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We proposed a novel tool-a dose linear energy transfer (LET)-volume histogram (DLVH)-and performed an exploratory study to investigate rectal bleeding in prostate cancer treated with intensity modulated proton therapy.The DLVH was constructed with dose and LET as 2 axes, and the normalized volume of the structure was contoured in the dose-LET plane as isovolume lines. We defined the DLVH index, DLv%(d,l) (ie, v% of the structure) to have a dose of ≥d Gy and an LET of ≥l keV/μm, similar to the dose-volume histogram index Dv%. Nine patients with prostate cancer with rectal bleeding (Common Terminology Criteria for Adverse Events grade ≥2) were included as the adverse event group, and 48 patients with no complications were considered the control group. A P value map was constructed by comparison of the DLVH indices of all patients between the 2 groups using the Mann-Whitney U test. Dose-LET volume constraints (DLVCs) were derived based on the P value map with a manual selection procedure facilitated by Spearman's correlation tests. The obtained DLVCs were further cross-validated using a multivariate support vector machine (SVM)-based normal tissue complication probability (NTCP) model with an independent testing data set composed of 8 adverse event and 13 control patients.We extracted 2 DLVC constraints. One DLVC was obtained, Vdose/LETboundary:2.5keVμmat 75 Gy to 3.2keVμmat8.65Gy1.27% (DLVC1), revealing a high LET volume effect. The second DLVC, V(72.2Gy,0keVμm)2.23% (DVLC2), revealed a high dose volume effect. The SVM-based NTCP model with 2 DLVCs provided slightly superior performance than using dose only, with an area under the curve of 0.798 versus 0.779 for the testing data set.Our results demonstrated the importance of rectal "hot spots" in both high LET (DLVC1) and high dose (DLVC2) in inducing rectal bleeding. The SVM-based NTCP model confirmed the derived DLVCs as good predictors for rectal bleeding when intensity modulated proton therapy is used to treat prostate cancer.

Azacitidine + venetoclax, decitabine + venetoclax, and low-dose cytarabine + venetoclax are now standard treatments for newly diagnosed older or unfit patients with acute myeloid leukemia (AML). Although these combinations are also commonly used in relapsed or refractory AML (RR-AML), clinical and molecular predictors of response and survival in RR-AML are incompletely understood. We retrospectively analyzed clinical and molecular characteristics and outcomes for 86 patients with RR-AML who were treated with venetoclax combinations. The complete remission (CR) or CR with incomplete hematologic recovery (CRi) rate was 24%, and the overall response rate was 31% with the inclusion of a morphologic leukemia-free state. Azacitidine + venetoclax resulted in higher response rates compared with low-dose cytarabine + venetoclax (49% vs 15%; P = .008). Median overall survival (OS) was 6.1 months, but it was significantly longer with azacitidine + venetoclax compared with low-dose cytarabine + venetoclax (25 vs 3.9 months; P = .003). This survival advantage of azacitidine + venetoclax over low-dose cytarabine + venetoclax persisted when patients were censored for subsequent allogeneic stem cell transplantation (8.1 vs 3.9 months; P = .035). Mutations in NPM1 were associated with higher response rates, whereas adverse cytogenetics and mutations in TP53, KRAS/NRAS, and SF3B1 were associated with worse OS. Relapse was driven by diverse mechanisms, including acquisition of novel mutations and an increase in cytogenetic complexity. Venetoclax combination therapy is effective in many patients with RR-AML, and pretreatment molecular characteristics may predict outcomes. Trials that evaluate novel agents in combination with venetoclax therapy in patients with RR-AML that have adverse risk genomic features are warranted.

Scientific presentations, usually given with slide presentation software such as PowerPoint™, are the most common method for disseminating knowledge to students and peers. Unfortunately, many are boring, text-heavy, and bullet point-riddled data dumps, with animations or cartoons that obscure or distract rather than clarify the message. These presentations, which we have all sat through and/or delivered, are often so dull that they are referred to as "death by PowerPoint™." In this paper, the authors intend to impart basic techniques for organizing and communicating information in the most effective, engaging, and actionable ways possible. We focus on three processes: generating ideas and outlining a talk, creating visually appealing uncluttered slides, and delivering an inspiring, practice-changing presentation. We also discuss considerations for a virtual presentation. We believe that even experienced speakers could benefit from reflecting on these recommendations and editing their slide presentations for clarity and simplicity.

Background Self-reported time spent standing has been associated with lower risk of mortality. No previous studies have examined this association using device-measured standing. Method This was a prospective cohort study of 5878 older (median age = 80 years), racial/ethnically diverse, community-dwelling women in the WHI Objective Physical Activity and Cardiovascular Health Study (OPACH). Women wore accelerometers for 1 week and were followed for mortality. The study applied previously validated machine learning algorithms to ActiGraph GT3X+ accelerometer data to separately measure time spent standing with and without ambulation. Cox proportional hazards models were used to estimate mortality risk adjusting for potential confounders. Effect modification by age, body mass index, moderate-to-vigorous physical activity, sedentary time, physical functioning, and race/ethnicity was evaluated. Results There were 691 deaths during 26 649 person-years of follow-up through March 31, 2018 (mean follow-up = 4.8 years). In fully adjusted models, all-cause mortality risk was lower among those with more standing without ambulation (quartile [Q] 4 vs Q1 HR = 0.63; 95% CI = 0.49–0.81, p-trend = .003) and more standing with ambulation (Q4 vs Q1 HR = 0.50; 95% CI = 0.35–0.71, p-trend < .001). Associations of standing with ambulation and mortality were stronger among women with above-median sedentary time (HR = 0.51; 95% CI = 0.38–0.68) compared to women with below-median sedentary time (HR = 0.80; 95% CI = 0.59–1.07; p-interaction = .02). Conclusions In this prospective study among older women, higher levels of accelerometer-measured standing were associated with lower risks of all-cause mortality. Standing is an achievable approach to interrupting prolonged sedentary time, and if not contraindicated, is a safe and feasible behavior that appears to benefit health in older ages.

The role of allogeneic hematopoietic cell transplant (allo-HCT) as consolidation after initial venetoclax therapy and the efficacy of venetoclax salvage therapy for relapse after allo-HCT in patients with acute myeloid leukemia (AML) are unclear. We conducted a retrospective study of patients with AML or myelodysplastic syndrome (MDS) who received venetoclax either before or after allo-HCT at Memorial Sloan Kettering Cancer Center and Yale University from 11 August 2016 to 16 November 2020. Among 39 heavily pretreated patients who received venetoclax before allo-HCT, median OS from allo-HCT was not reached after a median follow up of 12.5 months resulting in a 12-month OS estimate of 79.0%. In 37 patients who had received venetoclax-based combinations as salvage therapy after allo-HCT, the overall response rate was 32% with a median OS of 4.7 months (12-month OS estimate: 43.4%). Four patients underwent a second allo-HCT following venetoclax-based salvage therapy suggesting it as a potential salvage treatment option.

Background: Measurable residual disease (MRD) is a powerful prognostic factor in AML, including in prediction of outcomes post allogeneic stem cell transplant (alloSCT). However, genomic predictors of successful MRD eradication with chemotherapy prior to alloSCT are unclear. Objectives: Here we provide an integrated analysis of 233 patients (pts) who underwent induction chemotherapy with baseline next-generation sequencing (NGS) followed by serial immunophenotypic monitoring for MRD while patients received additional therapy and alloSCT. Methods: All pts who received anthracycline + cytarabine, +/- investigational agents at Memorial Sloan Kettering Cancer Center starting in April 2014 were retrospectively studied (A). 142 out of 233 pts subsequently underwent alloSCT after induction or additional therapy (A). Immunophenotypic MRD was identified in bone marrow aspirates (BMA) by multiparameter flow cytometry. Any level of residual disease was considered MRD+. Molecular analysis was obtained from pre-induction BMA by NGS using 28 or 49 or 400 gene panels. Results: Patient and treatment characteristics for all pts are detailed in panel (B). Induction chemotherapy resulted in an MRD-CR/CRi and MRD+CR/CRi in 29% and 23% of all pts, respectively (C). Additional therapy included consolidation (n=51), intensive re-induction/salvage (n=47) and non-intensive therapy (n=9). Of 83 AML pts with persistent AML and 58 pts with MRD+CR/CRi after induction (R1), 38/141 (27%) were able to be converted to MRD-CR/CRi. While 33/38 of pts went on to alloSCT after conversion to MRD-CR/CRi, 22 and 36 pts went to alloSCT with persistent AML and MRD+CR/CRi AML, respectively. We focused on pre-induction molecular predictors for achieving an MRD-CR/CRi response prior to transplant for the 142 pts who underwent alloSCT (D). Pts with a NPM1 (79%, Odds ratio [OR] 3.7, p=0.01), IDH1 (92%, OR 3.9, p=0.01) and KRAS (100%, OR 5.0, p=0.03) mutations achieved high rates of MRD-CR/CRi prior to alloSCT. In contrast, RUNX1 (28%, OR 0.2, p=0.01), TP53 (12%, OR 0.1, p=0.02) and SF3B1 (14%, OR 0.1, p=0.04) mutations predicted decreased odds of achieving MRD-CR/CRi prior to alloSCT despite induction and post-induction therapy. AlloSCT resulted in high rates of conversion from MRD+ and persistent disease to MRD negativity. Most pts who entered transplant with CR/CRi MRD+ (28/36, 76%) or persistent AML (14/22, 64%) cleared MRD by the first post-transplant BMA at a median of 32 days (E). Post-alloSCT follow-up indicated value in converting MRD+ to MRD- prior to alloSCT. There was no significant difference in post-transplant cumulative incidence of relapse (F) and OS (G) between early MRD-CR/CRi immediately following induction versus later conversion to MRD-CR/CRi with additional therapy prior to alloSCT. Despite initial post-transplant MRD clearance, pts who entered alloSCT with persistent AML or MRD+ had higher incidence of relapse (p=0.00037, F) and poorer post-transplant OS (p=0.013, G) compared to pts who entered alloSCT with MRD-. Pts with persistent disease prior to alloSCT had shorter duration of MRD- induced by alloSCT compared to pts with MRD-CR/CRi after induction or converted MRD-CR/CRi prior to alloSCT (p=0.0042, H). Importantly, duration of MRD negativity after alloSCT for patients who achieved MRD- prior to alloSCT was not affected by whether patients received induction +/- consolidation (I: treatment type 1-3 from B) vs. induction and salvage treatment for refractory AML (I: treatment type 4-6 from B). Conclusion: We show that transplanted AML pts with specific molecular mutations (RUNX1, SF3B1, and TP53) are unlikely to achieve MRD-CR/CRi after induction, consolidation or salvage therapy, while other mutations (NPM1, IDH1, KRAS) predict high rates of MRD- prior to alloSCT. Additional post-induction therapy may be advantageous for some MRD+ pts to achieve MRD- prior to alloSCT. Post-transplant OS is improved in pts who are MRD- at time of transplant, regardless of whether they required additional therapy beyond induction to achieve this state. AlloSCT is highly effective at eradicating MRD, but post-transplant MRD- is more durable in pts who are MRD- pre-alloSCT. Our results suggest that development of MRD-eradicating therapies has the potential to improve post-transplant outcomes and argues for innovative trials for pts with adverse molecular features currently unlikely to achieve MRD- pre alloSCT. Figure Disclosures Cai: Imago Biosciences, Inc.: Consultancy, Current equity holder in private company; DAVA Oncology: Honoraria. Geyer:Amgen: Research Funding. Glass:Gerson Lehman Group: Consultancy. Stein:Syros: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biotheryx: Consultancy; Bayer: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy, Research Funding; Seattle Genetics: Consultancy; Abbvie: Consultancy; Amgen: Consultancy; Celgene Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Levine:Gilead: Honoraria; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; Lilly: Consultancy, Honoraria; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy; Novartis: Consultancy; Prelude Therapeutics: Research Funding; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Morphosys: Consultancy; Roche: Consultancy, Honoraria, Research Funding. Gyurkocza:Actinium: Research Funding. Perales:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; MolMed: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Medigene: Membership on an entity's Board of Directors or advisory committees, Other; Servier: Membership on an entity's Board of Directors or advisory committees, Other; Omeros: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria; NexImmune: Membership on an entity's Board of Directors or advisory committees; Cidara Therapeutics: Other; Miltenyi Biotec: Research Funding; Kite/Gilead: Honoraria, Research Funding; Incyte Corporation: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Bellicum: Honoraria, Membership on an entity's Board of Directors or advisory committees. Abdel-Wahab:H3 Biomedicine Inc.: Consultancy, Research Funding; Janssen: Consultancy; Envisagenics Inc.: Current equity holder in private company; Merck: Consultancy. Papaemmanuil:Kyowa Hakko Kirin: Consultancy, Honoraria; Isabl: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; MSKCC: Patents & Royalties; Novartis: Consultancy, Honoraria; Illumina: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Prime Oncology: Consultancy, Honoraria. Giralt:KITE: Consultancy; NOVARTIS: Consultancy, Honoraria, Research Funding; OMEROS: Consultancy, Honoraria; AMGEN: Consultancy, Research Funding; TAKEDA: Research Funding; ACTINUUM: Consultancy, Research Funding; MILTENYI: Consultancy, Research Funding; CELGENE: Consultancy, Honoraria, Research Funding; JAZZ: Consultancy, Honoraria. Tallman:Glycomimetics: Research Funding; Rafael: Research Funding; Amgen: Research Funding; Bioline rx: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; KAHR: Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Rigel: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Membership on an entity's Board of Directors or advisory committees; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Research Funding; Orsenix: Research Funding; Cellerant: Research Funding; Abbvie: Research Funding. Goldberg:AROG: Research Funding; Aprea: Research Funding; ADC Therapeutics: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; Aptose: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Honoraria; Pfizer: Research Funding; Celularity: Research Funding.

The purpose of this review is to familiarize anesthesiologists with the role of the subspecialty of pediatric palliative care and its potential contributions to the management of children with complex and life-threatening illnesses, many of whom may present for operative or diagnostic procedures requiring anesthesia. Recent publications highlight the benefits of palliative care to patients and families experiencing a wide variety of conditions along the continuum from prenatal application to the management of young adults. Retrospective reports highlight improved quality of life, improved symptom control, an impact on place of care, and an increased rate of achieving the preferred place of death. Pediatric palliative care is a relatively new subspecialty of pediatrics which utilizes an interdisciplinary team approach to support patients and their families undergoing treatment for life-threatening or life-limiting conditions, addressing physical, emotional, and psychological needs to care for the whole patient.

A new version of the dual-system hypothesis is described. Consistent with earlier models, the improvisational subsystem of the instrumental system, which includes the occipital cortex, inferior temporal cortex, and medial temporal cortex, especially the hippocampus, directs the construction of visual representations of the world and constructs ad-hoc responses to novel targets. The habit system, which includes the occipital cortex; parietal cortex; premotor, supplementary motor, and ventrolateral areas of frontal cortex; and the basal ganglia, especially the caudate nucleus, encodes sequences of actions and generates previously successful actions to familiar targets. However, unlike in previous dual-system models, human cognitive activity involved in task performance is not exclusively associated with one system or the other. Rather, the two systems make it possible for people to learn a variety of skills that draw on the competencies of both systems. The collective effects of these skills define human cognition. So, in contrast with earlier versions of the dual-system hypothesis, which identified the habit system solely with procedural learning and implicit improvements in task performance, the model presented here attributes a direct role in declarative-memory tasks to the habit system. Furthermore, within the model, the computational competencies of the two systems are used to construct purposeful sequences of actions-that is, skills. Human cognition is the result of the performance of these skills. Thus, voluntary action is central to human cognition.

Following coverage by The ASCO Post, Jennifer L. Glass, PA-C, MS, of the Mayo Clinic dives into the major findings in prostate cancer from the 2019 ASCO Annual Meeting in Chicago and their implications for advanced practitioners.

The cell of origin of oncogenic transformation is a determinant of therapeutic sensitivity, but the mechanisms governing cell-of-origin–driven differences in therapeutic response have not been delineated. Leukemias initiating in hematopoietic stem cells (HSC) are less sensitive to chemotherapy and highly express the transcription factor MECOM (EVI1) compared with leukemias derived from myeloid progenitors. Here, we compared leukemias initiated in either HSCs or myeloid progenitors to reveal a novel function for EVI1 in modulating p53 protein abundance and activity. HSC-derived leukemias exhibit decreased apoptotic priming, attenuated p53 transcriptional output, and resistance to lysine-specific demethylase 1 (LSD1) inhibitors in addition to classical genotoxic stresses. p53 loss of function in Evi1lo progenitor-derived leukemias induces resistance to LSD1 inhibition, and EVI1hi leukemias are sensitized to LSD1 inhibition by venetoclax. Our findings demonstrate a role for EVI1 in p53 wild-type cancers in reducing p53 function and provide a strategy to circumvent drug resistance in chemoresistant EVI1hi acute myeloid leukemia. Significance: We demonstrate that the cell of origin of leukemia initiation influences p53 activity and dictates therapeutic sensitivity to pharmacologic LSD1 inhibitors via the transcription factor EVI1. We show that drug resistance could be overcome in HSC-derived leukemias by combining LSD1 inhibition with venetoclax. See related commentary by Gu et al., p. 1445. This article is highlighted in the In This Issue feature, p. 1426

Genetic studies have identified recurrent somatic mutations in acute myeloid leukemia (AML) patients, including in the Wilms’ tumor 1 (WT1) gene. The molecular mechanisms by which WT1 mutations contribute to leukemogenesis have not yet been fully elucidated. We investigated the role of Wt1 gene dosage in steady-state and pathologic hematopoiesis. Wt1 heterozygous loss enhanced stem cell self-renewal in an age-dependent manner, which increased stem cell function over time and resulted in age-dependent leukemic transformation. Wt1-haploinsufficient leukemias were characterized by progressive genetic and epigenetic alterations, including those in known leukemia-associated alleles, demonstrating a requirement for additional events to promote hematopoietic transformation. Consistent with this observation, we found that Wt1 depletion cooperates with Flt3-ITD mutation to induce fully penetrant AML. Our studies provide insight into mechanisms of Wt1-loss leukemogenesis and into the evolutionary events required to induce transformation of Wt1-haploinsufficient stem/progenitor cells.

INTRODUCTION Mixed phenotype (MP) is characteristic for de novo mixed phenotype acute leukemia (dnMPAL) but can also be seen in blast phase of myeloproliferative neoplasms (MPN-BP), myeloid/lymphoid neoplasms with eosinophilia & rearrangements, acute myeloid leukemia (AML) with recurrent cytogenetic abnormalities (AML-RCA) and secondary AML (sAML) including AML with myelodysplasia-related changes (AML-MRC) and therapy-related AML (t-AML). Although WHO classification excludes sAML with MP (sAML-MP) from dnMPAL, the significance of MP in the setting of sAML and their genetic landscape has not been studied. METHODS The MSKCC pathology data base was searched from 01/2014 to 09/2020 and a cohort of 125 patients with MP as defined per WHO 2016 classification was obtained. The clinical, morphologic, immunophenotypic, and cytogenetic/molecular results were reviewed. Patients with a diagnosis of AML-RCA, myeloid/lymphoid neoplasms with eosinophilia & rearrangements, MPN-BP, B-ALL with isolated MPO and myelodysplastic syndrome (MDS) were excluded (Fig 1). RESULTS 50 cases of sAML-MP (14 t-AML and 36 AML-MRC) were retrieved and compared to 42 dnMPAL cases and 100 sAML without MP (37 t-AML and 63 AML-MRC) (Table 1). The median age at diagnosis was 66 years which was higher than dnMPALs (44.5 yrs, p value Molecular studies (Table 2) revealed that the most commonly mutated genes in sAML-MP were RUNX1, DNMT3A, TP53 which were all present in a significantly higher frequency in comparison to dnMPAL. The frequency of RUNX1 mutation was higher in sAML-MP even in comparison to sAML without MP. Conversely, mutations in PHF6 frequently noted in dnMPALs were uncommon in sAML cohorts. Cluster analysis based on immunophenotyping of the 3 cohorts revealed 6 clusters (Fig 2) with separation between the 3 cohorts. Most of the dnMPAL with B/M phenotype formed a distinct tight cluster (cluster 3). Most of the sAML-MP were seen clustering together in cluster 4 whereas sAML without MP predominated in cluster 5 and 6. Cluster 1 revealed a mixture of dnMPAL and sAML-MP with predominantly T/M phenotype. The overall survival (OS) was inferior in the sAML-MP cohort in comparison to dnMPAL cohort (median survival: 6.77 vs 36.99 mths, p value 0.00005; Fig 3A) and was similar to the sAML without MP cohort (median survival: 6.77 vs 4.96 mths, p value 0.33; Fig 3A). In a multivariate analysis, model adjusted for the age and allotransplant did not explain difference in OS between these groups. Comparison of OS between the 6 clusters (Fig 3B) revealed better OS in clusters 2 and 3 that were enriched for dnMPAL. Cluster1 in spite of having a mixture of dnMPAL and sAML-MP did poorly. This could be driven by enrichment in DNMT3A mutations in the dnMPAL with T/M phenotype. Immunophenotypically distinct blast populations were flow cytometrically sorted in 4 cases of sAML-MP (2 each with T/M and B/M phenotype). While the majority of the genetic abnormalities were shared between populations with different lineages, a case with multiple mutations showed divergent KRAS/NRAS mutations showing clonal divergence as possible late event driving distinct lineage maturation (Table 3). CONCLUSION sAML-MP and sAML without MP are clinically similar and have biological overlaps with frequent somatic mutations in TP53, chromatin modifying genes and spliceosome-complex genes, which is different from dnMPAL. However, the frequency of RUNX1 mutation is higher in sAML-MP than sAML without MP suggesting its role in lineage infidelity. Though, cluster analysis based on immunophenotype separates the 3 cohorts into enriched clusters, there are still overlaps between sAML-MP and dnMPAL especially with a T/M phenotype as well as between sAML-MP and sAML without MP especially in TP53 mutated cases. Additional RNA seq and ATAC seq are currently being performed on flow cytometrically sorted distinct blast populations of the sAML-MP cases. Figure 1 Figure 1. Disclosures Galera: Paige.AI: Research Funding. Dogan: Physicians' Education Resource: Honoraria; Seattle Genetics: Consultancy; Takeda: Consultancy, Research Funding; EUSA Pharma: Consultancy; Roche: Consultancy, Research Funding; Peer View: Honoraria. Tallman: Syros: Membership on an entity's Board of Directors or advisory committees; NYU Grand Rounds: Honoraria; Kura: Membership on an entity's Board of Directors or advisory committees; Innate Pharma: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Biosight: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Membership on an entity's Board of Directors or advisory committees; KAHR: Membership on an entity's Board of Directors or advisory committees; Orsenix: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Rafael Pharmaceuticals: Research Funding; Glycomimetics: Research Funding; Biosight: Research Funding; Orsenix: Research Funding; Abbvie: Research Funding; Mayo Clinic: Honoraria; UC DAVIS: Honoraria; Northwell Grand Rounds: Honoraria; NYU Grand Rounds: Honoraria; Danbury Hospital Tumor Board: Honoraria; Acute Leukemia Forum: Honoraria; Miami Leukemia Symposium: Honoraria; New Orleans Cancer Symposium: Honoraria; ASH: Honoraria; NCCN: Honoraria. Levine: Celgene: Research Funding; Incyte: Consultancy; Roche: Honoraria, Research Funding; Morphosys: Consultancy; Imago: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy; Amgen: Honoraria; Gilead: Honoraria; Zentalis: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; Prelude: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees. Roshal: Physicians' Education Resource: Other: Provision of services; Celgene: Other: Provision of services; Auron Therapeutics: Other: Ownership / Equity interests; Provision of services. Glass: GLG: Consultancy. Xiao: Stemline Therapeutics: Research Funding.

Introduction: Acute Myeloid Leukemia (AML) is a biologically diverse disease. Expanded mutation panels and novel epigenetic assays are identifying an increasing number of putative AML subtypes beyond the traditional 'Good', 'Intermediate', and 'Poor' risk designations. Although these approaches show great promise, identifying the relevant underlying disease biology remains difficult. Single cell studies highlight this difficulty, showing dynamic interactions between multiple subclones, each with its own set of cooperating mutations interfering with normal hematopoiesis. We have previously shown that bulk ATAC data can be used to 'deconvolve' and identify hematopoietic state in AML samples. Here we extend this work, showing that this approach can be used to identify normal hematopoietic states with a high degree of accuracy. In addition, we show that AML samples that appear different in bulk actually contain overlapping lineage characteristics at the single cell level. Methods: Single cell ATAC-seq count files were downloaded from GSE74310, GSE96769 as well as corresponding bulk ATAC-seq count files from GSE74912, GSE96771. These data were generated by flow sorting normal specimens into well-known stages of hematopoiesis followed by either bulk or single cell ATAC-seq. A set of AML samples was processed by both single cell and bulk ATAC as well. Bulk ATAC data was normalized using DESeq2 followed by variance stabilizing transformation. Single cell data was processed and normalized using the Seurat pipeline with default parameters. A common peak atlas was created for each dataset, and peaks characteristic of each stage of hematopoiesis were selected using a modified Kruskal-Wallis statistic and optimized using a set of well-characterized in-vitro sample mixtures. Lineage deconvolution was performed using a non-negative least squares regression comparing each unknown sample to the set of normal hematopoietic states. Results: Dimensionality reduction of single cell ATAC-seq using uniform manifold approximation and projection (UMAP) largely recapitulates stages of hematopoiesis used to sort the samples (Figure 1a). Single cell lineage deconvolution is able to identify the purity of these populations more precisely (Figure 1b), with HSC, MPP, LMPP, CLP, GMP, MEP, and Monocytic stages showing relatively pure lineage characteristics. In contrast, the CMP stage appears to be composed of a heterogeneous population, as has been previously shown. Dimensionality reduction of bulk ATAC-seq data using Principle Component Analysis (PCA) illustrates distinct stages of hematopoiesis, and separates the AML samples into two groups (Figure 2c). To further analyze these groups, bulk lineage deconvolution was performed, showing that cluster 1 (purple) has a more differentiated appearance characterized by GMP and Monocyte lineages while cluster 2 also reflects earlier stages of hematopoiesis including HSC, MPP, and LMPP (Figure 2d). One sample from each cluster (highlighted in red in figure 2c,d) was evaluated using single cell ATAC-seq. Lineage deconvolution on the component cells illustrates substantial lineage characteristic overlap between subclones of these samples, with lineage based hierarchical clustering generating two clusters with mixed sample origin (Figure 2e). These clusters are separated into more and less differentiated lineage groups, with the cluster 2 sample cells more commonly having an HSC or MPP dominant lineage. However, some cluster 1 cells do have HSC or MPP lineage features as well, which is reflected by the poor association of cluster with sample (Fisher's exact p=0.8). Conclusions: Lineage deconvolution can be performed on single cell ATAC-seq data with a high degree of precision on normal samples and illustrates clonal lineage heterogeneity in malignant specimens not previously appreciated in bulk sequencing analysis. Analysis of greater numbers of samples and cells are needed to draw general conclusions, but the approach shows promise as a means of computationally identifying or sorting normal single cells and more precisely characterizing leukemias. Figure 1 Figure 1. Disclosures Melnick: Janssen Pharmaceuticals: Research Funding; Sanofi: Research Funding; Daiichi Sankyo: Research Funding; Epizyme: Consultancy; Constellation: Consultancy; KDAC Pharma: Membership on an entity's Board of Directors or advisory committees. Elemento: Johnson and Johnson: Research Funding; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Eli Lilly: Research Funding; Janssen: Research Funding; One Three Biotech: Consultancy, Other: Current equity holder; Champions Oncology: Consultancy; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Owkin: Consultancy, Other: Current equity holder; AstraZeneca: Research Funding. Levine: Lilly: Honoraria; Gilead: Honoraria; Janssen: Consultancy; Morphosys: Consultancy; Astellas: Consultancy; Roche: Honoraria, Research Funding; Incyte: Consultancy; Amgen: Honoraria; Celgene: Research Funding; Isoplexis: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Prelude: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Imago: Membership on an entity's Board of Directors or advisory committees; QIAGEN: Membership on an entity's Board of Directors or advisory committees. Glass: GLG: Consultancy.

Purpose We sought to evaluate the association between early-onset cardiac allograft vasculopathy (CAV) [ Methods Data from the Pediatric Heart Transplant Society (PHTS) for pediatric ( Results Over a median follow-up period of 4.1 (IQR 1.3 - 8.3) years, angiographic evidence of CAV was identified in 17% (885/5,075) of patients, 28% (252/885) of which were early-onset CAV. Compared to those who developed CAV >10 years post HT, patients with early-onset CAV were older at transplant (8.3 +/- 6.2 vs. 3.8 +/- 4.8 years, p Conclusion Following diagnosis, the progression of CAV & graft survival was similar for patients with early-onset CAV compared with CAV at other time points. These findings suggest that early-onset CAV does not necessarily represent a more aggressive phenotype of disease.

Introduction ite advances in anticoagulation management in pediatric VAD, thrombosis and stroke remain a major concern. For patients with thrombosis who fail intensification of standard anticoagulation options are limited. We present a case of thrombosis in a patient on Berlin Heart LVAD that was successfully treated with systemic alteplase. Case Report The patient is a 2 year old 13.6 kg girl (BSA 0.58) with dilated cardiomyopathy and left ventricular systolic heart failure necessitating placement of a Berlin Heart EXCOR LVAD as a bridge to heart transplantation. She was implanted with a 15mL pump at a rate of 100 beats/min. There were no intra-operative complications and anticoagulation was initiated with bivalirudin; target PTT 70-90 sec (achieved consistently by POD#3). On POD#5 she became febrile with rising inflammatory markers and broad spectrum antibiotic therapy was initiated. She then required escalating doses of bivalirudin for decreasing PTT values. After negative blood cultures for 24 hours, she completed a course of pulse steroids for possible inflammatory reaction on the pump. She was also started on aspirin and then dipyridamole. By POD#15, she had an escalating respiratory support requirement in the setting of a large pleural effusion and bivalirudin was held briefly for placement of a chest tube. Despite no obvious fibrin or clot in the device or cannulas, an echocardiogram a revealed a 5mm thrombus in the left ventricle adjacent to the inflow cannula. Given the position of the thrombus, clot extraction by surgical or cardiac catheterization was deemed not feasible due to limited ability to perform thrombectomy without embolization. Therefore, we treated medically with systemic alteplase while maintaining the patient on continuous bivalirudin infusion at lower PTTs. We achieved successful resolution of the thrombus by echocardiogram with no significant complications or neurologic deficits. The patient was supported by the Berlin Heart for 5 months until transplant with no other complications. On evaluation of the explanted heart there was no residual thrombus. A head CT performed after heart transplant for follow up of an unrelated concern revealed no evidence of intracranial hemorrhage or infarct. Summary Systemic tPA may be considered as a treatment option in patients with thrombosis on ventricular assist device in lieu of more invasive treatment options.

Purpose There are limited data reporting outcomes of children with trisomy 21 undergoing heart transplantation (HTx). This project reports the waitlist and post-HTx outcomes of children with trisomy 21. Methods This is a retrospective cohort study of children with trisomy 21 listed for or who underwent HTx at 11 centers within North America and internationally. Demographic, medical, surgical, and post-HTx outcome data were collected and described. Results Twenty-one patients with trisomy 21 were listed for HTx from 1992 through 2019 (median age 8.5y, 43% male, 52% white). High-risk or failed repair of congenital heart disease was the indication for transplant in 16 (76%) patients with atrioventricular septal defect being the most common (N=12). Other diagnoses included dilated cardiomyopathy in 4 patients (secondary to anthracyclines in 2) and myocarditis in 1 patient. A total of 18 (86%) patients survived to transplant. Of this group, support at the time of transplant included inotropes (N=10, 56%), ventricular assist device (N=7, 39%), and mechanical ventilation (N=2, 11%). No patients required post-operative extracorporeal membrane oxygenation or dialysis. All centers reported the use of standard immunosuppression without alterations secondary to trisomy 21. All transplanted patients survived to hospital discharge with a median post-HTx length of stay of 19d. With a median follow-up of 2.9y, 16 (89%) patients were alive, 5 (28%) had rejection within the 1st year post-HTx, 2 (11%) developed post-transplant lymphoproliferative disorder (PTLD), and 7 (39%) were hospitalized for infection within the 1st year post-HTx. For the two post-HTx mortalities, the causes of death were 1) coronary allograft vasculopathy at 22y post-HTx and 2) multiple episodes of rejection and PTLD at 1.5y post-HTx (Figure). Conclusion Waitlist and intermediate post-HTx outcomes are acceptable in children with trisomy 21. The presence of trisomy 21 should not be an absolute contraindication to heart transplantation in the appropriate clinical setting.

Made available in DSpace on 2018-11-26T17:56:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-05-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The South American lungfish Lepidosiren paradoxa is an obligatory air-breathing fish possessing well-developed bilateral lungs, and undergoing seasonal changes in its habitat, including temperature changes. In the present study we aimed to evaluate gas exchange and pulmonary breathing pattern in L paradoxa at different temperatures (25 and 30 degrees C) and different inspired O-2 levels (21,12,10, and 7%). Normoxic breathing pattern consisted of isolated ventilatory cycles composed of an expiration followed by 2.4 +/- 0.2 buccal inspirations. Both expiratory and inspiratory tidal volumes reached a maximum of about 35 ml kg(-1), indicating that L paradoxa is able to exchange nearly all of its lung air in a single ventilatory cycle. At both temperatures, hypoxia caused a significant increase in pulmonary ventilation ((V) over dot (E)), mainly due to an increase in respiratory frequency. Durations of the ventilatory cycle and expiratory and inspiratory tidal volumes were not significantly affected by hypoxia. Expiratory time (but not inspiratory) was significantly shorter at 30 degrees C and at all O-2 levels. While a small change in oxygen consumption ((V) over dot O-2) could be noticed, the carbon dioxide release ('CO2, P = 0.0003) and air convection requirement ((V) over dot (E)/(V) over dot O-2, P = 0.0001) were significantly affected by hypoxia (7% O-2) at both temperatures, when compared to normoxia, and pulmonary diffusion capacity increased about four-fold due to hypoxic exposure. These data highlight important features of the respiratory system of L. paradoxa, capable of matching O-2 demand and supply under different environmental change, as well as help to understand the evolution of air breathing in lungfish. (C) 2017 Elsevier Inc. All rights reserved. Sao Paulo State Univ, Coll Agr & Vet Sci, Sao Paulo, SP, Brazil Univ Fed Bahia, Master Program Anim Divers, Salvador, BA, Brazil Univ Sao Paulo, Fac Med Ribeirao Preto, Sao Paulo, Brazil Univ Sao Paulo, Sch Philosophy Sci & Literature Ribeirao Preto, Sao Paulo, Brazil Natl Inst Sci & Technol Comparat Physiol, Rio Claro, Brazil Sao Paulo State Univ, Coll Agr & Vet Sci, Sao Paulo, SP, Brazil FAPESP: 2008/57712-4 CNPq: 573921/2008-3 FAPESP: 2013/17606-9 FAPESP: 2014/12190-1 FAPESP: 12/17379-0

Genomic studies in acute myeloid leukemias (AML) have identified mutations that drive altered DNA methylation, including TET2 and IDH2. Here, we show that models of AML resulting from TET2 or IDH2 mutations combined with FLT3ITD mutations are sensitive to 5-azacytidine or to the IDH2 inhibitor AG-221, respectively. 5-azacytidine and AG-221 treatment induced an attenuation of aberrant DNA methylation and transcriptional output and resulted in a reduction in leukemic blasts consistent with antileukemic activity. These therapeutic benefits were associated with restoration of leukemic cell differentiation, and the normalization of hematopoiesis was derived from mutant cells. By contrast, combining AG-221 or 5-azacytidine with FLT3 inhibition resulted in a reduction in mutant allele burden, progressive recovery of normal hematopoiesis from non-mutant stem-progenitor cells, and reversal of dysregulated DNA methylation and transcriptional output. Together, our studies suggest combined targeting of signaling and epigenetic pathways can increase therapeutic response in AML. Significance: AMLs with mutations in TET2 or IDH2 are sensitive to epigenetic therapy through inhibition of DNA methyltransferase activity by 5-azacytidine or inhibition of mutant IDH2 through AG-221. These inhibitors induce a differentiation response and can be used to inform mechanism-based combination therapy. Cancer Discov; 7(5); 494–505. ©2017 AACR. See related commentary by Thomas and Majeti, p. 459. See related article by Yen et al., p. 478. This article is highlighted in the In This Issue feature, p. 443

The identity of the RNA binding proteins (RBPs) that govern cancer stem cell remains poorly characterized. The MSI2 RBP is a central regulator of translation of cancer stem cell programs. Through proteomics analysis of the MSI2 interacting RBP network and functional shRNA screening, we identified 24 genes required for in vivo leukemia and SYNCRIP was the most differentially required gene between normal and myeloid leukemia cells. SYNCRIP depletion increased apoptosis and differentiation while delaying leukemogenesis. Gene expression profiling of SYNCRIP depleted cells demonstrated a loss of the MLL and HOXA9 leukemia stem cell gene associated program. SYNCRIP and MSI2 interact indirectly though shared mRNA targets. SYNCRIP maintains HOXA9 translation and MSI2 or HOXA9 overexpression rescued the effects of SYNCRIP depletion. We validated SYNCRIP as a novel RBP that controls the myeloid leukemia stem cell program and propose that targeting these functional complexes might provide a novel therapeutic strategy in leukemia.

Background: Venetoclax combined with azacitidine (aza/ven), decitabine (dec/ven), and low-dose cytarabine (LDAC/ven) is commonly used off-label in relapsed/refractory (RR)-AML patients; however, predictors of response and survival are incompletely understood. Objectives: To report clinical outcomes of RR-AML patients (pts) treated with venetoclax combination therapy and to analyze molecular predictors of these outcomes. Methods: All pts with RR-AML, who received treatment with aza/ven, dec/ven or LDAC/ven from 08/2016 to 02/2020 at Memorial Sloan Kettering Cancer Center in New York City were included. The best response to therapy was determined using the 2017 European LeukemiaNet (ELN) response criteria. The overall response rate (ORR) was defined as the combination of the complete response (CR) + complete response with incomplete count recovery (CRi) + morphologic leukemia free state (MLFS) rate. Measurable residual disease (MRD) was assessed by multiparameter flow cytometric analysis of bone marrow aspirate samples and any level of residual disease was considered MRD+. Overall survival (OS) was calculated from cycle 1 day 1 of therapy until death or time of last follow-up. Results: A total of 86 pts were treated with venetoclax-based combination therapy (A). Median age was 67 years. More than half of the pts had received prior HMA therapy, 17% had received a prior allogeneic stem cell transplant. The majority of pts received aza/ven (41%) while 23% and 31% received dec/ven and LDAC/ven, respectively. Treatment trajectories of all patients are shown in the Swimmers' plot in panel (B). While the ORR was 31% and 24% of patients achieved a CR/CRi, 60% of pts were refractory to venetoclax therapy; 45% of responding pts eventually relapsed with a median follow up of 12 months (B, C). Oncoprint showing associations between molecular patterns and response to venetoclax combination therapy is shown in panel (D). Mutations in NPM1 (CR/CRi 46%, ORR 62%; OR 4.53, 95% CI 1.31-15.66, p=0.02) and IDH1 (CR/CRi 56%, ORR 67%; OR 5.3, 95% CI 1.21-23.24, p=0.03) but not in IDH2 (CR/CRi 40%, ORR 40%; OR 1.57, 95% CI 0.49-4.99, p=0.54) were associated with statistically significantly increased response rates. Adverse cytogenetics predicted lower odds of response (CR/CRi 11%, ORR 20%; OR 0.32, 95% CI 0.11-0.9, p=0.03). While TP53 (0%), NRAS/KRAS (20%/0%), SF3B1 (0%), ASXL1 (17%) and EZH2 (0%) mutations were associated with low response rates, due to small sample size, these associations were not statistically significant. Median OS for all pts was 6.1 months (95%CI: 4.9-10 month) (E). Pts who were treated with aza/ven had a median OS of 25 months (95% CI 5.8 months-NR), as compared to 5.4 months for patients treated with dec/ven (95% CI 3.9 months-NR; HR 1.63, p=0.2) and 3.9 months for patients treated with LDAC/ven (OS 3.9 months, 95% CI 2.5-8.3 months; HR 2.71, p=0.002). Presence of adverse cytogenetics, TP53 and KRAS/NRAS mutations was associated with poor OS (4.1 months, 95% CI 3.2-6.1 months) compared to a favorable OS when these features were absent (15 months, 95% CI 7.1 months-NA, p=0.001; F). In contrast, presence of mutations in IDH1/2, NPM1 or STAG2 was associated with prolonged OS (15 months, 95% CI 7.1 months-NA) whereas absence of these favorable mutations predicted poor OS (4.4 months, 95% CI 3.5-6.4 months, p=0.0051). Additionally, EZH2 (HR 4.13, p=0.01; H) and SF3B1 (HR 2.5, p=0.02; I) mutations predicted worse OS. Conclusion: Response rates to venetoclax combination therapy in RR-AML pts appeared lower than what has been reported for AML pts who receive venetoclax therapy as first line treatment. However, response rates were high and survival favorable for RR-AML pts with mutations in NPM1, IDH1 and STAG2. In contrast, RR-AML pts with high-risk molecular features such as adverse cytogenetics and mutations in TP53, KRAS/NRAS, EZH2 and SF3B1 had poor outcomes. Clinical trials combining aza/ven with novel therapeutics targeting specific adverse molecular characteristics are urgently needed to improve the outcomes of venetoclax therapy in RR-AML patients. Figure Disclosures Xiao: Stemline Therapeutics: Research Funding. Glass:Gerson Lehman Group: Consultancy. King:Abbvie: Other: advisory board. Abdel-Wahab:Merck: Consultancy; Janssen: Consultancy; Envisagenics Inc.: Current equity holder in private company; H3 Biomedicine Inc.: Consultancy, Research Funding. Levine:Novartis: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Lilly: Consultancy, Honoraria; Janssen: Consultancy; Astellas: Consultancy; Morphosys: Consultancy; Amgen: Honoraria; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Stein:Amgen: Consultancy; Astellas Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biotheryx: Consultancy; Abbvie: Consultancy; Seattle Genetics: Consultancy; Syndax: Consultancy, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Syros: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Tallman:UpToDate: Patents & Royalties; Novartis: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; KAHR: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Bioline rx: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Rafael: Research Funding; Glycomimetics: Research Funding; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Research Funding; Orsenix: Research Funding; Cellerant: Research Funding; Abbvie: Research Funding. Goldberg:Aptose: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Aprea: Research Funding; AROG: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celularity: Research Funding; Pfizer: Research Funding; Dava Oncology: Honoraria. OffLabel Disclosure: venetoclax therapy for the treatment for realpsed and treatment refractory AML

Background: Venetoclax (ven) combined with azacitidine (aza/ven), decitabine (dec/ven), and low-dose cytarabine (LDAC/ven) is approved as frontline therapy for older or otherwise unfit AML patients (pts) and also frequently used for pts with relapsed/refractory (RR)-AML. However, clinical outcomes of AML pts who receive an allogeneic stem cell transplant (alloSCT) after ven combination (ven combo) therapy are unclear. Methods: All AML pts who received treatment with aza/ven, dec/ven or LDAC/ven as either initial induction or for RR disease at Memorial Sloan Kettering Cancer Center from 08/2016 to 02/2020 and underwent a subsequent allo-SCT were included. The response to ven therapy prior to alloSCT was determined using the 2017 European LeukemiaNet response criteria. The overall response rate was defined as the combination of the complete response (CR) + complete response with incomplete count recovery (CRi) + morphologic leukemia free state (MLFS) rate. Measurable residual disease (MRD) was assessed by multiparameter flow cytometric analysis of bone marrow aspirate samples. Any level of residual disease was considered MRD+. Overall survival (OS) after alloSCT was calculated from the day of graft infusion until death or time of last follow-up. Results: A total of 130 pts were treated with ven combo therapy with 18 pts (13.8% of all pts) receiving a subsequent alloSCT. Median age was 65 years (A). While 3 pts received a ven combo as the first treatment for AML, 15 pts had RR-AML. Aza/ven was most commonly used (72%) and the majority of pts (83%) received 1-2 cycles of ven therapy prior to alloSCT. For 7, 6, 4 and 1 pts the donor was a matched related, a matched unrelated, a mismatched unrelated, or haploidentical; only one pt had received a prior alloSCT for AML (B). Unmodified peripheral blood stem cells (66%) and reduced intensity conditioning regimens (77%) were most commonly used. In pts who proceeded to an alloSCT, 12/18 pts achieved a response after ven combination therapy: 4/18 MRD-CR/CRi, 4/18 MRD+CR/CRi, 4/18 MLFS; 6/18 pts had persistent disease (C and D). It is important to note that these response rates were specific to patients who received an alloSCT after ven combination therapy. While pts with DNMT3A, NPM1, IDH1/2 and FLT3 mutations had a high response rate to ven therapy prior to alloSCT, none of the pts with TP53 or NRAS mutations achieved a response prior to alloSCT (E). Only DNMT3A mutations were statistically significantly associated with a high response rate prior to alloSCT (ORR 100%, CR/CRi 63%, p=0.01). AlloSCT was able to convert pts with MRD+ or persistent disease into an MRD- state in 50% of cases (F). With a median follow up of 10 months, the median OS was not reached; 56% of pts relapsed after alloSCT. Median time to relapse for all pts was 18 months (95% CI 4 months-not reached [NR]). Disease status prior to alloSCT was a powerful predictor of post-transplant outcomes. Pts who achieved CR, CRi, or MLFS with ven therapy prior to alloSCT had significantly prolonged OS (median OS not reached) compared with pts who had persistent disease prior to alloSCT (median OS 7 months, 95% CI 2.14 months-NR; p=0.029; G). The poor OS for pts with persistent disease prior to alloSCT was mainly driven by a high incidence of relapse for these pts compared to pts who achieved a response to ven combos (H). The median time to relapse after alloSCT was 17.9 months (95% CI 6.1 months-NR) for pts who achieved CR, CRi, or MLFS prior to alloSCT, and only 3.3 months (95% CI 1.9 months-NR, p=0.052) for pts with persistent disease prior to alloSCT. While 67% of pts experienced grade I-II acute graft versus host disease (aGVHD), only 1 pt was found to have grade III aGVHD and no pt experienced grade IV aGVHD. Median time to aGVHD after alloSCT was 1.4 months (95% CI 0.89 months-NR). Conclusion: Venetoclax combination therapy can bridge some AML pts to subsequent alloSCT. Most pts who underwent alloSCT received reduced-intensity conditioning given their advanced age and comorbidities. Pts who achieved CR, CRi, or MLFS with ven-based therapy prior to alloSCT had more favorable outcomes after alloSCT. With more widespread use of venetoclax, we anticipate that alloSCT following good responses to venetoclax combination therapy will become more common in pts with AML. Longer-term studies are needed to determine durability of post-alloSCT responses following pre-alloSCT venetoclax combination therapy. Figure Disclosures Ponce: Kadmon: Membership on an entity's Board of Directors or advisory committees; Generon: Membership on an entity's Board of Directors or advisory committees; Ceramedix: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding. Young:Amgen: Other: Common stock; Merck: Other: Common stock; Pfizer: Other: Common stock. Gyurkocza:Actinium: Research Funding. Chan:AbbVie: Current equity holder in publicly-traded company; Bristol-Myers Squibb: Current equity holder in publicly-traded company. Xiao:Stemline Therapeutics: Research Funding. Glass:Gerson Lehman Group: Consultancy. King:Abbvie: Other: advisory board. Cai:Imago Biosciences, Inc.: Consultancy, Current equity holder in private company; DAVA Oncology: Honoraria. Stein:Celgene Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Abbvie: Consultancy; Seattle Genetics: Consultancy; Syndax: Consultancy, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Biotheryx: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Astellas Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Giralt:GSK: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Actinuum: Other: Advisory board, Research Funding; Amgen: Other: Advisory Board, Research Funding; OMEROS: Research Funding; Jensenn: Membership on an entity's Board of Directors or advisory committees, Research Funding; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; TAKEDA: Membership on an entity's Board of Directors or advisory committees, Research Funding; JAZZ Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; MILTENYI: Research Funding; PFIZER: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; SPECTRUM Pharma: Membership on an entity's Board of Directors or advisory committees; KITE: Membership on an entity's Board of Directors or advisory committees. Perales:Omeros: Honoraria, Membership on an entity's Board of Directors or advisory committees; Medigene: Membership on an entity's Board of Directors or advisory committees, Other; NexImmune: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees, Other; Cidara Therapeutics: Other; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; MolMed: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria; Miltenyi Biotec: Research Funding; Kite/Gilead: Honoraria, Research Funding; Incyte Corporation: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Bellicum: Honoraria, Membership on an entity's Board of Directors or advisory committees. Tallman:Novartis: Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; Orsenix: Research Funding; ADC Therapeutics: Research Funding; UpToDate: Patents & Royalties; Abbvie: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; Glycomimetics: Research Funding; Rafael: Research Funding; Amgen: Research Funding; Bioline rx: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; KAHR: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Membership on an entity's Board of Directors or advisory committees; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees. Goldberg:Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; Aptose: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Research Funding; Aprea: Research Funding; AROG: Research Funding; Celularity: Research Funding; Pfizer: Research Funding; Dava Oncology: Honoraria.

Background: Mixed phenotype acute leukemia is a rare disease characterized by an expanded blast population exhibiting multiple lineage features. It is a diagnosis of exclusion, and we and others have observed that AML-MRC or therapy-related AML cases may also exhibit similar features. We therefore sought to determine whether multi-lineage expression is present in de-novo and therapy-related MDS and whether it has an impact on clinical outcomes. Methods: We reviewed pathology, flow cytometry, cytogenetic, and molecular reports from patients seen at Memorial Sloan Kettering Cancer Center between 1996 and 2020 and identified 472 patients diagnosed with MDS using a combination of custom natural language processing tools and manual review. Cox proportional hazards modeling was performed to assess the contribution of patient characteristics, pathology, flow cytometric, cytogenetic, and molecular characteristics to overall survival (OS). Fisher's exact testing was used to assess the association of individual features. Results: We found that an abnormal myeloid lineage signature was associated with poorer OS after adjusting for age, cytogenetics, and molecular features (HR=2.3, p Conclusion: Routine flow cytometric and molecular assessment of diagnostic bone marrows can provide added prognostic value in MDS. Identification of an abnormal myeloid lineage is associated with higher risk disease, while identification of abnormal plasma cell features is associated with lower risk disease. SRSF2 mutation is also associated with abnormal plasma cell features, but does not contribute independently to OS. Abnormal T-cell features may also provide prognostic value, although assessment in a larger cohort will be necessary. We confirm previous findings indicating that TP53 mutation is associated with higher risk disease while SF3B1 mutation is associated with better outcomes characteristic of MDS-RARS. In addition, our data suggests that IDH1 and EZH2 are associated with poorer outcomes and that IDH2 is associated with lower risk disease. Figure 1 Disclosures Tallman: Bioline rx: Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Abbvie: Research Funding; Cellerant: Research Funding; Orsenix: Research Funding; ADC Therapeutics: Research Funding; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; Glycomimetics: Research Funding; Rafael: Research Funding; Oncolyze: Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Novartis: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; KAHR: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees. Levine:Morphosys: Consultancy; Astellas: Consultancy; Janssen: Consultancy; Lilly: Consultancy, Honoraria; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Gilead: Honoraria; Novartis: Consultancy; Amgen: Honoraria; Roche: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Xiao:Stemline Therapeutics: Research Funding. Glass:Gerson Lehman Group: Consultancy.

Purpose Plastic bronchitis is a rare post Fontan complication with limited treatment options. Heart transplantation has evolved as a potential curative option, but outcomes have not been well-defined. This study aims to assess contemporary waitlist and post-transplant outcomes in patients with plastic bronchitis. Methods All Fontan patients were identified in the Pediatric Heart Transplant Society database (2010-2018). Waitlist and post-transplant outcomes were compared between Fontan patients with and without plastic bronchitis. Competing outcomes and Kaplan Meier analyses were used to assess the impact of plastic bronchitis on waitlist and post-transplant survival. A secondary analysis excluded those with protein losing enteropathy (PLE) from the comparison cohort. Results Of 645 Fontan patients listed for heart transplant, 69 (8%) had plastic bronchitis. At listing, patients with plastic bronchitis were younger (9.2 vs 10.6 years, p=0.02), but had no other differences at baseline including hemodynamic data. Fewer Fontan patients with plastic bronchitis were listed in the more recent era (46 [15.4%] in 2010-2014 vs. 23 [6.6%] in 2015-2018, p Conclusion Contrary to prior reports, in this large series plastic bronchitis did not have a negative impact on survival to or after heart transplantation in Fontan patients. A 50% reduction in listing in the most recent era suggests improvement in medical management of this rare and life-threatening condition.

Key Points Question Is there an association between light-intensity physical activity and incident mobility disability among older women? Findings In this cohort study of 5735 postmenopausal, community-dwelling women without mobility limitation, the risk of incident mobility disability over 6 years of follow-up was 22%, 40%, and 40% lower for women in the second, third, and fourth quartiles of daily mean light-intensity physical activity, respectively, compared with the lowest quartile. Meaning These results suggest that recommendations to increase light-intensity physical activity have the potential to improve prospects for preserving mobility among older women., This cohort study evaluates data from 40 clinical sites across the US to assess the association of light-intensity physical activity and incident mobility disability among older women., Importance Almost 1 in 4 women older than 65 years is unable to walk 2 to 3 blocks, and mobility disability is a key factor associated with loss of independence. Lack of moderate to vigorous–intensity physical activity is associated with mobility disability, but whether lighter physical activity is associated with mobility disability is unknown. Objective To determine the association of light-intensity physical activity and incident mobility disability among older women. Design, Setting, and Participants This prospective cohort study included women enrolled in the Objectively Measured Physical Activity and Cardiovascular Health study, an ancillary study of the Women’s Health Initiative, between March 2012 and April 2014, with follow-up through March 31, 2018. The Women’s Health Initiative was a population-based, multisite study that recruited from 40 clinical sites across the US. Participants in the present analysis included 5735 of 7058 ambulatory, community-dwelling women aged 63 years and older who returned an accelerometer with usable data, were free of mobility disability, and had follow-up data on mobility status. Data were analyzed from August 2018 to May 2019. Exposures Light-intensity physical activity, defined as movement requiring energy expenditure between 1.6 and 2.9 metabolic equivalents, captured using an accelerometer over 7 days. Main Outcomes and Measures Incident mobility disability, defined as the first self-reported inability to walk 1 block or up a flight of stairs at annual follow-up, and persistent incident mobility disability, defined as incident mobility loss that persisted through the end of follow-up. Results A total of 5735 participants were included for primary analysis of all incident mobility disability (mean [SD] age, 78.5 [6.6] years [range, 63-97 years]; 2811 [49.0%] White participants). Compared with women in the lowest quartile of light-intensity physical activity, lower risk of incident mobility disability was observed in quartile 2 (multivariable hazard ratio [HR], 0.78; 95% CI, 0.67-0.90), quartile 3 (HR, 0.60; 95% CI, 0.51-0.71), and quartile 4 (HR, 0.60; 95% CI, 0.51-0.71) (P

Highlights • GPR119, a putative fat sensor, is a potential target for metabolic disease. • KO of GPR119 in murine L-cells reduced GLP-1 response to fat in vivo. • Primary L-cells secreted GLP-1 in response to GPR119 agonists. • GPR119 agonists increased L-cell cAMP, with greatest efficacy in the colon. • Our data support the use of GPR119 agonists to raise GLP-1 levels., Aims/hypothesis Glucagon-like peptide-1 (GLP-1) is an incretin hormone derived from proglucagon, which is released from intestinal L-cells and increases insulin secretion in a glucose dependent manner. GPR119 is a lipid derivative receptor present in L-cells, believed to play a role in the detection of dietary fat. This study aimed to characterize the responses of primary murine L-cells to GPR119 agonism and assess the importance of GPR119 for the detection of ingested lipid. Methods GLP-1 secretion was measured from murine primary cell cultures stimulated with a panel of GPR119 ligands. Plasma GLP-1 levels were measured in mice lacking GPR119 in proglucagon-expressing cells and controls after lipid gavage. Intracellular cAMP responses to GPR119 agonists were measured in single primary L-cells using transgenic mice expressing a cAMP FRET sensor driven by the proglucagon promoter. Results L-cell specific knockout of GPR119 dramatically decreased plasma GLP-1 levels after a lipid gavage. GPR119 ligands triggered GLP-1 secretion in a GPR119 dependent manner in primary epithelial cultures from the colon, but were less effective in the upper small intestine. GPR119 agonists elevated cAMP in ∼70% of colonic L-cells and 50% of small intestinal L-cells. Conclusions/interpretation GPR119 ligands strongly enhanced GLP-1 release from colonic cultures, reflecting the high proportion of colonic L-cells that exhibited cAMP responses to GPR119 agonists. Less GPR119-dependence could be demonstrated in the upper small intestine. In vivo, GPR119 in L-cells plays a key role in oral lipid-triggered GLP-1 secretion.

Measurable residual disease is associated with inferior outcomes in patients with acute myeloid leukemia (AML). Measurable residual disease monitoring enhances risk stratification and may guide therapeutic intervention. The European LeukemiaNet working party recently came to a consensus recommendation incorporating leukemia associated immunophenotype-based different from normal approach by multi-color flow cytometry for measurable residual disease evaluation. However, the analytical approach is highly expertise-dependent and difficult to standardize. Here we demonstrate that loss of plasmacytoid dendritic cell differentiation after 7+3 induction in AML is highly specific for measurable residual disease positivity (specificity 97.4%) in a uniformly treated patient cohort. Moreover, loss of plasmacytoid dendritic cell differentiation as determined by a blast-to-plasmacytoid dendritic cell ratio >10 was strongly associated with inferior overall and relapse-free survival (RFS) [Hazard ratio 2.79, 95% confidence interval (95%CI): 0.98-7.97; P=0.077) and 3.83 (95%CI: 1.51-9.74; P=0.007), respectively), which is similar in magnitude to measurable residual disease positivity. Importantly, measurable residual disease positive patients who reconstituted plasmacytoid dendritic cell differentiation (blast/ plasmacytoid dendritic cell ratio

It has been proposed that spontaneous panic attacks are the outcome of the misfiring of an evolved suffocation alarm system. Evidence gathered in the last years is suggestive that the dorsal periaqueductal gray (dPAG) in the midbrain harbors a hypoxia-sensitive suffocation alarm system. We here investigated whether facilitation of 5-HT-mediated neurotransmission within the dPAG changes panic-like defensive reactions expressed by male Wistar rats submitted to a hypoxia challenge (7% O2), as observed in other animal models of panic. Intra-dPAG injection of 5-HT (20 nmol), (±)-8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT) (8 nmol), a 5-HT1A receptor agonist, or (±)-2,5-dimethoxy-4-iodo amphetamine hydrochloride (DOI) (16 nmol), a preferential 5-HT2A agonist, reduced the number of upward jumps directed to the border of the experimental chamber during hypoxia, interpreted as escape attempts, without affecting the rats' locomotion. These effects were similar to those caused by chronic, but not acute, intraperitoneal administration of the antidepressant fluoxetine (5-15 mg/kg), or acute systemic administration of the benzodiazepine receptor agonist alprazolam (1-4 mg/kg), both drugs clinically used in the treatment of panic disorder. Our findings strengthen the view that the dPAG is a key encephalic area involved in the defensive behaviors triggered by activation of the suffocation alarm system. They also support the use of hypoxia-evoked escape as a model of respiratory-type panic attacks.

Although there are a number of psychological and practical obstacles to reporting professional boundary violations, physicians must learn their ethical and legal obligations to report misconduct and seek assistance when needed.

Alterations in the intracellular levels of calcium are a common response to cell death stimuli in animals and fungi and, particularly, in the Neurospora crassa response to staurosporine. We highlight the importance of the extracellular availability of Ca2+ for this response. Limitation of the ion in the culture medium further sensitizes cells to the drug and results in increased accumulation of reactive oxygen species (ROS). Conversely, an approximately 30-fold excess of external Ca2+ leads to increased drug tolerance and lower ROS generation. In line with this, distinct staurosporine-induced cytosolic Ca2+ signaling profiles were observed in the absence or presence of excessive external Ca2+. High-throughput RNA sequencing revealed that different concentrations of extracellular Ca2+ define distinct transcriptional programs. Our transcriptional profiling also pointed to two putative novel Ca2+-binding proteins, encoded by the NCU08524 and NCU06607 genes, and provides a reference dataset for future investigations on the role of Ca2+ in fungal biology.

Prior studies have shown that the cell of origin of acute myeloid leukemia (AML) initiation is an important determinant of therapeutic sensitivity. MLL-rearranged leukemias in which the fusion is acquired in hematopoietic stem cells (HSC) are less sensitive to chemotherapy and express high levels of the oncogenic transcription factor, Evi1, when compared to leukemias which are initiated in granulocyte-macrophage progenitors (GMP). However, the mechanisms governing how cell-of-origin modulates therapeutic response have not been delineated. Here, we describe a functional link that ties therapeutic sensitivity of MLL-AF9 leukemias to both chemotherapy and pharmacologic inhibitors of lysine-specific demethylase 1 (LSD1) - currently under investigation in clinical trials - to AML cell of origin via a novel mechanism that modulates p53 protein stability and activity through Evi1. This Evi1-dependent mechanism revealed a therapeutic vulnerability in the resistant HSC-derived leukemias that could be targeted with the BCL2 inhibitor venetoclax, which overcame resistance when combined with LSD1 inhibition. Murine HSC-derived MLL-AF9 leukemias exhibited markedly reduced sensitivity to the LSD1 inhibitor, IMG-7289, when compared to GMP-derived MLL-AF9 leukemias, in vitro and in vivo. Consistent with previously published reports, HSC-derived leukemias exhibited several hundred-fold higher expression of EVI1 mRNA relative to GMP-derived leukemias in both mouse and human MLL-rearranged leukemia models; the differential EVI1 expression mirrors expression patterns of this key hematopoietic regulator in normal HSCs and GMPs. H3K27me3 chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) revealed that the EVI1 locus is silenced by Polycomb repression in GMP-derived leukemias, and this repression was also observed in EVI1low primary AML patient samples. Knockdown of Evi1 via shRNA sensitized HSC-derived leukemias to LSD1 inhibition. In vitro assays revealed induction of apoptosis in GMP-derived leukemias - but not in HSC-derived leukemias - after treatment with IMG-7289. HSC-derived leukemias also exhibited decreased apoptotic priming assessed through functional BH3 profiling assays as well as blunted p53 transcriptional output. These data suggested that cell-of-origin led to differential p53 activity and therapeutic response in AMLs driven by the same fusion gene initiated from different stem/progenitor populations. The diminished p53 activity in HSC-derived leukemias was associated with reduced p53 protein abundance both at steady-state and after LSD1 inhibitor treatment compared to GMP-derived leukemias. Quantification of p53 mRNA did not show differential p53 expression, including after LSD1 inhibition, in HSC vs. GMP-derived leukemias implicating post-transcriptional regulatory mechanisms that underlie this cell-of-origin mediated phenotype. We found that modulation of Evi1 expression resulted in altered p53 protein stability: specifically, (1) shRNA-mediated knockdown of Evi1 in HSC-derived leukemias increased p53 protein stability and (2) overexpression of Evi1 blunted doxorubicin-induced p53 protein stability. Moreover, p53 loss-of-function in Evi1low GMP-derived MLL-AF9 leukemias induced resistance to LSD1 inhibition. By contrast, Evi1high HSC-derived leukemias exposed to the BCL2 inhibitor venetoclax in vivo were sensitized to LSD1 inhibition, resulting in enhanced apoptosis and greater reductions in disease burden, observations that we observed in patients with Evi1high AML treated with venetoclax. Our findings describe how the cell of origin of p53 wild-type cancers can differentially modulate p53 function and therapeutic response and provide a mechanistic rationale for therapies aimed to circumvent this resistance mechanism. Disclosures Cai: Imago Biosciences, Inc.: Consultancy. Goldberg:Celgene: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Pfizer: Research Funding; Arog Pharmaceuticals: Research Funding; ADC Therapeutics: Research Funding; American Society of Clinical Oncology: Research Funding; Abbvie: Consultancy; Abbvie: Research Funding; American Society of Hematology: Research Funding; DAVA Oncology: Honoraria. Stein:Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Tallman:Daiichi-Sankyo: 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, Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; ADC Therapeutics: Research Funding; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; UpToDate: Patents & Royalties; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Cellerant: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; UpToDate: Patents & Royalties; Cellerant: Research Funding; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellerant: Research Funding; Biosight: Research Funding; Biosight: Research Funding; ADC Therapeutics: Research Funding; ADC Therapeutics: Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Research Funding; Cellerant: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellerant: Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biosight: Research Funding; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Delta Fly Pharma: 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, Research Funding; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Biosight: Research Funding; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lowe:ORIC pharmaceuticals: Consultancy, Equity Ownership; Mirimus: Consultancy, Equity Ownership; Constellation Pharma: Consultancy, Equity Ownership; Petra Pharmaceuticals: Consultancy, Equity Ownership; PMV Pharmaceuticals: Consultancy, Equity Ownership; Faeth Therapeutics: Consultancy, Equity Ownership; Blueprint Medicines: Consultancy, Equity Ownership. Rienhoff:Imago Biosciences: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Levine:Loxo: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; Amgen: Honoraria; Celgene: Consultancy, Research Funding; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Roche: Consultancy, Research Funding; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armstrong:AstraZeneca: Research Funding; Epizyme, Inc.: Consultancy, Equity Ownership; Imago Biosciences, Inc.: Consultancy, Equity Ownership; Cyteir Therapeutics: Consultancy, Equity Ownership; C4 Therapeutics: Consultancy, Equity Ownership; Syros Pharmaceuticals: Consultancy, Equity Ownership; OxStem Oncology: Consultancy, Equity Ownership; Accent Therapeutics: Consultancy, Equity Ownership; Mana Therapeutics: Consultancy, Equity Ownership; Novartis: Research Funding; Janssen: Research Funding.

Background: Myeloid and lymphoid leukemias each have distinct diagnostic algorithms, treatment paradigms, and therapeutic options. In mixed phenotype acute leukemia (MPAL), myeloid and lymphoid immunophenotypes are expressed simultaneously. MPAL is rare, accounting for fewer than 5% of all leukemias, and carries a poor prognosis. Currently, there is no standard of care for treatment of this disease and therapeutic options tailored to this group of patients are lacking. Additionally, the biological mechanisms underlying lineage infidelity are poorly understood. Here we seek to establish more precise biological characterization of this disease and have identified a new subgroup of patients with Mixed Phenotype Acute Myeloid Leukemia (AML-MP). Methods: We developed a novel natural language processing pipeline and used it to review 830 patient records, representing all acute leukemia patients treated at Memorial Sloan Kettering Cancer Center since 2015. We identified those with ambiguous lineages based on multiparameter flow cytometry data and integrated next-generation sequencing, cytogenetics, and clinical information. Statistical methods are outlined with the associated results below. Results: Among the 830 patient records reviewed, 54 (6.5%) patients with mixed lineage characteristics were identified. Of these, 26 (48%) carried a formal diagnosis of MPAL while 28 (52%) carried a diagnosis of AML with myelodysplasia related changes (AML-MRC) or therapy related AML (t-AML). Among the cases expressing multiple lineages, 34 (25%) had B/Myeloid features (11 MPAL, 23 AML-MRC), 17 (13%) had T/Myeloid features (13 MPAL, 4 AML-MRC), 3 (0.7%) had B/T/Myeloid lineage (2 MPAL, 1 AML-MRC). Only 8 patients received prior chemotherapy at our institution > 1 year prior to diagnosis and were classified as t-AML. As a control group, we also identified 79 patients with AML-MRC exhibiting myeloid lineage alone. We pooled the mixed-lineage and myeloid-only AML-MRC cases and performed k-means clustering into 2 groups using all available molecular features (Figure 1a). Although TP53 was enriched within AML cases (p Among patients with mixed lineage characteristics, we performed a cox proportional hazards analysis and found that RUNX1 and SRSF2 mutations were predictors of better overall survival (OS) after adjusting for age, cytogenetics, and diagnosis type (age > 60: p < 0.01, high risk cytogenetics: p Among AML-MRC myeloid only cases, ASXL1 mutation was associated with improved OS and TET2 mutation showed a trend toward poorer OS after adjusting for age and cytogenetic risk (age>60: p=0.01, ASXL1: p=0.02, TET2: p=0.06; overall: p < 0.01, Figure 1b, right). Cytogenetic risk did not independently contribute to the model (high risk: p=0.3, intermediate risk: p=0.55). We also manually reviewed flow cytometry results from 64 patients diagnosed with MPAL or diagnosed with t-AML or AML-MRC despite expressing markers of multiple lineages (Mixed Phenotype AML). Using a k-nearest neighbor approach, we found that immunophenotype identified two distinct patient populations - one largely Mixed Phenotype AML, and the other with a mixture of MPAL and Mixed Phenotype AML cases (p Conclusions: Genomic characteristics improve prognostication for patients with MPAL, with RUNX1 and SRSF2 predicting a more favorable OS and TP53 not predictive of worse OS; this is in contrast to current stratifications of patients with de novo AML. While TP53 is enriched among AML cases, it does not distinguish AML-MRC from MPAL. Additionally, AML-MRC patients with ASXL1 mutations had improved overall survival after adjusting for cytogenetic risk. Some cases that are categorized as AML-MRC and t-AML by WHO criteria should be considered Mixed Phenotype AML and may be better classified into one of two distinct immunophenotypic subsets. Our study and model provide a more refined biological classification and prognostic schema for patients with MPAL and AML-MP; further validation of these observations is required in other data sets. Figure 1 Disclosures Roshal: Auron Therapeutics: Equity Ownership, Other: Provision of services; Celgene: Other: Provision of Services; Physicians' Education Resource: Other: Provision of services. Levine:Prelude Therapeutics: Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Gilead: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Loxo: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; Amgen: Honoraria. Tallman:Indy Hematology Review: Honoraria; Hematology Oncology of Indiana: Honoraria; Salzberg Weill Cornall MSKCC Seminar in Hematologic Malignancies: Honoraria; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; International Conference in Leukemia: Honoraria; 14th Annual Miami Cancer Meeting: Honoraria; Amgen: Consultancy; Orsenix: Membership on an entity's Board of Directors or advisory committees, Research Funding; UpToDate: Patents & Royalties; Mayo Clinic: Honoraria; New Orleans Summer Cancer Conference: Honoraria; ADC Therapeutics: Research Funding; Danbury Hospital Tumor Board: Honoraria; Arog Pharmaceuticals: Research Funding; Nohla: Membership on an entity's Board of Directors or advisory committees, Research Funding; KAHR: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bioline: Membership on an entity's Board of Directors or advisory committees, Research Funding; University of Oklahoma Medical Center: Honoraria; Cellerant Therapeutics: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees.

Background: Activating mutations of NRAS and KRAS genes are common in newly diagnosed acute myeloid leukemia (AML), occurring in 11-16% and 4-5% of patients, respectively. RAS mutations are frequently acquired at time of progression from MDS to AML and are associated with poor survival. Next generation sequencing (NGS) at diagnosis and during complete remission has shown that RAS mutations have high clearance rates with induction chemotherapy. In the CALGB 8525 study, RAS-mutant younger patients (age Methods: We retrospectively reviewed the charts of patients with newly diagnosed AML treated at Memorial Sloan Kettering Cancer Center between January 1, 2014 to May 15, 2019. Patients with pathologic confirmation of AML and treatment with induction chemotherapy were included. Age < 18 years old, treatment with a pediatric induction regimen, a diagnosis of biphenotypic AML, unknown RAS mutation status at diagnosis, or treatment an outside institution were criteria for exclusion. All patients underwent NGS from a diagnostic bone marrow aspirate (BMA) with MiSeq or MSK-IMPACT platforms. Mutations present with a variant allele frequency (VAF) ≥ 1% were retained. Response was evaluated per ELN 2017 criteria. Immunophenotypic MRD was identified in BMA by multiparameter flow cytometry. Any level of residual disease was considered MRD+. Baseline characteristics were evaluated by Fisher's exact test and Wilcoxon rank sum tests. Kaplan-Meier estimates were used to summarize OS and EFS. Multivariable cox regression, including time-dependent variables was performed on univariate factors with p Results: 202 patients, including 162 WT and 40 RAS mutant met inclusion criteria for further analysis. Mutations in NRAS and KRAS occurred in 14%, and 8% of patients, respectively with 6 patients having co-occurring NRAS and KRAS mutations. At baseline, the RAS mutant AML cohort had a significantly greater proportion of patients with AML-MRC and a trend toward fewer patients receiving allogeneic stem cell transplant. (Table 1.) Cytogenetic abnormalities were similar among RAS and WT patients. Sequencing at diagnosis revealed an increased frequency of FLT3 TKD, RUNX1, TET2, WT1, and ETV6 mutations and a decreased frequency of FLT3-ITD and TP53 mutations in the RAS mutant cohort. Response rates and MRD negative remission rates to induction chemotherapy were similar between RAS and WT AML patients (Table 2). With a median follow up of 25 months among survivors, RAS mutant AML was associated with a significant decrease in median EFS (4.9 vs. 11.4 months, p< 0.01) and a near significant decrease in median OS (12 vs. 30.1 months, p=0.057) (Figure 1 and 2). After controlling for variables with p Conclusions: In summary, the presence of RAS mutations in patients with AML receiving induction chemotherapy was associated with decreased overall and event free survival. RAS mutant AML was enriched among patients with AML-MRC and prior myeloid neoplasms, which was also associated with decreased survival. Lastly, treatment with chemotherapy led to a high rate of RAS mutation clearance in responders that persisted at the time of disease relapse. The poor prognosis of RAS mutant AML despite RAS mutation clearance suggests that other therapies are needed in combination with chemotherapy to improve outcomes in this high-risk population. Disclosures Cai: Imago Biosciences, Inc.: Consultancy. Viny:Hematology News: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Other: Sponsored travel. Goldberg:American Society of Clinical Oncology: Research Funding; Abbvie: Research Funding; ADC Therapeutics: Research Funding; American Society of Hematology: Research Funding; DAVA Oncology: Honoraria; Pfizer: Research Funding; Arog Pharmaceuticals: Research Funding; Abbvie: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Celgene: Consultancy. Tallman:BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Consultancy; Cellerant: Research Funding; ADC Therapeutics: Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees. Stein:Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees.

OBJECTIVE: The effect of chronic ethanol exposure on chemoreflexes has not been extensively studied in experimental animals. Therefore, this study tested the hypothesis that known ethanol-induced autonomic, neuroendocrine and cardiovascular changes coincide with increased chemoreflex sensitivity, as indicated by increased ventilatory responses to hypoxia and hypercapnia. METHODS: Male Wistar rats were subjected to increasing ethanol concentrations in their drinking water (first week: 5% v/v, second week: 10% v/v, third and fourth weeks: 20% v/v). At the end of each week of ethanol exposure, ventilatory parameters were measured under basal conditions and in response to hypoxia (evaluation of peripheral chemoreflex sensitivity) and hypercapnia (evaluation of central chemoreflex sensitivity). RESULTS: Decreased respiratory frequency was observed in rats exposed to ethanol from the first until the fourth week, whereas minute ventilation remained unchanged. Moreover, we observed an increased tidal volume in the second through the fourth week of exposure. The minute ventilation responses to hypoxia were attenuated in the first through the third week but remained unchanged during the last week. The respiratory frequency responses to hypoxia in ethanol-exposed rats were attenuated in the second through the third week but remained unchanged in the first and fourth weeks. There was no significant change in tidal volume responses to hypoxia. With regard to hypercapnic responses, no significant changes in ventilatory parameters were observed. CONCLUSIONS: Our data are consistent with the notion that chronic ethanol exposure does not increase peripheral or central chemoreflex sensitivity.

We hypothesized that hydrogen sulphide (H2 S), acting specifically in the anteroventral preoptic region (AVPO - an important integrating site of thermal and cardiorespiratory responses to hypoxia in which H2 S synthesis has been shown to be increased under hypoxic conditions), modulates the hypoxic ventilatory response.To test this hypothesis, we measured pulmonary ventilation (V˙E) and deep body temperature of rats before and after intracerebroventricular (icv) or intra-AVPO microinjection of aminooxyacetate (AOA; CBS inhibitor) or Na2 S (H2 S donor) followed by 60 min of hypoxia exposure (7% O2 ). Furthermore, we assessed the AVPO levels of H2 S of rats exposed to hypoxia. Control rats were kept under normoxia.Microinjection of vehicle, AOA or Na2 S did not change V˙E under normoxic conditions. Hypoxia caused an increase in ventilation, which was potentiated by microinjection of AOA because of a further augmented tidal volume. Conversely, treatment with Na2 S significantly attenuated this response. The in vivo H2 S data indicated that during hypoxia the lower the deep body temperature the smaller the degree of hyperventilation. Under hypoxia, H2 S production was found to be increased in the AVPO, indicating that its production is responsive to hypoxia. The CBS inhibitor attenuated the hypoxia-induced increase in the H2 S synthesis, suggesting an endogenous synthesis of the gas.These data provide solid evidence that AVPO H2 S production is stimulated by hypoxia, and this gaseous messenger exerts an inhibitory modulation of the hypoxic ventilatory response. It is probable that the H2 S modulation of hypoxia-induced hyperventilation is at least in part in proportion to metabolism.

The medullary raphé is an important component of the central respiratory network, playing a key role in CO2 central chemoreception. However, its participation in hypoxic ventilatory responses is less understood. In the present study, we assessed the role of nucleus raphé obscurus (ROb), and specifically 5-HT neurons confined in the ROb, on ventilatory and thermoregulatory responses to hypoxia. Chemical lesions of the ROb were performed using either ibotenic acid (non-specific lesion; control animals received PBS) or anti-SERT-SAP (5-HT specific lesion; control animals received IgG-SAP). Ventilation (V˙E; whole body plethysmograph) and body temperature (Tb; data loggers) were measured during normoxia (21% O2, N2 balance) and hypoxia exposure (7% O2, N2 balance, 1h) in conscious adult rats. Ibotenic acid or anti-SERT-SAP-induced lesions did not affect baseline values of V˙E and Tb. Similarly, both lesion procedures did not alter the ventilatory or thermoregulatory responses to hypoxia. Although evidence in the literature suggests a role of the rostral medullary raphé in hypoxic ventilatory responses, under the present experimental conditions our data indicate that caudal medullary raphé (ROb) and its 5-HT neurons neither participate in the tonic maintenance of breathing nor in the ventilatory and thermal responses to hypoxia.

We performed cytosine methylation sequencing on genetically diverse patients with acute myeloid leukemia (AML) and found leukemic DNA methylation patterning is primarily driven by nonpromoter regulatory elements and CpG shores. Enhancers displayed stronger differential methylation than promoters, consisting predominantly of hypomethylation. AMLs with dominant hypermethylation featured greater epigenetic disruption of promoters, whereas those with dominant hypomethylation displayed greater disruption of distal and intronic regions. Mutations in IDH and DNMT3A had opposing and mutually exclusive effects on the epigenome. Notably, co-occurrence of both mutations resulted in epigenetic antagonism, with most CpGs affected by either mutation alone no longer affected in double-mutant AMLs. Importantly, this epigenetic antagonism precedes malignant transformation and can be observed in preleukemic LSK cells from Idh2R140Q or Dnmt3aR882H single-mutant and Idh2R140Q/Dnmt3aR882H double-mutant mice. Notably, IDH/DNMT3A double-mutant AMLs manifested upregulation of a RAS signaling signature and displayed unique sensitivity to MEK inhibition ex vivo as compared with AMLs with either single mutation. Significance: AML is biologically heterogeneous with subtypes characterized by specific genetic and epigenetic abnormalities. Comprehensive DNA methylation profiling revealed that differential methylation of nonpromoter regulatory elements is a driver of epigenetic identity, that gene mutations can be context-dependent, and that co-occurrence of mutations in epigenetic modifiers can result in epigenetic antagonism. Cancer Discov; 7(8); 868–83. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 783

OBJECTIVE: Chemoreceptors play an important role in the autonomic modulation of circulatory and ventilatory responses to changes in arterial O2 and/or CO2. However, studies evaluating hemodynamic responses to hypoxia and hypercapnia in rats have shown inconsistent results. Our aim was to evaluate hemodynamic and respiratory responses to different levels of hypoxia and hypercapnia in conscious intact or carotid body-denervated rats. METHODS: Male Wistar rats were submitted to bilateral ligature of carotid body arteries (or sham-operation) and received catheters into the left femoral artery and vein. After two days, each animal was placed into a plethysmographic chamber and, after baseline measurements of respiratory parameters and arterial pressure, each animal was subjected to three levels of hypoxia (15, 10 and 6% O2) and hypercapnia (10% CO2). RESULTS: The results indicated that 15% O2 decreased the mean arterial pressure and increased the heart rate (HR) in both intact (n = 8) and carotid body-denervated (n = 7) rats. In contrast, 10% O2 did not change the mean arterial pressure but still increased the HR in intact rats, and it decreased the mean arterial pressure and increased the heart rate in carotid body-denervated rats. Furthermore, 6% O2 increased the mean arterial pressure and decreased the HR in intact rats, but it decreased the mean arterial pressure and did not change the HR in carotid body-denervated rats. The 3 levels of hypoxia increased pulmonary ventilation in both groups, with attenuated responses in carotid body-denervated rats. Hypercapnia with 10% CO2 increased the mean arterial pressure and decreased HR similarly in both groups. Hypercapnia also increased pulmonary ventilation in both groups to the same extent. CONCLUSION: This study demonstrates that the hemodynamic and ventilatory responses varied according to the level of hypoxia. Nevertheless, the hemodynamic and ventilatory responses to hypercapnia did not depend on the activation of the peripheral carotid chemoreceptors.

Made available in DSpace on 2018-11-26T16:54:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-09-22 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Preconditioning can induce a cascade of cellular events leading to neuroprotection against subsequent brain insults. In this study, we investigated the chronic effects of hypoxic preconditioning on spontaneous recurrent seizures (SRS), neuronal death, and spatial memory performance in rats subjected to pilocarpine (Pilo)-induced status epilepticus (SE). Rats underwent a short hypoxic episode (7% O-2 + 93% N-2; 30 min on two consecutive days) preceding a 4-h SE (HSE group). Control groups were rats submitted to SE only (SE), rats subjected to hypoxia only (H) or normoxia-saline (C). Animals were monitored for the occurrence of SRS, and spatial memory performance was evaluated in the radial-arm maze. Hippocampal sections were analyzed for cell death and mossy fiber sprouting at 1 or 60 days after SE. Compared to SE group, HSE had increased SE latency, reduced number of rats with SRS, reduced mossy fiber sprouting at 60 days, and reduced cell death in the hilus and the CA3 region 1 and 60 days after SE. Additionally, HSE rats had better spatial memory performance than SE rats. Our findings indicated that short hypoxic preconditioning preceding SE promotes long-lasting protective effects on neuron survival and spatial memory. (C) 2016 IBRO. Published by Elsevier Ltd. All rights reserved. Univ Sao Paulo, Ribeirao Preto Sch Med, Dept Neurosci & Behav Sci, BR-14049900 Ribeiaro Preto, Brazil Univ Sao Paulo, Ribelaro Preto Sch Med, Dept Physiol, BR-14049900 Ribeirao Preto, Brazil Sao Paulo State Univ, Araraquara Sch Dent, Dept Physiol & Pathol, Sao Paulo, SP, Brazil Univ Fed Rio Grande do Norte, Inst Brain, BR-59056450 Natal, RN, Brazil Sao Jose Rio Preto Med Sch, Dept Mol Biol, BR-1509000 Sao Jose Do Rio Preto, Brazil Sao Paulo State Univ, Araraquara Sch Dent, Dept Physiol & Pathol, Sao Paulo, SP, Brazil FAPESP: 2005/56447-7 FAPESP: 2010/51515-2 CNPq: 476250/2013-7 CNPq: 466995/2014-8

The medullary raphe (MR) is a putative central chemoreceptor site, contributing to hypercapnic respiratory responses elicited by changes in brain PCO2/pH. Purinergic mechanisms in the central nervous system appear to contribute to central chemosensitivity. To further explore the role of P2 receptors within the rostral and caudal MR in relation to respiratory control in room air and hypercapnic conditions, we performed microinjections of PPADS, a non-selective P2X antagonist, in conscious rats. Microinjections of PPADS into the rostral or caudal MR produced no changes in the respiratory frequency, tidal volume and ventilation in room air condition. The ventilatory response to hypercapnia was attenuated after microinjection of PPADS into the rostral but not in the caudal MR when compared to the control group (vehicle microinjection). These data suggest that P2X receptors in the rostral MR contribute to the ventilatory response to CO2, but do not participate in the tonic maintenance of ventilation under room air condition in conscious rats.

The control of pulmonary ventilation in South American lungfish Lepidosiren paradoxa is poorly understood. Interactions between temperature and hypoxia are particularly relevant due to large seasonal variations of its habitat. Therefore, we tested the hypothesis that the ventilatory responses to aerial hypoxia of Lepidosiren are highly dependent on ambient temperature. We used a pneumotachograph to measure pulmonary ventilation (VE), tidal volume (VT) and respiratory frequency (fR) during normoxic (21% O2) and hypoxic (12%, 10% and 7% O2) conditions at two temperatures (25 and 35 °C). Blood gases, arterial PO2 (PaO2), arterial PCO2 (PaCO2) and arterial pH (pHa) were also evaluated. At 25 °C, VE increased significantly at 10% and 7% hypoxic levels when compared to the control value (21% O2). At 35 °C, all hypoxic levels elicited a significant increase of VE relative to control values. VE is augmented mostly by increases of respiratory frequency (fR), and there were significant interactions (p

Serotonergic (5-HT) neurons in the nucleus raphe obscurus (ROb) are involved in the respiratory control network. However, it is not known whether ROb 5-HT neurons play a role in the functional interdependence between central and peripheral chemoreceptors. Therefore, we investigated the role of ROb 5-HT neurons in the ventilatory responses to CO2 and their putative involvement in the central–peripheral CO2 chemoreceptor interaction in unanaesthetised rats. We used a chemical lesion specific for 5-HT neurons (anti-SERT-SAP) of the ROb in animals with the carotid body (CB) intact or removed (CBR). Pulmonary ventilation (V E), body temperature and the arterial blood gases were measured before, during and after a hypercapnic challenge (7% CO2). The lesion of ROb 5-HT neurons alone (CB intact) or the lesion of 5-HT neurons of ROb+CBR did not affect baseline V E during normocapnic condition. Killing ROb 5-HT neurons (CB intact) significantly decreased the ventilatory response to hypercapnia (p

Background: MRD is a powerful prognostic factor in AML. Emerging data indicate that allogeneic stem cell transplant (alloSCT) with MRD results in outcomes equivalently poor to alloSCT with morphologic AML (Araki et al., JCO 2016). Genomic predictors of MRD are unclear, and relative efficacy of therapies for MRD remains elusive. Objectives: Here we provide an integrated analysis of responses for 163 patients (pts) who underwent induction chemotherapy with baseline next-generation sequencing (NGS) followed by serial immunophenotypic monitoring for MRD. Methods:163 patients starting in April 2014 who underwent induction chemotherapy at Memorial Sloan Kettering Cancer Center were retrospectively studied. All received anthracycline + cytarabine, with or without investigational agents. Immunophenotypic MRD was identified in bone marrow aspirates (BMA) by multiparameter flow cytometry. Any level of residual disease was considered MRD+. Molecular analysis was obtained from pre-induction BMA by NGS using 28 or 49 gene panels. Cytogenetics/FISH were performed using standard techniques. Results: Patient characteristics are in Table 1. 7/163 (4.9%) died within 30 days of induction.153 pts had BM biopsy after induction prior to further therapy. 124/153 underwent flow after induction. 65/124 (52.4%) achieved CR/CRi after induction alone, 31/124 (25%) MRD+CR/CRi, and 34/124 (27.4%) MRD-CR/CRi. Pre-induction molecular analysis from 126 suggests that certain cytogenetic and molecular abnormalities correlate with achievement of MRD-CR. (Figure 1) Only 2/25 (8%) with RUNX1, 0/13 with SF3B1, and 0/11 with TP53 mutations achieved MRD-CR/CRi as best response after 1 cycle of induction. Only 3 additional RUNX1, 2 SF3B1, and 0/11 TP53 achieved MRD-CR/CRi as best response after a second cycle of therapy. In contrast, 7/8 with CBF AML (inv16 and no KIT mutation, n=4) or (t(8;21), n=3) achieved MRD-CR/CRi (n=5) or CR without flow (n=2) after 1 cycle of induction. 91/163 (55.8%) underwent alloSCT following induction or additional therapy. Post-alloSCT follow-up indicates potential value in converting MRD+ to MRD-. 84/91 were evaluable for MRD with flow cytometry prior to alloSCT. 41/84 (48.8%) were MRD-, 30/84 (35.7%) MRD+, and 13/84 (15.4%) persistent AML. 13/41 (31.7%) MRD-pre-alloSCT were MRD- post-induction. 28/41 (68.2%) MRD+ or persistent AML converted to MRD- prior to alloSCT following additional therapy. 23/29 MRD+CR/CRi pts after induction were intermediate/unfavorable and therefore transplant candidates. 19/23 MRD+CR/CRi intermediate/unfavorable underwent transplant (9 without post-induction therapy, 10 after consolidation), while 4 did not proceed to transplant due to relapse after induction (n=1), relapse after consolidation (n=2), and patient preference. There was no significant difference in post-transplant OS between early MRD-CR immediately following induction and later conversion to MRD-CR prior to alloSCT (Figure 1B). Post-transplant analysis reveals that most pts who enter transplant with persistent AML (n=13) or MRD+ (n=30) clear MRD (30/43, 69.7%) by the first post-transplant BM (median 32 days, Figure 1C). Despite initial post-transplant MRD clearance, pts who entered alloSCT with persistent AML or MRD+ had poorer post-transplant OS compared to pts who entered alloSCT with MRD- (p=0.02, Figure 1D). Conclusion: Our data show that AML pts with specific molecular mutations (RUNX1, SF3B1, and TP53) are unlikely to achieve MRD-CR/CRi after induction chemotherapy. We further show that additional therapy such as consolidation may be advantageous for some MRD+ pts to achieve MRD-CR prior to alloSCT, although others remain resistant to MRD clearance. Post-transplant OS is improved in pts who are MRD- at time of transplant, regardless of whether they required additional therapy beyond induction to achieve this state. Our results suggest that development of MRD-eradicating therapies after AML induction has the potential to improve post-transplant outcomes. Disclosures Goldberg: AROG: Research Funding; Pfizer: Research Funding; Celgene: Consultancy. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria. Perales:Takeda: Other: Personal fees; Merck: Other: Personal fees; Abbvie: Other: Personal fees; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees and Clinical trial support; Novartis: Other: Personal fees. Tallman:ADC Therapeutics: Research Funding; Daiichi-Sankyo: Other: Advisory board; Orsenix: Other: Advisory board; Cellerant: Research Funding; BioSight: Other: Advisory board; AROG: Research Funding; AbbVie: Research Funding.

Background Measurable residual disease (MRD) is associated with inferior outcomes in patients with acute myeloid leukemia (AML). MRD monitoring enhances risk stratification and may guide therapeutic intervention. Post-induction MRD is frequently cleared with further therapy and the clearance may lead to better outcomes. In contrast, persistent MRD is associated with poor outcomes. At present it is not possible to predict which patients are likely to clear MRD with further therapy. Here we report a simple, objective, widely applicable and quantitative MFC approach using the ratio of blast/PDC to predict persistent MRD and poor outcomes in AML. Patients and Methods A cohort of 136 adult patients with a confirmed diagnosis of AML by WHO criteria who underwent standard induction therapy at a single center between 4/2014 and 9/2017 was initially included. 69 patients achieved complete morphologic remission (36 MRD-neg. and 33 MRD-pos.). MRD status was assessed by MFC using a different from normal (DfN) approach. PDC were quantified as the percent of total WBC by flow cytometry based on low side scatter, moderate CD45, CD303, bright CD123 and HLA-DR expression. Results The proportion of PDC was markedly decreased in patients with AML (≥20% blasts) (N=136) with a median of 0.016% (interquartile range IQR: 0.0019%-0.071%, Figure 1A), more than 10-fold lower than observed in normal controls (median 0.23%, IQR 0.17%-0.34%) (N=20). While there was no difference between MRD-neg. and normal control groups (median 0.31%, IQR: 0.17%-0.49%; vs. 0.28%, IQR: 0.17%-0.34%), MRD-pos. group had significantly reduced PDC proportion compared to the control (median 0.074%, IQR: 0.022%-0.33%, Wilcoxon rank sum, p=0.019). In an attempt to achieve better separation and to eliminate possible effects of hemodilution, the ratio of blast/PDC was calculated by using the proportions of blasts and PDCs out of total WBCs as quantitated by flow cytometry. A cut-off threshold of the blast/PDC ratio of 10 was chosen to separate each group (Figure 1B). Importantly, a ratio cut-off of 10 had a corresponding specificity of 97.4% for predicting MRD positivity status. MRD positivity was significantly associated with inferior overall survival (OS) and relapse-free survival (RFS) in our study cohort (OS HR 4.11 (95% CI: 1.30-13.03), p=0.016; RFS HR 4.20 (95% CI: 1.49-11.82), p=0.007, Figure 1C and D). The 2-year cumulative incidence of relapse in the MRD-neg. group compared to MRD-pos. group was 10% (95% CI: 2-24%) vs. 37% (95% CI: 18-56%, p=0.014). Importantly, blast/PDC ratio ≥10 was also strongly associated with inferior OS and RFS (OS HR 3.12 (95% CI: 1.13-8.60), p= 0.028; RFS HR 4.05 (95% CI: 1.63-10.11), p=0.003, Figure 1E and F), which is similar in magnitude to MRD positivity. Furthermore, MRD-pos. patients with blast/PDC ratio Conclusion We have established an objective and quantitative MFC method to risk stratify post induction AML patients by risk for relapse, MRD clearance and likelihood of survival. Loss of PDC correlates with residual leukemia, is highly specific for MRD positivity in post-induction patients, and strongly predicts poorer overall survival and higher likelihood of relapse. Loss of PDC also predicts persistent MRD in post-induction MRD-pos. patients despite further therapy, suggesting that MRD-pos. patients with normal PDC may benefit from further therapy prior to transplant, while MRD-pos. patients with loss of PDC may not. Figure 1. Figure 1. Disclosures Goldberg: AROG: Research Funding; Pfizer: Research Funding; Celgene: Consultancy. Geyer:Dava Oncology: Honoraria. Levine:Isoplexis: Equity Ownership; C4 Therapeutics: Equity Ownership; Gilead: Honoraria; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Prelude: Research Funding; Imago: Equity Ownership; Roche: Consultancy, Research Funding; Loxo: Consultancy, Equity Ownership; Celgene: Consultancy, Research Funding; Novartis: Consultancy; Epizyme: Patents & Royalties; Janssen: Consultancy, Honoraria. Tallman:BioSight: Other: Advisory board; AROG: Research Funding; AbbVie: Research Funding; Cellerant: Research Funding; ADC Therapeutics: Research Funding; Orsenix: Other: Advisory board; Daiichi-Sankyo: Other: Advisory board.

Acute Myeloid Leukemia (AML) is a biologically diverse disease with subtypes that can be identified through integrated cytogenetic, mutational, and epigenetic characterization. Mutations in epigenetic regulators such as IDH1, IDH2, and DNMT3A are among the most common gene mutations found in AML (Cancer Genome Atlas Research et al. 2013; Papaemmanuil et al. 2016). Targeted inhibitors of IDH1 and IDH2 have recently been approved by the FDA for the treatment of AML (DiNardo et al. 2018; Stein et al. 2015). In addition, IDH/DNMT3A dual mutant AMLs exhibit a distinct epigenetic signature and are characterized by increased in vitro sensitivity to MEK inhibition (Glass et al. 2017). Here we explore the clinical course of AML patients with IDH1, IDH2, and DNMT3A mutations treated at Memorial Sloan Kettering Cancer Center (MSKCC) between 2012 and 2018. Treatment regimens included standard chemotherapy, as well as investigational therapies such as IDH1, IDH2, and pan-IDH inhibitors (Table 1). Using overall survival as our endpoint, we assessed contributions of WHO risk, age, treatment regimen, and gene mutations using multivariate Cox Proportional Hazards modeling. Among intermediate risk patients, we found that the presence of concurrent IDH1/DNMT3A mutations (n=16) conferred the highest mortality risk compared to DNMT3A mutation alone (HR=2.31; p=0.046). In contrast, IDH2/DNMT3A mutated AML conferred similar mortality risk compared to DNMT3A mutation alone (HR=1.07; p=0.85). When compared to intermediate risk IDH1/DNMT3A mutated AML, intermediate risk IDH2/DNMT3A mutated AML had a lower risk of mortality (HR=0.345; p=0.038), (Fig. 1). This adverse risk of IDH1/DNMT3A mutations persists after adjusting for IDH inhibitor therapy (HR=2.42; p=0.05). In multivariate analysis, IDH1 mutation and use of low intensity therapeutic regimens conferred an increased risk of mortality after adjusting for WHO risk (HR=2.25; p=0.016 and HR=1.71; p=0.036, respectively). Induction chemotherapy provided an overall survival benefit after adjusting for WHO Risk and the presence of IDH1, IDH2, DNMT3A, or dual IDH/DNMT3A mutations (HR=0.50; p Overall, our findings suggest that AML with dual IDH1/DNMT3A mutations constitutes a higher risk disease entity. Further study of targeted treatment modalities such as dual IDH1/MEK inhibition or combined chemotherapy/IDH1 inhibition may be helpful in the treatment of these higher risk AML patients. Disclosures Levine: Isoplexis: Equity Ownership; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Imago: Equity Ownership; Gilead: Honoraria; Janssen: Consultancy, Honoraria; C4 Therapeutics: Equity Ownership; Novartis: Consultancy; Epizyme: Patents & Royalties; Loxo: Consultancy, Equity Ownership; Prelude: Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Tallman:AbbVie: Research Funding; Cellerant: Research Funding; Orsenix: Other: Advisory board; BioSight: Other: Advisory board; AROG: Research Funding; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding. Goldberg:Abbvie: Research Funding; AROG: Research Funding; Pfizer: Research Funding; Celgene: Research Funding. Stein:Pfizer: Consultancy; Celgene: Consultancy; Bayer: Consultancy; Daiichi Sankyo: Consultancy; Agios: Consultancy; Novartis: Consultancy.

We studied staurosporine-induced cell death in the filamentous fungus Neurospora crassa . The generation of reactive oxygen species during the process appears to be an important signaling event, since addition of the antioxidant glutathione prevents the effects of staurosporine on fungal growth. Selected mutants with mutations in respiratory chain complex I are extremely sensitive to the drug, stressing the involvement of complex I in programmed cell death. Following this finding, we determined that the complex I-specific inhibitor rotenone used in combination with staurosporine results in a synergistic and specific antifungal activity, likely through a concerted action on intracellular glutathione depletion. Paradoxically, the synergistic antifungal activity of rotenone and staurosporine is observed in N. crassa complex I mutants and in Saccharomyces cerevisiae , which lacks complex I. In addition, it is not observed when other complex I inhibitors are used instead of rotenone. These results indicate that the rotenone effect is independent of complex I inhibition. The combination of rotenone and staurosporine is effective against N. crassa as well as against the common pathogens Aspergillus fumigatus and Candida albicans , pointing to its usefulness as an antifungal agent.

e21534Background: Uveal melanoma (UM) is characterized by a long latency period between the time of initial diagnosis and the development of metastatic disease, with a limited understanding of geno...