pts on other FLT3 inhibitors. Median OS was 9.82 mo for GIL+AZA and 8.87 mo for AZA (HR 0.916 [95% CI 0.529, 1.585]; P=.753). Patient subgroups with improved OS with GIL+AZA vs AZA included pts with ECOG PS 0-1 (HR 0.811 [95% CI 0.409, 1.608]) and high FLT3-ITD allelic ratio ≥0.5 (HR 0.580 [95% CI 0.285, 1.182]). Median EFS was 0.03 mo in both arms (HR 1.175 [95% CI 0.764, 1.807]; P=.459). In sensitivity analyses, median EFS with events based on composite CR (CRc; CR+CRi+CRp) was 5.03 mo for GIL+AZA and 3.29 mo for AZA (HR 0.924 [95% CI 0.576, 1.482]; P=.767). Although CR rates for both arms were similar (16.2% vs 14.3%), CRc rates were significantly higher for GIL+AZA vs AZA (58.1 vs 26.5%, difference 31.4% [95% CI 13.1, 49.7]; P No substantial differences in GIL trough concentrations at steady state (C trough) were seen between GIL+AZA and GIL alone (prior to arm removal). However, on Cycle 1 Day 15, median GIL C trough was 579 ng/mL (GIL+AZA and GIL arms) in contrast to C trough of 279 ng/mL observed with GIL monotherapy in the ADMIRAL trial in pts with R/R AML. Reasons for this difference are being evaluated. No apparent relationship was seen between C trough and response rates/grade of thrombocytopenia or neutropenia. Conclusions: In this trial of pts with ND FLT3mut+ AML ineligible for IIC, GIL+AZA led to significantly higher CRc rates but similar OS vs AZA alone. Pts with ECOG PS 0-1 and high FLT3-ITD allelic ratio appeared to have greater benefit with GIL+AZA. No new safety signals were seen. Curiously, C trough values in pts with ND FLT3mut+ AML ineligible for IIC were 2-fold greater than in pts with R/R FLT3mut+ AML. These results support the safety, tolerability, and activity of GIL+AZA vs AZA. Disclosures Wang: GlaxoSmithKline: Consultancy, Honoraria, Other: Advisory Board; Genentech: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Other: Advisory Board; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Pfizer: Consultancy, Honoraria, Other: Advisory Board, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Advisory board; Mana Therapeutics: Consultancy, Honoraria; Kite Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Kura Oncology: Consultancy, Honoraria, Other: Advisory board, steering committee, Speakers Bureau; Stemline Therapeutics: Consultancy, Honoraria, Other: Advisory board, Speakers Bureau; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; DAVA Oncology: Consultancy, Speakers Bureau; Rafael Pharmaceuticals: Other: Data safety monitoring committee; Gilead: Consultancy, Honoraria, Other: Advisory board; Daiichi Sankyo: Consultancy, Honoraria, Other: Advisory board; PTC Therapeutics: Consultancy, Honoraria, Other: Advisory board; Genentech: Consultancy; MacroGenics: Consultancy. Montesinos: Stemline/Menarini: Consultancy; Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Forma Therapeutics: Consultancy; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Glycomimetics: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Tolero Pharmaceutical: Consultancy; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Agios: Consultancy; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas Pharma, Inc.: Consultancy, Honoraria, Other: Advisory board, Research Funding, Speakers Bureau. Minden: Astellas: Consultancy. Lee: Korean Society of Hematology: Membership on an entity's Board of Directors or advisory committees; Astellas Pharma, Inc.: Consultancy, Honoraria, Other: Advisory board; AbbVie: Honoraria, Other: Advisory board. Heuser: Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding; BergenBio: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Tolremo: Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer Pharma AG: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding. Naoe: Fuji Film: Other: Study funding for JAGSE; Astellas Pharma, Inc.: Consultancy, Other: Study funding for JAGSE; Bristol-Myers: Honoraria; Nippon Shinyaku: Honoraria; Daichi Sankyo: Other: Study funding for JAGSE; Otsuka Pharma: Honoraria. Chou: Abbvie: Honoraria, Other: Advisory Board, Research Funding; Celgene: Honoraria, Other: Advisory Board, Research Funding; IQVIA: Honoraria, Other: Advisory Board; Pfizer: Honoraria, Other: Advisory Board; Novartis: Honoraria, Other: Advisory Board; Bristol Myers Squibb: Honoraria, Research Funding; Kirin: Honoraria, Research Funding. Laribi: IQONE: Other: Personal Fees; Takeda: Other: Personal Fees, Research Funding; AstraZeneca: Other: Personal Fees; BeiGene: Other: Personal Fees; Astellas Phama, Inc.: Other: Personal Fees; Jansen: Research Funding; AbbVie: Other: Personal Fees, Research Funding; Le Mans Hospital: Research Funding; Novartis: Other: Personal Fees, Research Funding. Esteve: Jazz: Consultancy; Novartis: Research Funding; Bristol Myers Squibb/Celgene: Consultancy; Pfizer: Consultancy; Abbvie: Consultancy; Novartis: Consultancy, Research Funding; Astellas: Consultancy. Altman: ALZ Oncology: Research Funding; Amgen: Research Funding; Biosight: Consultancy, Other: Travel fees, Research Funding; Kartos: Research Funding; Boehringer Ingelheim: Research Funding; Daiichi Sankyo: Consultancy; Syros: Consultancy; BMS: Research Funding; GlycoMimetics: Other: Participation on an advisory board; Aprea: Research Funding; Kura: Research Funding; Kura Oncology: Consultancy; AbbVie: Consultancy, Other: Advisory Board, Research Funding; Theradex: Consultancy, Other: Advisory boards; Fujifilm: Research Funding; Astellas: Consultancy, Other: Advisory Board, Research Funding; Immunogen: Research Funding. Havelange: Astellas Pharma, Inc.: Consultancy; Novartis: Honoraria, Other: Travel fees, Participation on an advisory board; BMS: Other: Travel fees, Participation on an advisory board; Incyte: Other: Advisory board; Abbvie: Other: Advisory board. Watson: Astellas Pharma, Inc.: Consultancy; Roche, Amgen: Other: Travel support. Patkowska: Astellas Pharma, Inc.: Consultancy, Other: Travel fees; Pfizer: Other: Travel fees; Jazz Pharmaceuticals: Other: Travel fees; Angelini Pharma: Honoraria, Other: Travel fees; Novartis: Honoraria, Other: Travel fees; Bristol-Myers Squibb: Other: Travel fees; AMGEN: Honoraria; Servier: Honoraria, Other: Travel fees; KCR US, Inc.: Consultancy. Liu: Astellas Pharma, Inc.: Current Employment. Wu: Astellas: Current Employment. Philipose: Astellas Pharma Global Development: Current Employment. Hill: Astellas Pharma Global Development: Current Employment; Ligacept, LLC: Current holder of individual stocks in a privately-held company, Other: Stockholder. Gill: Astellas Pharma Global Development: Current Employment. Rich: Astellas Pharma Global Development, Inc.: Current Employment. Tiu: Astellas Pharma, Inc.: Current Employment.

Background: The presence of measurable residual disease (MRD) after achievement of remission with induction therapy is a prognostic marker of relapse risk in patients with acute myeloid leukemia (AML). Gilteritinib is an oral FLT3 inhibitor approved as a single agent for the treatment of patients with FLT3-mutated (FLT3mut+) relapsed or refractory AML. Evaluation of gilteritinib in the front-line setting is under way. We evaluated FLT3 internal tandem duplication (FLT3-ITD) mutation clearance using two different thresholds and correlated mutation clearance with survival outcomes in patients with newly diagnosed AML ineligible for intensive chemotherapy who were treated with front-line gilteritinib plus azacitidine (AZA) or either agent alone in the phase 2/3 LACEWING trial. Methods: Adult patients with newly diagnosed FLT3mut+ AML ineligible for intensive induction chemotherapy received 28-day cycles of once-daily gilteritinib plus AZA in the Safety Cohort (80 or 120 mg/day gilteritinib plus 75 mg/m 2 AZA, Days 1-7) and in Arm AC (120 mg/day gilteritinib plus 75 mg/m 2 AZA, Days 1-7), gilteritinib (120 mg/day) alone in Arm A, or AZA (75 mg/m 2, Days 1-7) alone in Arm C. A subset of patients who had a best overall response of composite complete remission (CRc; defined as the sum of patients who achieved complete remission with or without complete hematologic or platelet recovery) and who had bone marrow-derived DNA samples available at baseline and at least one additional post-baseline timepoint were assessed for FLT3-ITD mutation clearance using next-generation sequencing. An Illumina ® sequencing platform was used to quantify FLT3-ITD and total FLT3 alleles. The FLT3-ITD variant allelic frequency (VAF) was defined as the ratio of FLT3-ITD to total FLT3 frequency. Data were analyzed using two different mutation clearance thresholds, FLT3-ITD VAF Results: The median age of patients enrolled in LACEWING was 77 years (range, 59-90), with 73% of patients aged >75 years. Although baseline characteristics of the overall LACEWING population were generally well balanced across treatment arms, higher proportions of patients treated with gilteritinib plus AZA (47%) or gilteritinib alone (59%) had an Eastern Cooperative Oncology Group (ECOG) performance status of ≥2 compared with patients treated with AZA alone (33%). Overall, 40 patients who achieved CRc and had sufficient DNA samples from bone marrow aspirates obtained at baseline and at least one additional post-baseline timepoint were included in the analysis (Safety Cohort, n=8; Arm A, n=7; Arm AC, n=17; and Arm C, n=8). Across both thresholds, the proportions of patients with FLT3 mutation clearance did not markedly differ between patients treated with gilteritinib or AZA (Table). In patients who received gilteritinib, FLT3-ITD mutation clearance using either threshold was associated with a similar increase in median overall survival (OS) compared to patients who did not achieve mutation clearance (Figure). Conclusions: Regardless of MRD threshold, rates of MRD negativity were not substantially different between newly diagnosed FLT3mut+ AML patients ineligible for intensive induction chemotherapy who received gilteritinib alone, gilteritinib plus AZA, or AZA alone. Advanced age coupled with a worse baseline ECOG performance score at baseline may have compromised treatment response and achievement of FLT3 mutation clearance in patients treated with gilteritinib. The mutation clearance thresholds used in this analysis showed similar median OS in patients who received gilteritinib. Figure 1 Figure 1. Disclosures Wang: Pfizer: Consultancy, Honoraria, Other: Advisory Board, Speakers Bureau; Genentech: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria, Other: Advisory Board; Novartis: Consultancy, Honoraria, Other: Advisory Board; Kura Oncology: Consultancy, Honoraria, Other: Advisory board, steering committee, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Takeda: Consultancy, Honoraria, Other: Advisory board; Kite Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Stemline Therapeutics: Consultancy, Honoraria, Other: Advisory board, Speakers Bureau; Mana Therapeutics: Consultancy, Honoraria; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; DAVA Oncology: Consultancy, Speakers Bureau; Rafael Pharmaceuticals: Other: Data safety monitoring committee; Gilead: Consultancy, Honoraria, Other: Advisory board; Daiichi Sankyo: Consultancy, Honoraria, Other: Advisory board; PTC Therapeutics: Consultancy, Honoraria, Other: Advisory board; Genentech: Consultancy; MacroGenics: Consultancy. Altman: Kartos: Research Funding; Theradex: Consultancy, Other: Advisory boards; Biosight: Consultancy, Other: Travel fees, Research Funding; Daiichi Sankyo: Consultancy; AbbVie: Consultancy, Other: Advisory Board, Research Funding; BMS: Research Funding; Amgen: Research Funding; Astellas: Consultancy, Other: Advisory Board, Research Funding; Fujifilm: Research Funding; ALZ Oncology: Research Funding; Immunogen: Research Funding; GlycoMimetics: Other: Participation on an advisory board; Syros: Consultancy; Kura Oncology: Consultancy; Boehringer Ingelheim: Research Funding; Aprea: Research Funding; Kura: Research Funding. Minden: Astellas: Consultancy. Wu: Astellas: Current Employment. Rich: Astellas Pharma Global Development, Inc.: Current Employment. Hill: Ligacept, LLC: Current holder of individual stocks in a privately-held company, Other: Stockholder; Astellas Pharma Global Development: Current Employment.

Background: Midostaurin is approved for FLT3 mutant-positive (FLT3+) acute myeloid leukemia (AML), however efficacy has also been observed in a subpopulation of FLT3 mutant-negative AML, suggesting that FLT3 mutation is not the only determinant conferring midostaurin sensitivity. Casado et al previously described phosphoprotein signatures significantly associated with ex vivo responses to midostaurin in primary AML blasts (Casado et al Leukaemia 2018). In the current study, we tested whether our signatures could group FLT3+ patients based on clinical responses to midostaurin plus chemotherapy. Methods: FLT3+ bone marrow (BM) and peripheral blood (PB) specimens were collected at diagnosis, post-treatment and relapse (n=54 cases) from the Leukemia Tissue Bank at Princess Margaret Cancer Centre. All patients in this study were treated with standard chemotherapy plus midostaurin. Protein/phosphoprotein-signatures for BM and PB samples were analysed independently. Case-studies with multiple post-treatment time-points or relapse events following second line treatments were also analysed. Peptides (proteomics) and enriched phosphopeptides (phosphoproteomics) were quantified using liquid chromatography - tandem mass spectrometry. A classification machine learning (ML) algorithm was trained to group patients based on response to treatment as a function of protein/phosphoprotein-signature status. Other features (e.g. genetic mutations, HSC-transplant) were also analysed. Differential survival analysis between patient groups was carried out with Kaplan-Meier and Log Rank test methods. Pathways upregulated in post-treatment or relapse specimens, particularly from those cases that responded poorly to chemo + midostaurin (i.e. early relapse / refractory disease) were investigated using enrichment statistical methods including kinase-substrate enrichment analysis (KSEA) and gene ontology analysis and identified as potential mechanisms of resistance. Statistical significance of enrichment was determined using parametric methods and p-values adjusted for multiple testing using the Benjamini-Hochberg method. Results: ML models were developed based on the ex-vivo phosphoproteomics signatures described in the Casado et al study, from which we trained a predictive model (model 1). Patients positive for model 1 exhibited a survival probability of 243 weeks, compared to 126 weeks in signature negative patients (averages by geometric mean, Log Rank p = 9.88e-05). As the patients in the current study received chemotherapy, in addition to midostaurin, we identified a new phosphoproteomic signature consisting of 26 phospho-sites which partially overlapped with the ex-vivo signature. Patients positive for this new phosphoproteomic signature showed a markedly longer survival time than negative patients (269 vs 76 weeks, Log Rank p = 1.30e-05 for PB and 241 vs 56, Log Rank p = 2.13e-09 for BM specimens, Table). A proteomic signature was also identified in the current study. Positive patients showed a longer survival time than negative patients (330 vs 173 weeks, Log Rank p = 5.0e-04 for PB and 460 vs 156, Log Rank p = 5.2e-06 for BM specimens, Table), however this was less differentiating than the phosphoproteomic signature. Pathways upregulated in post-treatment or relapse specimens from early relapse or refractory cases were associated with molecular functions such as cell proliferation, anti-apoptosis, non-homologous end-joining, transcriptional regulation, spliceosome and cytoskeleton remodelling. Conclusions: We have identified protein and phosphoprotein signatures with the potential to further stratify AML for midostaurin treatment. Phosphoproteomic signatures differentiated according to response better than the proteomic signatures. Pathways upregulated in relapse/refractory cases may have a role in resistance and this will be determined in follow up studies. Analysis will also be performed on FLT3 mutant-negative cases to validate the signatures and elucidate mechanisms of resistance in this group. Disclosures Veiga Nobre: Kinomica Ltd.: Current Employment. Minden: Astellas: Consultancy. Gribben: Janssen: Honoraria, Research Funding; AZ: Honoraria, Research Funding; Abbvie: Honoraria; BMS: Honoraria; Gilead/Kite: Honoraria; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutics: Honoraria. Britton: Kinomica Ltd.: Current Employment, Current equity holder in publicly-traded company.

BACKGROUND: Isocitrate dehydrogenase 2 (IDH2) mutations are found in about 10-15% of acute myeloid leukemia (AML) cases. Enasidenib (ENA) is a first-in-class mutant IDH2 inhibitor that induces differentiation of IDH2-mutated leukemic cells. However, the clinical efficacy of single agent ENA therapy in relapsed or refractory (R/R) AML is limited, underscoring the need for combination therapy. Preclinical studies have shown that IDH-mutated leukemic cells are particularly sensitive to BCL2 inhibition with venetoclax (VEN), a finding supported by clinical studies of VEN combination therapies. We previously demonstrated that the combination of ENA and VEN can be more effective than each drug alone, in reducing leukemic burden in patient-derived xenograft models of IDH2-mutated AML. Here, we report preliminary safety and efficacy results of an ongoing open-label, single-arm, phase Ib/II trial (NCT04092179) of ENA in combination with VEN in patients with IDH2-mutated myeloid malignancies. METHODS: Patients 18 years of age or above with IDH2-mutated R/R AML or high-risk MDS/MPN and an ECOG performance status of 0-2 were eligible. Patients previously treated with an IDH2 or BCL2 inhibitor were excluded. Participants received VEN continuously starting on cycle 1 day 1 with a 3-day ramp-up to a target dose of 400 mg daily (dose level 0) in cohorts 1 and 2. ENA was administered at 100 mg daily continuously starting on cycle 1 day 15. Each cycle was 28 days. Concurrent use of a moderate or strong CYP3A4 inhibitor was allowed with 50% dose reduction of VEN after completion of the first 2 cycles at 100% target dose. Interruptions of VEN and/or ENA were permitted for management of adverse events (AEs). Primary endpoints were 1) safety and tolerability and 2) overall response rate (ORR) defined as complete remission (CR) + CR with incomplete blood count recovery (CRi) + morphologic leukemia-free state (MLFS) + partial remission (PR) by revised IWG criteria. Secondary endpoints include pharmacokinetic (PK) profiles of VEN, duration of response, overall survival (OS), and IDH2 mutant allele burden in bone marrow by ddPCR. RESULTS: The study opened to recruitment in November of 2020. As of July 28, 2021 (data cutoff), 11 patients were enrolled on study; 10 with R/R AML and 1 with very-high risk MDS by IPSS-R. Six patients had a R140Q mutation, and 5 had a R172K mutation. Median age was 72 years (range: 32 - 80); 6 patients were male. Participants had received a median of 2 prior lines of therapies (range: 1 - 4). Six of 10 AML patients had primary refractory disease. The MDS patient experienced secondary azacitidine failure. Key treatment emergent grade ≥ 3 AEs regardless of attribution were: febrile neutropenia (n=3), intracranial hemorrhage (n=3), lung infection (n=2), other infection (n=2), elevated AST/ALT (n=2), sepsis (n=1), leukocytosis (n=1), TRALI (n=1), and small bowel obstruction (n=1). No cases of differentiation or tumor lysis syndrome were observed. No patients discontinued the study due to AEs. The addition of ENA, a known inhibitor of CYP3A4, did not significantly affect the PK profiles of VEN. Nine of the AML patients completed at least 1 cycle of treatment and are considered evaluable for efficacy. One AML patient died from intracranial hemorrhage prior to completion of cycle 1. Median duration of observation is 3.5 months. Of the evaluable patients, CR was achieved in 2 patients (22%) and CRi in 3 patients (33%) for an ORR of 55% (Fig. 1). Median number of cycles to response was 3. All responders remain in remission and on study with a median of 6 cycles received to date (range: 3 - 8). Of the remaining 4 patients, 2 patients (22%) remain on study with stable disease, and 2 patients (22%) experienced progressive disease and died (one after 7 cycles and the other after 1 cycle). Median OS for the entire cohort has not been reached. A sustained reduction in mutant IDH2 allele frequency in bone marrow correlated with response (Fig. 2). For the MDS patient, no response was observed after 1 cycle of treatment. CONCLUSIONS: VEN in combination with ENA is a well-tolerated regimen with no dose-limiting toxicities observed at the current dose level. The preliminary efficacy of this combination is encouraging with an ORR of 55% in evaluable R/R AML patients, with some responders achieving deep molecular remissions. Patient enrollment in dose-escalation cohorts is ongoing. Figure 1 Figure 1. Disclosures Chan: AbbVie: Research Funding; BMS: Research Funding. Gupta: Sierra Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Constellation Pharma: Consultancy, Honoraria; Incyte: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Roche: Consultancy; BMS-Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy. Maze: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene-BMS: Honoraria; Takeda: Research Funding; PharmaEssentia: Research Funding; Kronos Bio: Research Funding. Minden: Astellas: Consultancy. Schimmer: Takeda Pharmaceuticals: Consultancy, Research Funding; Medivir AB: Research Funding; Novartis: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Otsuka Pharmaceuticals: Consultancy, Honoraria; UHN: Patents & Royalties. Schuh: AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlycoMimetics: Research Funding; Kite/Gilead: Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees; Teva: Honoraria, Membership on an entity's Board of Directors or advisory committees. Yee: Pfizer: Membership on an entity's Board of Directors or advisory committees; Forma Therapeutics: Research Funding; F. Hoffmann La Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Onconova: Research Funding; Bristol-Myers Squibb/Celgene: Membership on an entity's Board of Directors or advisory committees; Otsuka: Membership on an entity's Board of Directors or advisory committees; Paladin: Membership on an entity's Board of Directors or advisory committees; Geron: Research Funding; MedImmune: Research Funding; Jazz: Research Funding; TaiHo: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria; Astex: Membership on an entity's Board of Directors or advisory committees, Research Funding. DiNardo: Agios/Servier: Consultancy, Honoraria, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; AbbVie: Consultancy, Research Funding; Novartis: Honoraria; Foghorn: Honoraria, Research Funding; Forma: Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; ImmuneOnc: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. OffLabel Disclosure: Enasidenib is approved for the treatment of relapsed or refractory AML as single agent. Venetoclax, in combination with azacitidine or low-dose cytarabine, is approved for the treatment of newly diagnosed AML patients who are 75 years or older, or who have comorbidities that preclude use of intensive induction chemotherapy.

Background: Spleen tyrosine kinase (SYK) is a component of both lymphoid and myeloid cell signaling pathways and has been implicated in the pathogenesis of a subset of acute myeloid leukemia (AML) defined by dysregulated expression of the HOXA9 and MEIS1 transcription factors. Entospletinib (ENTO) is an oral, selective SYK inhibitor that is acceptably tolerated when administered with intensive induction and consolidation in newly diagnosed AML patients. In a phase 2 study, following induction with cytarabine and daunorubicin (7+3) plus ENTO, higher rates of complete response (CR) or CR with incomplete hematologic recovery (CRi) were observed in patients with rearrangements of the KMT2A (MLL) gene (MLL-r) and mutations of the nucleophosmin 1 (NPM1) gene, both of which are associated with aberrant expression of HOXA9 and MEIS1, as compared to patients without these mutations. In an exploratory analysis, patients with HOXA9/MEIS1 expression levels above the median experienced superior overall survival (OS) as compared to patients with expression levels below the median. In the AGILITY trial, we hypothesize that the addition of ENTO to intensive induction/consolidation in newly diagnosed patients with NPM1-mutated AML will improve the rate of CR without evidence of measurable residual disease (MRD-negative CR) post-induction and duration of event-free survival (EFS). Methods: AGILITY will be a global, multi-center, double-blind, placebo-controlled trial of ENTO in combination with cytarabine plus daunorubicin or idarubicin induction (7+3) and age-adjusted high-dose cytarabine (HiDAC) consolidation in newly diagnosed AML patients aged 18-75 years who are candidates for intensive induction and harbor a documented NPM1 mutation based on local or central mutation testing. Patients with co-mutated FLT3 (internal tandem duplication or tyrosine kinase domain) and for whom midostaurin with 7+3 is indicated are excluded. Patients will be stratified based on age ( An independent data-monitoring committee will monitor emerging safety and efficacy data from this trial on an ongoing basis. Disclosures Byrd: Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Newave: Membership on an entity's Board of Directors or advisory committees. Cortes: Bristol Myers Squibb, Daiichi Sankyo, Jazz Pharmaceuticals, Astellas, Novartis, Pfizer, Takeda, BioPath Holdings, Incyte: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bio-Path Holdings, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Sun Pharma: Consultancy, Research Funding. Minden: Astellas: Consultancy. Oellerich: Roche: Consultancy; Gilead: Research Funding; Kronos Bio, Inc.: Consultancy; Merck KGaA: Consultancy, Research Funding. Stein: Syros Pharmaceuticals, Inc.: Consultancy; Daiichi Sankyo: Consultancy; PinotBio: Consultancy; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Jazz Pharmaceuticals: Consultancy; Foghorn Therapeutics: Consultancy; Blueprint Medicines: Consultancy; Gilead Sciences, Inc.: Consultancy; Abbvie: Consultancy; Janssen Pharmaceuticals: Consultancy; Genentech: Consultancy; Syndax Pharmaceuticals: Consultancy; Agios Pharmaceuticals, Inc: Consultancy; Novartis: Consultancy; Astellas: Consultancy. Elder: PharPoint Research, Inc.: Current Employment. Kumar: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Bray: Kronos Bio, Inc.: Consultancy. DiMartino: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Stock: Pfizer: Consultancy, Honoraria, Research Funding; amgen: Honoraria; agios: Honoraria; jazz: Honoraria; kura: Honoraria; kite: Honoraria; morphosys: Honoraria; servier: Honoraria; syndax: Consultancy, Honoraria; Pluristeem: Consultancy, Honoraria. OffLabel Disclosure: Entospletinib is an investigational therapy

Introduction : Peripheral blood samples of healthy individuals may harbour detectable mutations in genes recurrently mutated in myeloid malignancies, a situation referred to as clonal hematopoiesis (CH). Risk factors for CH include increasing age as well as previous exposure to cytotoxic therapy. CH has been associated with an increased risk of overall mortality, including in the setting of autologous stem cell transplantation (ASCT) for non-Hodgkin lymphoma (Gibson et al, JCO, 2017). The excess mortality is largely driven by cardio-vascular disease, but may also be additionally attributable to an increased risk of myeloid malignancies that arise through the selection of CH subclones. Herein, we aimed to investigate the prognostic implications of CH after ASCT in an independent and diversified, large cohort of lymphoma patients using ultra-deep, highly sensitive error-correction sequencing. Methods : DNA was obtained from 420 residual apheresis products obtained from patients who had undergone autologous stem cell transplantation for lymphoma at the Princess Margaret Cancer Center between 2002 and 2018. Target DNA sequences corresponding to regions recurrently mutated in myeloid neoplasms (affecting n = 36 genes) were captured using single molecule molecular inversion probes (smMIPs) that incorporate molecular tagging. Single nucleotide variants and short insertions and deletions were identified using SmMIP-tools (Medeiros et al, bioRxiv, 2021), which implements a series of steps including probabilistic modeling of allele-specific error rates and generation of consensus sequences to suppress next-generation sequencing-associated errors. Given the high sensitivity and precision of our method, we did not prespecify a variant allele fraction cut-off. Results : All patients had relapsed/refractory lymphoma, except for 98 (23.3%) mantle cell lymphoma patients and one patient with extranodal NK/T-cell lymphoma where ASCT was part of frontline management. The most common conditioning regimens were high-dose melphalan and etoposide (77.5%) and high-dose melphalan and Ara-C (16.4%). We identified 275 high-confidence mutations in 181 out of 420 patients (43.1%), with 64 of these 181 patient samples (35.4%) having more than one mutation. The median age was higher in patients with CH than in patients without (55 years vs. 51, P = 0.002). The most frequently mutated gene were PPM1D (11.9%), followed by TET2 (11.4%), DNMT3A (8.8%), ASXL1 (5.2%) and TP53 (4.5%). The lymphoma subtype with the highest prevalence of CH was T-cell lymphoma (CH found in 72.2% of cases), followed by transformed indolent lymphoma (51.4%), mantle cell lymphoma (47.5%), diffuse large B-cell lymphoma (40.4%) and Hodgkin lymphoma (33.3%). While there was no difference in the number of CD34+ cells infused for patients with and without CH, the median time to neutrophil engraftment and the median time to platelet engraftment were significantly longer in patients with CH (11 days vs. 10 days, P = 0.025; and 14 days vs. 13 days, P < 0.001, respectively). The median follow-up of living patients was 4.2 years. Patients with CH had inferior 5-year OS from the time of first relapse (38.9% vs. 45.5%, P = 0.037) and from the time of ASCT (51.2% vs. 59.1%, P = 0.017, see figure). Five-year OS from ASCT was 47.5% vs. 53.7% in patients with 1 mutation and > 1 mutation, respectively, compared to 59.1% in patients without CH (P = 0.005). The presence of CH did not have an impact on the risk of post-ASCT relapse. In multivariate Cox regression analysis in which CH and age (as a continuous variable) were included, CH remained significantly associated with adverse OS post-ASCT (HR 1.39, 95% 1.02-1.91, P = 0.038). Only seven patients out of 420 (1.7%) developed a therapy-related myeloid neoplasm (TMN). The cumulative incidence of TMN was not significantly increased in patients with CH (10-year cumulative incidence 3.3% vs. 3.0% in those without CH, P = 0.433). Conclusions : Our results show that CH was associated with delayed neutrophil and platelet engraftment. Moreover, CH conferred an increased risk of death after ASCT that was not explained by lymphoma relapse. The risk of TMN was low in our cohort and CH was not a risk factor for TMN, an observation that is distinct from prior observations (e.g. Gibson et al, JCO, 2017 and Husby et al, Leukemia, 2020). Our results raise the possibility that the risk of TMN may be modulated by factors other than CH. Figure 1 Figure 1. Disclosures Minden: Astellas: Consultancy. Kuruvilla: Janssen: Honoraria, Research Funding; Antengene: Honoraria; AstraZeneca: Honoraria, Research Funding; Amgen: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Karyopharm: Honoraria, Other: Data and Safety Monitoring Board; Pfizer: Honoraria; AbbVie: Honoraria; TG Therapeutics: Honoraria; Medison Ventures: Honoraria; Merck: Honoraria; Gilead: Honoraria; BMS: Honoraria; Roche: Honoraria, Research Funding; Seattle Genetics: Honoraria. Crump: Roche: Research Funding; Epizyme: Research Funding; Kyte/Gilead: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Prica: Kite Gilead: Honoraria; Astra-Zeneca: Honoraria. Chen: Beigene: Membership on an entity's Board of Directors or advisory committees; Astrazeneca: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy. Kridel: Gilead Sciences: Research Funding.

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. Reinstating immunological control of AML is highly desirable to eradicate chemotherapy-resistant clones and provide long-term disease control. We recently identified bone marrow (BM) microenvironmental transcriptomic profiles that stratify patients with newly diagnosed AML into an immune-infiltrated and an immune-depleted subtype and that refine the accuracy of survival prediction beyond that afforded by current prognosticators (Vadakekolathu J et al., 2020). We have also shown that CD8 + T cells from patients with AML exhibit features of immune exhaustion and senescence (IES), including heightened expression of killer cell lectin-like receptor subfamily G member 1 (KLRG1) and B3GAT1 (encoding CD57) (Knaus H et al., 2018). Whether deranged T-cell functions affect the likelihood of responding to antitumor therapy, including immune checkpoint blockade (ICB), is an outstanding question in AML. In the current study, we analyzed 183 BM samples collected longitudinally at time of AML onset, response assessment and disease relapse from multiple cohorts of patients with AML treated with standard-of-care induction chemotherapy, and from 33 elderly AML patients with newly diagnosed or chemotherapy-refractory/relapsed AML treated with azacitidine, and the PD-1 checkpoint inhibitor pembrolizumab (NCT02845297). Primary patient specimens and associated clinical data were obtained via informed consent in accordance with the Declaration of Helsinki on research protocols approved by the Institutional Review Boards of the participating Institutions. RNA (150-200 ng) was extracted from BM aspirates and was processed on the nCounter FLEX analysis system (NanoString Technologies, Seattle, WA) using the PanCancer Immune profiling panel, as previously published (Vadakekolathu J et al., 2020). The correlation between transcriptomic features of IES, clinical characteristics, therapeutic response and patient outcome was validated using publicly available RNA-sequencing and NanoString data from 1,698 patients with AML, including samples from the TCGA-AML (n=147 cases), Beat-AML Master Trial (n=264 cases, of which 240 with survival data and 195 with chemotherapy response data) and Children's Oncology Group (COG)-TARGET AML series (n=145 cases). We initially showed that, compared with their non-senescent CD8 +CD57 -KLRG1 - counterpart, senescent CD8 +CD57 +KLRG1 + T cells are functionally impaired in terms of their ability to effect AML-blast killing mediated by an anti-CD33/CD3 bi-specific T-cell engager antibody construct (kindly provided by Amgen, USA; effector/target [E/T] ratio = 1:5). We then used gene set enrichment analysis (GSEA) to derive a transcriptomic signature of IES encompassing natural killer (NK)-cell and stem-like CD8 + T-cell markers, and showed that IES states correlate with lymphoid infiltration, adverse-risk molecular lesions (TP53 and RUNX1 mutations), experimental gene signatures of leukemia stemness (LSC17 score; Ng et al., 2016) and poor outcome in response to standard induction chemotherapy (Fig. 1A). In independent validation cohorts of children and adults with AML, the IES score was higher at baseline in patients with primary induction failure (following a standard 2 cycles of chemotherapy) compared with complete remission, increased in post-chemotherapy BM specimens, and predicted survival with greater accuracy than the ELN cytogenetic risk classifier (Fig. 1B). In the immunotherapy setting, high IES scores at baseline defined a checkpoint blockade-unresponsive AML tumor microenvironment and correlated with significantly shorter overall survival (9.1 versus 15.56 months in patients with high and low IES scores, respectively; HR = 3.32 (95% CI = 1.19-9.25); log-rank P = 0.021; Fig. 1C). Finally, the IES-related gene set also predicted for long-term outcomes and objective responses, based on RECIST criteria, to single-agent nivolumab or pembrolizumab, or combination anti-PD-1 + anti-CTLA-4, in 106 patients with melanoma (PRJEB23709 and GSE93157 series), a tumor type known to derive durable clinical benefit from ICB (Fig. 1D). Our findings encourage the pursuit of immune senescence reversal as a strategy to functionally reinvigorate T cells and could inform the delivery of ICB and other T cell-targeting immunotherapies to patients who are likely to benefit. Figure 1 Figure 1. Disclosures Radojcic: Syndax Pharmaceuticals: Research Funding; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Allakos: Membership on an entity's Board of Directors or advisory committees. Minden: Astellas: Consultancy. Tasian: Aleta Biotherapeutics: Consultancy; Gilead Sciences: Research Funding; Kura Oncology: Consultancy; Incyte Corporation: Research Funding.

Simple Summary Persistence of infused cells is an important factor that dictates the outcome of adoptive cellular therapy (ACT). DNT therapy is a novel form of ACT with promising result in treating relapsed or refractory AML in preclinical and early clinical studies. However, in vivo kinetics of human DNTs in cancer-bearing host have not been previously investigated. This study was the first to investigate the persistence of DNTs and ways to improve it in patient-derived xenograft models. DNTs persistence was observed up to 50 days in various organs of leukemia-bearing hosts. However, the detected DNT level was low while significant level of persisting AMLs was observed. To improve the in vivo persistence and therapeutic efficacy of DNTs, we expanded DNTs in the presence of an PI3Kδ inhibitor, idelalisib (Ide). Ide treatment of healthy donor-derived DNTs promoted early memory subsets and improved overall fitness, reducing exhaustion while improving viability. These Ide-induced attributes led to prolonged persistence of DNTs, resulting in superior anti-leukemic activity in vivo. Further, Ide-treated DNTs improved the durability of the treatment response. Collectively, our study highlights the importance of DNT persistence and Ide-mediated improvements in the overall fitness of DNTs, which promote longer persistence in vivo and better treatment outcome. Abstract The double negative T cell (DNT) is a unique subset of T cells with potent anti-leukemic potential. Previously, DNT therapy has been shown to effectively target AML cells in patient-derived xenograft (PDX) models. Further, a recently completed phase I/IIa clinical study demonstrated the safety, feasibility, and potential efficacy in AML patients that relapsed after allogeneic hematopoietic stem cell transplantation. However, the persistence and durability of DNT-mediated anti-leukemic response is less well understood. In this study, we characterized the in vivo persistence of DNTs in PDX models. Further, we improved the efficacy and durability of DNT-mediated activity with phosphoinositide 3-kinase delta (PI3Kδ) inhibition. Mechanistically, DNTs treated with the PI3Kδ inhibitor, Idelalisib (Ide), exhibited early memory phenotype with superior viability and proliferative capacity but less cell exhaustion. Collectively, the findings from this study support the use of Ide-treated DNTs to improve its therapeutic outcome.

Flexible bronchoscopy with bronchoalveolar lavage (BAL) is commonly performed in immunocompromised patients. Nevertheless, it remains unclear whether bronchoscopy with BAL leads to changes in medical management or is associated with procedural complications among critically ill acute leukemia (AL) patients. We evaluated 71 AL patients who underwent diagnostic bronchoscopy with BAL in the intensive care unit (ICU) between 1 January 2007 and 31 December 2012. We recorded baseline characteristics, vital signs (before, during, and after the procedure), changes in medical management following the procedure, and procedural complications. Using a multivariable logistic regression model, we explored the relationship between patient characteristics and whether bronchoscopy changed management or caused complications. Patient characteristics included as predictors in the regression model were age, sex, immunosuppression status (those undergoing active chemotherapy), and the Acute Physiology And Chronic Health Evaluation II score. The most common indication for ICU admission was respiratory failure (51 patients, 72%), followed by sepsis (14 patients, 20%). Overall, the results obtained from bronchoscopy with BAL were associated with a change in management in 32 patients (45%), most commonly a change in antimicrobial therapy as a result of an infectious pathogen being identified (17 patients, 24%). Complications were documented in nine patients (13%) and included post-procedural hypoxia (six patients, 8%), the need for intubation (one patient, 9% of non-intubated patients), and tracheal perforation (one patient, 1%). No clinically significant changes in patient vital signs were observed during or immediately following the procedure. Patient characteristics did not predict whether bronchoscopy was associated with changes in medical management or procedural complications in multivariable analyses. Flexible bronchoscopy with BAL is relatively safe and helps to guide medical management among patients with AL admitted to the ICU.

Introduction: CFI-400945 is a first-in-class, potent, selective, orally active inhibitor of Polo-like kinase 4 (PLK4) (Ki=0.26nM), a master regulator of centriole duplication, necessary for genomic integrity (Mason et al. Cancer Cell 2014; 26:163-76). CFI-400945 has activity in leukemia cell lines and primary leukemia samples including those with complex karyotype, inversion 3 and monosomy 7 (Minden. personal communications). This suggests that CFI-400945 may provide an effective treatment of patients with AML. The objectives of this phase 1 trial was to establish the safety, tolerability, and recommend phase II dose (RP2D) of CFI-400945 in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Methods: Patients with relapsed/refractory AML or MDS and patients with untreated AML who refused induction chemotherapy or who are not candidates for intensive chemotherapy were eligible. Dose escalation followed a standard 3+3 design with a starting dose of 64 mg orally once daily. Plasma levels of CFI-400945 free base were measured on Days 1, 2, & 29 of Cycle 1 and Day 15 on all subsequent cycles. Peripheral blood and/or bone marrow were obtained at baseline, Day 8 of Cycle 1 and Day 1 of each subsequent cycle prior to dosing for pharmacodynamic monitoring. Safety assessments using the NCI CTCAE version 4.03 were performed. Results: From May 2018 to June 2019, nine patients have been enrolled on study across three pre-defined dose levels (64 mg [n=3], 96 mg [n=4], and 128 mg [n=2]). Three patients had untreated AML, five patients had relapsed/refractory AML and one patient had myelodysplastic syndrome/myeloproliferative disorder (MDS/MPN). Patient characteristics at diagnosis are outlined in Table 1. Six (67%) patients had baseline high throughput sequencing; the most frequent mutations were TP53 (33%), TET2 (33%), KRAS (33%) and DNMT3A (33%). A total of 20 cycles were administered with a median of 1 cycle (range, 0 to 7 cycles). The most common non-hematological drug related toxicities of any grade, which occurred in over 20%, were diarrhea (44%), headache (44%), colitis (33%), vomiting (33%), bilirubin increase (22%), dizziness (22%), fatigue (22%), and nausea (22%). One patient on the 96 mg dose level was not evaluable for DLT and hence, replaced. Both patients treated at the 128 mg/day dose level developed DLTs, consisting of grade 3 colitis and grade 5 sepsis and colitis. Pharmacokinetic profile indicated low interpatient variability between patients. Maximum exposure did not correlate with toxicity Six patients were evaluable for disease response. Two (33%) achieved complete remission (CR), 3 pts (50%) had stable disease (with one patient having a 78% reduction in marrow blast count). The patient with MDS/MPN who did not complete 1 cycle of therapy progressed to AML (Figure 1). Both patients who obtained a CR had an early response within 2 cycles. One CR has been durable for 218 days with no measurable residual disease (MRD) by flow cytometry. The additional patient, who obtained a CR with incomplete platelets recovery, with subsequent best response of CR, had a sustained response for 91 days before relapse was confirmed by bone marrow examination (Figure 1). Conclusion: Single agent CFI-400945 has activity in patients with poor risk AML. The RP2D in this population is 96 mg once daily. Dose expansion is occurring at the RP2D level. A phase 2 study with CFI-400945 single agent or in combination study with azacitidine or decitabine is planned. Disclosures Leber: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Lundbeck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda/Palladin: Honoraria, Membership on an entity's Board of Directors or advisory committees; Treadwell: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Otsuka Pharmaceutical: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bray:Treadwell Therapeutics: Current Employment; TIO Discovery: Current Employment. Gupta:Pfizer: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Research Funding. Maze:Novartis: Honoraria; Pfizer: Consultancy; Takeda: Research Funding. McNamara:Novartis: Honoraria. Schimmer:Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock ; Takeda: Honoraria, Research Funding; Novartis: Honoraria.

BACKGROUND: Multiparameter flow cytometry (MFC) has increasingly been used for measurable residual disease (MRD) assessment in patients with acute myeloid leukemia (AML), while next-generation sequencing (NGS)-based MRD monitoring tool is in clinical development for its application. Clonal hematopoiesis (CH), in which leukemia-associated somatic mutations gene are present in individuals with no apparent hematologic disease, adds a challenge in the detection of MRD. In patients with AML, CH could be potentially pre-leukemic, while persistent mutations in DNMT3A, TET2 orASXL1 (DTA) in remission marrow are usually removed from the analysis of residual leukemic cells. However, reports suggest that persistent DTA mutations in remission may be correlated with an increased relapse risk. In the patients with DTA mutations, the use of NGS for MRD monitoring is limited or modified due to the presence of CH clone in the remission marrow. We evaluated whether MFC-MRD can be adjunctive to predict the risk of AML relapse in this population of 221 patients with DTA mutation (DNMT3A (n=123), ASXL1 (n=56) or TET2 (n=100). METHODS: The present study evaluated long-term outcomes in AML patients who achieved first complete remission (CR1) and compared outcomes according to MFC-based MRD status (was defined as negative if patients achieved 0.1 or less) assessed at the time of CR1. A total of 435 patients diagnosed with AML and treated with induction chemotherapy between 2015 and 2018 were included. MFC-MRD was assessed in 336 patients in CR1 (77%). NGS was performed using samples obtained at the time of initial diagnosis and used for mutational subgroup classification. Overall survival (OS) was calculated as the date of CR1 to the date of death and censored on the date of the last follow-up. Relapse-free survival (RFS) was defined as the time from the date of CR1 to the date of relapse or death from any cause. Cumulative incidence of relapse (CIR) and non-relapse mortality (NRM) were calculated considering competing risk. The Kaplan-Meier method using a log-rank test and a multivariate Cox proportional hazard model was used for analyses of time-to-event endpoints. For CIR and NRM, Gray test was performed for the risk factors and the Fine-Gray model was adopted for the multivariate model. RESULTS: According to the MFC-MRD status, i.e., the group with positive MRD (MRDpos; n=118, 35%) vs. those with negative MRD (MRDneg; n=218, 65%), we evaluated OS, RFS, and CIR. The MFC-MRDneg group showed better OS at 2 years 67.0% than the MFC-MRDpos group 40.7% (p We divided the groups according to the number of induction treatment courses, AML type, cytogenetics risk, and age ( Also, we evaluated MFC-MRD status at CR by mutational profile subgroup. Long-term outcomes such as OS, RFS, CIR or NRM were compared by the mutational subgroup. It consistently showed a trend of superior OS, RFS and lower risk of CIR in patients with MFC-MRDneg compared to MFC-MRDposTab1. Of interest, in the subgroup of patients carrying any DTA mutations (n=221), those with MFC-MRDneg (n=103) showed better OS (HR 1.61 [1.01-2.55%]; p=0.042), RFS (HR 1.66 [1.06-2.61%]; p=0.026) and CIR (HR 1.99[1.03-3.83%]; p=0.04) compared to those MFC-MRDpos (n=64; Fig 1). Multivariate analysis confirmed that the MFC-MRDneg is an independent prognostic factor in patients with DTAmutwith respect to OS: MFC-MRDpos (HR 1.63, p=0.04) and age (≥60; HR 2.04, p=0.008) for OS; for RFS, MFC-MRDpos (HR 1.71, p=0.02) and age (≥60; HR 2.32, p= 0.001); for CIR, MFC-MRDpos (HR 2.31, p=0.01) and HCT (HR 0.14, p= Conclusion: These findings suggest that in AML patients with DTAmut, MFC-MRD status at the time of remission assessment can be a tool for MRD assessment when NGS-based MRD assessment is limited. Further study is strongly warranted to reach a clearer conclusion with multiple cohorts. Disclosures Schimmer: Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock . Tierens:Amgen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas Pharma: Membership on an entity's Board of Directors or advisory committees. McNamara:Novartis: Honoraria. Maze:Pfizer: Consultancy; Novartis: Honoraria; Takeda: Research Funding. Gupta:Pfizer: Consultancy; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Research Funding.

Introduction: Acute myeloid leukemia (AML) is driven by a subpopulation of leukemia stem cells (LSCs), which possess properties such as quiescence and self-renewal that are linked to therapy resistance and relapse. The LSC17 score was derived from genes differentially expressed between functionally validated LSC+ and LSC- fractions from 78 AML patients and is strongly associated with survival and response to standard therapy. A critical advantage of the LSC17 test over cytogenetic and molecular analysis is its rapid turnaround time (24-48h on a NanoString platform), providing clinicians with a rapid and powerful tool for upfront risk stratification. We have developed a clinical assay for the LSC17 score validated in a CAP/CLIA-lab setting. Methods: We conducted a prospective, multicenter validation and feasibility study to test the prognostic value of the LSC17 assay under real-world conditions in AML patients treated with curative intent. Patients with a possible new diagnosis of AML were eligible. Patients with a confirmed diagnosis of acute promyelocytic leukemia were excluded from analysis. Standard prognostic markers including cytogenetics, molecular studies and targeted sequencing using a standard AML panel were performed in parallel to the LSC17 score. Treatment was administered according to physician preference, based on patient history and results of standard prognostic assays, when available. Survival data was censored on June 14th, 2020. Results: 381 patients were recruited to the study between June 2016 and March 2020. 4 patients were excluded for quality control reasons (one sample had insufficient RNA and three samples failed quality control checks). 103 were excluded as they had alternative diagnoses. 84 patients were excluded because they did not receive intensive chemotherapy. LSC17 scores ranged from 0 to 1.25, and were classified as high or low according to the median score of 0.51 from a previously validated reference cohort (Ng et al, Nature 2016). Of the 190 patients included in this analysis, 84 had a low LSC17 score and 106 had a high LSC17 score. The median age was 61 years (range 18-79); 86 (45%) were female. When stratified according to ELN 2017 criteria, 48 (27%), 51 (29%), and 77 (44%) patients had favorable, intermediate, and adverse risk disease, respectively. Low LSC17 score was associated with normal cytogenetics (high vs low, 33% vs 58%; P We first considered response to induction chemotherapy (Table 1). 141 patients had standard induction chemotherapy with 3+7, 40 had Flag-IDA and 9 had CPX-351. High score patients had inferior responses to 3+7 with only 59% achieving complete remission (CR) after 1 cycle of chemotherapy compared to 96% of low score patients; responses for LSC17 high score patients were better in the Flag-IDA group with 80% achieving CR after 1 cycle. When considering overall CR rates after 2 cycles of induction, patients with a high LSC17 score were less likely to achieve CR (high vs low, 87% vs 98%; P=0.02). However, this difference was predominantly observed in patients treated with 3+7 (87% vs 99% CR rate in high vs low score patients, respectively); response rates to Flag-IDA were not significantly different between the 2 groups. Measurable residual disease (MRD) monitoring by flow cytometry was performed at the time of CR in 135 (71%) patients enrolled at Princess Margaret Cancer Centre. Patients with a high LSC17 score were significantly more likely to have MRD compared to low score patients (46% vs 10% respectively, P Conclusion: AML patients with a high LSC17 score have inferior outcomes following 3+7 induction chemotherapy. The LSC17 score should be considered as a tool to identify and stratify high-risk patients to alternative upfront therapies such as Flag-IDA. A risk adapted study is planned to validate these results. Disclosures Gupta: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Research Funding. Maze:Novartis: Honoraria; Takeda: Research Funding; Pfizer: Consultancy. McNamara:Novartis: Honoraria. Schimmer:Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock ; Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria. Leber:Takeda/Palladin: Honoraria, Membership on an entity's Board of Directors or advisory committees; Treadwell: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Otsuka Pharmaceutical: Honoraria, Membership on an entity's Board of Directors or advisory committees; Lundbeck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Tierens:Amgen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas Pharma: Membership on an entity's Board of Directors or advisory committees. Wang:Trilium therapeutics: Patents & Royalties: There is an existing license agreement between TTI and University Health Network and J.C.Y.W. may be entitled to receive financial benefits further to this license and in accordance with UHN's intellectual property policies. .

Background and objectives: CPX-351 is a liposomal formulation of daunorubicin and cytarabine in a fixed synergistic ratio of 5:1. It has been approved by both the FDA and EMA for use in high risk AML including therapy related AML (t-AML) and AML with myelodysplastic related change (AML-MRC). When compared with '3+7', CPX-351 resulted in superior OS (9.56 vs 5.96 months) and overall response rate (47.7% v 33.3%; P = .016; Lancet JCO 2018). However, this is little data of CPX-351 in a real world setting. The primary objective of our multi-centre study was to assess outcomes and associated toxicities of CPX-351 in these high risk AML patients. Methods: Patients aged 18 and over who were treated with CPX-351 and who met the WHO criteria for t-AML and AML-MRC were included in the study. Retrospective data was collected from 6 centres throughout Canada. Targeted sequencing was performed on DNA samples using the TruSight Myeloid Sequencing Panel isolated from peripheral blood or bone marrow samples at diagnosis. Overall survival (OS) and progression free survival (PFS) rates were calculated using the Kaplan-Meier method. Results: Fifty patients treated with CPX-351 were identified from six centres. Baseline characteristics are seen in Table 1. Cytogenetics was available in 45 (90%) patients and an abnormal karyotype was seen in 30 (60%) patients. Monosomal (33%) and complex (28%) karyotype were the most common chromosomal abnormalities seen. Targeted sequencing was performed on 64% patients (32/50) and the average number of mutations was 2(0-7). RUNX1 was the most commonly mutated gene found in 22% (7/32), followed by SRSF2 in 19% (6/32) with ASXL1 and NRAS both at 16% (5/32). Other commonly mutated genes were NPM1, IDH2, DNMT3a and TP53 all occurred at a frequency of 12% (4/32). Of the 10 patients (20%) that were FLT3 mutated (80% ITD and 20% TKD), 5 patients received the FLT3 inhibitor midostaurin in combination with CPX-351. Assessing treatment responses, 50% (25/50) of patients achieved a complete remission (CR) or a complete remission with incomplete recovery (CRi). Median neutrophil recovery (≥500/μl) was 32 days (range 18-68) and median platelet recovery (≥50 000/μl) was 34 days (range 19-70). Proven or probable fungal infection was seen in 10 (20%) patients. 30 day mortality was 4% (2/50) and 60-day mortality was 10% (5/50). The median CR duration was short at 7.17 months. Of the 25 patients who did not achieve a CR, 6 (24%) had prior azacitidine use for MDS suggesting CPX-351 may not have activity in this group of patients. Median follow up was 7.43 months (range 0.2 to 18 months). OS was 44% at 12 months (0.28-0.58 95% CI) and 29% at 18 months (Fig 1; 0.13-0.46 95% CI). PFS was 28% at 12 months (0.15-0.43 95% CI) and 18% at 18 months (0.06-0.39 95% CI). There were no differences in OS when stratified by ELN risk (p=0.25), adverse risk cytogenetics (p=0.1485), or poor risk mutations such as FLT3-ITD (p=0.29), RUNX1 (p=0.123) or ASXL1 (p=0.06). This is consistent with previous studies, which suggest that CPX-351 overcomes the poor prognosis associated with these mutations. 18 (36%) patients received an allogeneic stem cell transplant (ASCT) in CR1. Patients who received a allogeneic transplant had significant improvement of OS of 62% at 18 months compared to those who did not receive a transplant of 14.5% at 18 months (Fig 2; p=0.0008). Conclusion Our study reveals that CPX-351 produces high remission rates in patients with AML with a similar efficacy and toxicity profile seen in the Phase 3 trial data (Lancet JCO 2018). However, response rates are short and therefore CPX-351 is most effective when used as a bridge for allogeneic stem cell transplantation in this high risk population. Disclosures Assouline: AbbVie: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Takeda: Research Funding; BeiGene: Consultancy, Honoraria, Research Funding. Brandwein:Celgene: Honoraria; Astellas: Honoraria; Taiho: Honoraria; Roche: Honoraria; Jazz Pharmaceuticals: Honoraria; Pfizer: Honoraria; Amgen: Honoraria. Gupta:Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Research Funding; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Maze:Pfizer: Consultancy; Takeda: Research Funding; Novartis: Honoraria. McNamara:Novartis: Honoraria. Sanford:Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees. Schimmer:Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock ; Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria.

Background: CFI-400945 is a potent, selective, orally administered, first-in-class inhibitor of the serine/threonine kinase, Polo-like kinase 4 (PLK4). PLK4 is a highly conserved master regulator of centriole duplication, and is critical for maintenance of genomic integrity. Aberrant expression of PLK4 results in a number of effects including the centrosome amplification often seen in aneuploid cancers, pointing to a potentially causative role for PLK4 in genome instability and cancer progression. A Phase 1 study has been completed evaluating CFI-400945 as a monotherapy in solid tumors, showing a tolerable safety profile and promising signs of activity. As acute myeloid leukemia (AML) is a disease characterized by genomic instability, it is of significant interest as a potential indication for the clinical evaluation of CFI-400945. pre-clinical studies, CFI-400945 showed potent activity towards leukemia cell lines and primary human samples in vitro, as well as marked efficacy in two subcutaneous models of leukemia, specifically the MV4-11 FLT3-ITD AML. A Phase 1 trial in AML was initiated at the Princess Margaret Cancer Center in 2018, and of six patients evaluable for response, two (33%) achieved complete remission (CR), and 3 patients (50%) had stable disease (with one patient having a 78% reduction in marrow blast count) [re: Murphy et al, ASH 2020]. These promising results have led to a plan for an expanded trial examining CFI-400945 in AML, particularly focused on complex karyotype (CK). Study Design and Methods: The study will have 4 parts, Part 1A (1A): a single agent dose escalation portion, Part 1B (1B): a food effect portion once the MTD of 1A is determined, and combinations with azacitidine (2A), and decitabine (2B). For parts 1A and 1B, patients with relapsed and/or refractory AML, MDS, or CMML after >1 prior therapy will be included. Patients with MDS or CMML must have progressed or had a lack of response after at least 4 cycles of hypomethylating agents. For parts 2A and 2B, patients should have relapsed and/or refractory AML or untreated MDS or CMML. Untreated patients who decline or are ineligible for intensive therapy may be included. The study will use a standard 3 + 3 design. The maximum tolerated dose (MTD) will be defined as the dose level where the number of dose limiting toxicities (DLTs) is Biomarker Selection and companion Diagnostics: No biomarker based pre-selection of patients. PD evaluations will include blast reduction and markers of mitosis. Study Treatment and Endpoints: Part 1: Each cycle will be 28 days (21 days on/7 days off). Starting does of CFI-400945 will be 32mg po. Once the MTD of 1A is determined, 1B will explore the food effect of a high fat meal on the PK of CFI-400945 at the MTD. 2A and 2B will explore the combination of CFI-400945 with standard dose of either azacitidine (2A) or decitabine (2B). The starting dose of CFI-400945 will be 32mg po for 21 days on/7 off. The DLTs will be defined as NCI CTCAE V5.0 Grade >3 related non-hematologic event(s) occurring during cycle 1 or prolonged pancytopenia in the presence of a hypocellular bone marrow > day 42 without evidence of disease. The efficacy endpoints for AML, MDS, and CMML include the overall response rate, and the CR rate per standard criteria. The aim of the food effect part of the study is the asses the effect of high fat food on the PK of CFI-400945. The safety endpoint is the incidence of treatment emergent adverse events. PK endpoints include evaluations of parameters such as half-life, AUC, etc. Exploratory endpoints include eval of minimal residual disease, genomic alterations and other molecular features associated with response and biological effects of PLK4 inhibition. Disclosures Jonas: Amgen: Consultancy, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; AbbVie: Consultancy, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; GlycoMimetics: Consultancy, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; Sigma Tau: Research Funding; Jazz: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Takeda: Consultancy; Tolero: Consultancy; Treadwell: Consultancy; Forty Seven: Research Funding; Accelerated Medical Diagnostics: Research Funding; AROG: Research Funding; Daiichi Sankyo: Research Funding; F. Hoffmann-La Roche: Research Funding; Forma: Research Funding; Genentech/Roche: Research Funding; Hanmi: Research Funding; Incyte: Research Funding; LP Therapeutics: Research Funding; Pfizer: Research Funding; Pharmacyclics: Research Funding. Bixby:GlycoMimetics: Research Funding. Brandwein:Pfizer: Honoraria; Amgen: Honoraria; Celgene: Honoraria; Astellas: Honoraria; Taiho: Honoraria; Roche: Honoraria; Jazz Pharmaceuticals: Honoraria. Michelson:Treadwell Therapeutics: Consultancy. Bray:TIO Discovery: Current Employment; Treadwell Therapeutics: Current Employment. Roberts-Thomson:Treadwell Therapeutics: Current Employment. Borthakur:BioTherix: Consultancy; FTC Therapeutics: Consultancy; Nkarta Therapeutics: Consultancy; Treadwell Therapeutics: Consultancy; PTC Therapeutics: Consultancy; Argenx: Consultancy; Oncoceutics: Research Funding; Xbiotech USA: Research Funding; Polaris: Research Funding; AstraZeneca: Research Funding; BMS: Research Funding; BioLine Rx: Research Funding; Jannsen: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Incyte: Research Funding; BioLine Rx: Consultancy; PTC Therapeutics: Research Funding; Curio Science LLC: Consultancy; Cyclacel: Research Funding; GSK: Research Funding.

Upfront therapy for newly diagnosed patients with acute myeloid leukemia (AML) includes intensive induction chemotherapy with curative intent, low dose chemotherapy, best supportive care, and clinical trials. The choice between these therapies is influenced by multiple factors including age, cytogenetic and molecular mutations, and performance status. In our single payer provincial health care system, induction chemotherapy and clinical trials are only offered at a small number of specialized quaternary care centers with geographically large catchment areas. As a result, some patients are required to travel long distances for their appointments, which may constitute a barrier to care, especially among elderly patients. We therefore asked whether distance from the quaternary center influences the choice of care for AML. We reviewed the records of patients ≥18 years of age diagnosed with AML from 2015-2017 and assessed at our quaternary care center in Toronto, Canada. We compared upfront therapy choice and survival between patients living close versus distant from the cancer center (empirically defined as 50km) and stratified by age. A total of 675 patients were assessed by our quaternary center for a new diagnosis of AML during the timeframe studied. Of those patients, 477 (71%) patients lived ≤50km, and 198 (29%) patients lived >50km from the quaternary center. The overall median distance from patient residence to the quaternary center was 33.2km (range: 1-1791km), and the median distance of patients in the >50km group was 93km (range: 50.2-1791km). Age, sex, baseline Eastern Cooperative Oncology Group Performance Status (ECOG), and cytogenetic risk were not significantly different between the two groups. There were no differences in the proportion of patients receiving induction chemotherapy or clinical trial as upfront therapy between patients living close versus distant from the quaternary center, even when stratified for age ≥70 years. There was no difference in overall survival between patients living ≤50km versus >50km from the quaternary center either overall, or when stratified by age. In conclusion, geographic distance from treatment center does not appear to impact choice of upfront therapy, access to clinical trials, or clinical outcomes in this study of newly diagnosed patients with AML treated in a single payer environment. Disclosures Gupta: Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Research Funding; Pfizer: Consultancy; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees. Maze:Novartis: Honoraria; Pfizer: Consultancy; Takeda: Research Funding. McNamara:Novartis: Honoraria. Schimmer:Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock .

7019 Background: Midostaurin is approved for FLT3 mutant-positive (FLT3+) acute myeloid leukemia (AML), however efficacy has also been observed in a subpopulation of FLT3 mutant-negative AML, suggesting that FLT3 mutation is not the only determinant in conferring midostaurin sensitivity. We previously described a phosphoproteomic signature significantly elevated in primary AML blasts that responded to midostaurin ex vivo (Casado et al Leukaemia 2018). This signature includes phosphorylation sites on protein kinase C delta (a midostaurin off-target) and its substrate GSK3A. In this study, we tested whether these phospho-signatures could group FLT3+ patients based on clinical responses to midostaurin plus chemotherapy. Methods: We obtained FLT3+ bone marrow (BM) and peripheral blood (PB) diagnosis specimens (n=56 cases) from the Leukemia Tissue Bank at Princess Margaret Cancer Centre. These patients were treated with standard chemotherapy plus midostaurin. Phospho-signatures quantified using mass spectrometry were analysed with a classification machine learning algorithm to group patients based on response to treatment as a function of phospho-signature status. Other features (e.g. genetic mutations, HSC-transplant) were also analysed. Differential survival analysis was carried out with Kaplan-Meier and Log Rank test methods. Phospho-signatures for BM and PB samples were analysed independently. Results: A first ML model was developed based on the signature described in the Casado et al study. Patients positive for this signature exhibited a survival probability of 243 weeks, compared to 126 weeks in signature negative patients (averages by geometric mean, Log Rank p = 9.88e-05). As the patients in the current study received chemotherapy, in addition to midostaurin, we also identified a new signature consisting of 26 phospho-sites (model 2), which partially overlapped with the first model. Patients positive for model 2 signature showed a markedly longer survival time than negative patients (269 vs 76 weeks, Log Rank p = 1.30e-05 for PB and 241 vs 56, Log Rank p = 2.13e-09 for BM specimens, Table). No other features separated survival as clearly as model 2. Conclusions: We have identified phospho-signatures with the potential to further stratify FLT3+ AML for midostaurin treatment. The presence of PRKCD signalling components in signatures provides a rationale for midostaurin activity in sensitive cases. Analysis will also be performed on FLT3 mutant-negative cases to validate the signature in this group.[Table: see text]

In myelofibrosis (MF), driver mutations in JAK2, MPL, or CALR impact survival and progression to blast phase, with the greatest risk conferred by triple-negative status. Subclonal mutations, including mutations in high–molecular risk (HMR) genes, such as ASXL1, EZH2, IDH1/2, and SRSF2 have also been associated with inferior prognosis. However, data evaluating the impact of next-generation sequencing in MF patients treated with JAK1/2 inhibitors are lacking. Using a 54-gene myeloid panel, we performed targeted sequencing on 100 MF patients treated with ruxolitinib (n = 77) or momelotinib (n = 23) and correlated mutational profiles with treatment outcomes. Ninety-nine patients had at least 1 mutation identified, 46 (46%) had 2 mutations, and 34 (34%) patients had ≥3 mutations. Seventy-nine patients carried a mutation in JAK2V617F, 14 patients had mutations in CALR, 6 patients had an MPL mutation, and 2 patients were triple negative. No mutation was significantly associated with spleen or anemia response. A high Dynamic International Prognostic Scoring System score and pretreatment transfusion dependence were associated with a shorter time to treatment failure (TTF), and this association retained significance on multivariable analysis. Patients with ASXL1 (hazard ratio [HR], 1.86; P = .03) and EZH2 mutations (HR, 2.94; P = .009) and an HMR profile (HR, 2.06; P = .01) had shorter TTF. On multivariate analysis, ASXL1 or EZH2 mutations were independently associated with shorter TTF and overall survival. These findings help identify patients unlikely to have a durable response with current JAK1/2 inhibitors and provide a framework for future studies.

In the cytogenetically normal population of AML (CN-AML), FLT3-ITD-positive and wild-type NPM1 is correlated with a worse outcome, and FLT3-ITD-negative with NPM1-mut is correlated with a better outcome. This leaves a large subpopulation of CN-AML patients without NPM1 or FLT3-ITD mutations with heterogeneous outcomes with overall survivals (OS) ranging from several weeks to years. Therefore, new prognostic markers are needed to better risk stratify this subset of patients.The retrospective study included 60 de novo adult AML patients diagnosed at our institution with normal karyotype, no FLT3-ITD or NPM1 mutations, and who did not receive allogeneic hematopoietic stem cell transplantations. We investigated the prognostic significance of immunophenotypic markers and clinical laboratory features in this double-negative population.Older age (60) and CD4 expression (14%) were significantly correlated with shorter event-free survival (EFS) (P0.001, P = 0.016, respectively). Expression of CD56 (12%), as well as lack of CD34 expression (19% of the cases), was also associated with a worse EFS (P = 0.048, P = 0.028, respectively). On multivariable analysis, CD4 expression and old age (60) were identified as independent predictors for worse EFS (P = 0.016; P = 0.001, respectively) and OS (P = 0.048; P = 0.028, respectively).Our results indicate that CD4 expression and older age are adverse prognostic factors in wild-type NPM1, FLT3-ITD-negative CN-AML.