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It is increasingly becoming clear that cancers are a symbiosis of diverse cell types and tumor clones. Combined single-cell RNA sequencing, flow cytometry, and immunohistochemistry studies of the innate immune compartment in the bone marrow of patients with acute myeloid leukemia (AML) reveal a shift toward a tumor-supportive M2-polarized macrophage landscape with an altered transcriptional program, with enhanced fatty acid oxidation and NAD + generation. Functionally, these AML-associated macrophages display decreased phagocytic activity and intra–bone marrow coinjection of M2 macrophages together with leukemic blasts strongly enhances in vivo transformation potential. A 2-day in vitro exposure to M2 macrophages results in the accumulation of CALR low leukemic blast cells, which are now protected against phagocytosis. Moreover, M2-exposed “trained” leukemic blasts display increased mitochondrial metabolism, in part mediated via mitochondrial transfer. Our study provides insight into the mechanisms by which the immune landscape contributes to aggressive leukemia development and provides alternatives for targeting strategies aimed at the tumor microenvironment.

Breakpoint cluster region-Abelson (BCR-ABL) negative myeloproliferative neoplasms (MPNs) are chronic myeloid neoplasms initiated by the acquisition of gene mutation(s) in a haematopoietic stem cell, leading to clonal expansion and over-production of blood cells and their progenitors. MPNs encompass a spectrum of disorders with overlapping but distinct molecular, laboratory and clinical features. This includes polycythaemia vera, essential thrombocythaemia and myelofibrosis. Dysregulation of the immune system is key to the pathology of MPNs, supporting clonal evolution, mediating symptoms and resulting in varying degrees of immunocompromise. Targeting immune dysfunction is an important treatment strategy. In the present review, we focus on the immune landscape in patients with MPNs - the role of inflammation in disease pathogenesis, susceptibility to infection and emerging strategies for therapeutic immune modulation. Further detailed work is required to delineate immune perturbation more precisely in MPNs to determine how and why vulnerability to infection differs between clinical subtypes and to better understand how inflammation results in a competitive advantage for the MPN clone. These studies may help shed light on new designs for disease-modifying therapies.

While it is increasingly becoming clear that cancers are a symbiosis of diverse cell types and tumor clones, the tumor microenvironment (TME) in acute myeloid leukemias (AML) remains poorly understood. Here, we uncover the functional and prognostic relevance of an M2-polarized macrophage compartment. Intra bone marrow co-injection of M2d-macrophages together with leukemic blasts that fail to engraft on their own now induce fatal leukemia in mice. Even a short-term two-day in vitro exposure to M2d macrophages can “train” leukemic blasts after which cells are protected against phagocytosis, display increased mitochondrial metabolism and improved in vivo homing, resulting in full-blown leukemia. Single-cell RNAseq analysis of AML associated macrophages revealed metabolic-related pathways such as Fatty Acid Oxidation and NAD+ generation as therapeutical targetable vulnerabilities. Our study provides insight into the mechanisms by which the immune landscape contributes to aggressive leukemia development and provides alternatives for effective targeting strategies.

Azacitidine and enasidenib are two therapies available for treatment of acute myelogenous leukemia (AML), and the mechanisms of action of these drugs involve alteration of aberrant DNA methylation. We hypothesized that a combination of these agents could have interactive effects on DNA methylation and enhance differentiation in mIDH2 cells. Combination treatment enhanced cellular differentiation in TF-1 cells overexpressing IDJ2R140Q through increased hemoglobinization and increased hemoglobin γ RNA expression compared with the effects of single agents. Furthermore, in primary AML samples (IDH2R140Q or R172K), combination treatment reduced CD34+ cells and increased CD15+ cells to a greater extent than attained with single agents. To explore the mechanism of enhanced differentiation with combination treatment, the TF-1 epigenome was analyzed by profiling 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) DNA methylation changes. Enasidenib treatment alone increased 5hmC, consistent with reactivation of ten-eleven-translocation (TET) enzyme activity. Compared with treatment with azacitidine alone, combination treatment reduced 5mC levels at greater numbers of sites and these loci were significantly enriched in regions with increased 5hMC (25.8% vs. 7.4%). Results are consistent with a model in which enasidenib-mediated reactivation of ten-eleven-translocation enzymes cooperates with azacitidine-mediated inhibition of DNA methyltransferase enzymes, leading to greater reductions in DNA methylation and enhanced erythroid differentiation.

Outcomes in acute myeloid leukemia (AML) are dependent on patient- and disease-characteristics, treatment, and socioeconomic factors. AML outcomes between resource-constrained and developed countries have not been compared directly. We analyzed two cohorts: from São Paulo state, Brazil (USP, n = 312) and Oxford, United Kingdom (OUH, n = 158). USP cohort had inferior 5-year overall survival compared with OUH (29% vs. 49%, adjusted-p=.027). USP patients have higher early-mortality (23% vs. 6% p

The 2017 European LeukemiaNet 2017 acute myeloid leukemia (AML) risk stratification (ELN2017) is widely used for risk-stratifying patients with AML. However, its applicability in low- and middle-income countries is limited because of a lack of full cytogenetic and molecular information at diagnosis. Here, we propose an alternative for risk stratification (the Adapted Genetic Risk [AGR]), which permits cytogenetic or molecular missing data while retaining prognostic power. We first analyzed 167 intensively treated patients with nonacute promyelocytic leukemia AML enrolled in São Paulo, Brazil (Faculdade de Medicina da Universidade de São Paulo), as our training data set, using ELN2017 as the standard for comparison with our AGR. Next, we combined our AGR with clinical prognostic parameters found in a Cox proportional hazards model to create a novel scoring system (survival AML score, SAMLS) that stratifies patients with newly diagnosed AML. Finally, we have used 2 independent test cohorts, Faculdade de Medicina de Ribeirão Preto (FMRP; Brazil, n = 145) and Oxford University Hospitals (OUH; United Kingdom, n = 157) for validating our findings. AGR was statistically significant for overall survival (OS) in both test cohorts (FMRP, P = .037; OUH, P = .012) and disease-free survival in FMRP (P = .04). The clinical prognostic features in SAMLS were age (>45 years), white blood cell count (30.0 × 103/μL), and low albumin levels (

Isocitrate dehydrogenase (IDH) 1 and 2 mutations result in overproduction of D-2-hydroxyglutarate (2-HG) and impaired cellular differentiation. Ivosidenib, a targeted mutant IDH1 (mIDH1) enzyme inhibitor, can restore normal differentiation and results in clinical responses in a subset of patients with mIDH1 relapsed/refractory (R/R) acute myeloid leukemia (AML). We explored mechanisms of ivosidenib resistance in 174 patients with confirmed mIDH1 R/R AML from a phase 1 trial. Receptor tyrosine kinase (RTK) pathway mutations were associated with primary resistance to ivosidenib. Multiple mechanisms contributed to acquired resistance, particularly outgrowth of RTK pathway mutations and 2-HG–restoring mutations (second-site IDH1 mutations, IDH2 mutations). Observation of multiple concurrent mechanisms in individual patients underscores the complex biology of resistance and has important implications for rational combination therapy design. This trial was registered at www.clinicaltrials.gov as #NCT02074839

Mutations in the gene encoding isocitrate dehydrogenase 2 (IDH2) occur in several types of cancer, including acute myeloid leukemia (AML). In model systems, mutant IDH2 causes hematopoietic differentiation arrest. Enasidenib, a selective small-molecule inhibitor of mutant IDH2, produces a clinical response in 40% of treated patients with relapsed/refractory AML by promoting leukemic cell differentiation. Here, we studied the clonal basis of response and acquired resistance to enasidenib treatment. Using sequential patient samples, we determined the clonal structure of hematopoietic cell populations at different stages of differentiation. Before therapy, IDH2-mutant clones showed variable differentiation arrest. Enasidenib treatment promoted hematopoietic differentiation from either terminal or ancestral mutant clones; less frequently, treatment promoted differentiation of nonmutant cells. Analysis of paired diagnosis/relapse samples did not identify second-site mutations in IDH2 at relapse. Instead, relapse arose by clonal evolution or selection of terminal or ancestral clones, thus highlighting multiple bypass pathways that could potentially be targeted to restore differentiation arrest. These results show how mapping of clonal structure in cell populations at different stages of differentiation can reveal the response and evolution of clones during treatment response and relapse.

With immune therapies on the rise, an in-depth understanding of the immunological changes in leukemic bone marrow (BM) niches becomes indispensable. Being an crucial part of the tumor microenvironment (TME) in solid tumours, tumour-associated macrophages are often associated with poor prognosis (Bruni et al. 2020). Yet, in acute myeloid leukaemia (AML) the role of macrophages has not been thoroughly studied. The expression of the M2-markers CD163 and CD206 in the AML BM cell population predicted poor clinical outcome. We identified that this expression emerges from a more mature (CD45 midSSC highHLA-DR +CD14 +CD16 +/-) myeloid cell population (hereafter called AML-associated macrophages - AAM) and not from the leukemic blasts. By employing flow cytometry analysis (FACS) we noted a decrease in the expression of the M1-marker (CD80) and an increase of the M2-markers CD163/CD206on AAM (n=70) compared to healthy donors (HD, n=10). Unsupervised clustering based on the CD163/CD206 levels detected on AAM generated 4 distinct clusters, whereby patients within the CD163 low/CD206 low cluster displayed better overall survival than the other clusters. In vitro, the co-culture of HD-derived M1 macrophages and AML primary/cell lines reduced AML growth via apoptosis induction and cell cycle arrest, while M2-macrophages promoted AML survival and phagocytosis/drug-resistance when treated with FLT3/BCL2 inhibitors. Primary AML cells were also able to repolarize M1- into M2-macrophages, suggesting that leukemic cells actively remodel their microenvironment. Next, we evaluated the impact of M2-macrophages on leukemogenesis in a patient derived xenograft (PDX) model, using the notoriously difficult to engraft primary Acute Promyelocytic Leukaemia (APL) cells (n=7 patient samples). Intra-BM injection of M2-macrophages and retro-orbital transplant of primary APL cells induced full-blown APL in NSGS mice. More strikingly, ex vivo culture of APL cells on M2-macrophages (48h) was sufficient to "train" these cells to engraft and induce fatal APL. Maintenance of self-renewal was shown in a secondary transplant and an enhanced frequency of leukemic stem cells was assessed by in vivo LTC-IC assays. To identify the biological changes acquired by leukemic blasts, we performed RNA sequencing comparing AML/APL samples at diagnosis to cells that were "trained" (48 h) on M2-macrophages or on MS5 mesenchymal BM stromal cells. Gene ontology and gene set enrichment analysis on the genes up-regulated upon M2 co-culture were significantly enriched for cell migration, cell cycle progression and oxidative phosphorylation (OXPHOS) signatures. In line with our RNAseq data, we noted improved in vivo homing of primary APL cells to the BM within 18 h post-transplant upon ex vivo M2 co-culture compared to diagnosis (n=7 APL blasts). Concurrently, we detected increased levels of surface protein expression Integrin-α4 (CD49d) and -α5 (CD49e) on APL/AML blast cells after M2 exposure. The CD49d expression remained high in primary and secondary transplants. Using seahorse measurements, we confirmed the increased respiration capacity (basal and maximum) of primary AML/APL cells (n=7) after exposure to M2 macrophages compared to MS5.FACS analysis revealed that M2-macrophages were able to transfer more mitochondria than MS5 cells to primary AML cells, which could underlie the observed increase in OXPHOS mitochondrial metabolism. Treatment with Etomoxir (50 µM), prevented the gain in functional respiration when AML blast were co-cultured on M2-macrophages, while no changes were observed for MS5 co-cultures, suggesting increased fatty acid oxidation to drive the OXPHO-like state. Finally, we noted that training on M2 macrophages significantly increased colony formation and endowed the cells with long term proliferation in liquid cultures for over 30 days. Overall, we reveal that the frequency of M2-macrophages is up-regulated in a subgroup of AML patients representing a group with poor prognosis. M2 macrophages can support leukemic growth and therapy-resistance, and support fatal APL in PDX models. Even an in vitro exposure to M2 macrophages suffices to alter adhesion, homing and metabolic characteristics of leukemic blasts to allow efficient engraftment and fatal leukemogenesis. Our study uncovers how the TME can contribute to leukemic transformation which provides alternative avenues for therapeutic interventions. Disclosures Silveira: BMS/Celgene: Research Funding; Servier/Agios: Research Funding; Abbvie: Speakers Bureau; Astellas: Speakers Bureau. Quek: BMS/Celgene: Research Funding; Servier/Agios: Research Funding. Mota: Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Technopharma: Speakers Bureau; Bristol Myer Squibb: Speakers Bureau; Bayer: Speakers Bureau; Pfizer: Speakers Bureau; AstraZeneca: Speakers Bureau; Astellas: Speakers Bureau; Ipsen: Speakers Bureau; Amgen: Speakers Bureau.

Differentiation arrest in acute myeloid leukaemia (AML) results in accumulation of leukaemic progenitors (L-Prog) and bone marrow failure. Mutant isocitrate dehydrogenase enzyme produces d-2-hydroxyglutarate (2HG), which inhibits α-ketoglutarate-dependent dioxygenases, including Jumonji histone demethylases (JKDM) and TET2, but how this causes AML is unclear. Inhibitors of mutant IDH enzyme (mIDHi) restore differentiation in IDH-mutant (mIDH) AML (Amatangelo et al., 2018). Here, we studied transcriptional networks involved using single-cell (SC) gene expression (GEX) and transcription factor (TF) motif accessibility in primary AML treated with the mIDH2 inhibitor enasidenib (ENA) and found that ENA activates cell cycle (CC) and pro-differentiation programmes through increased promoter accessibility of granulocyte-monocyte (GM)-TF targets. We treated patient L-Prog in vitro with ENA or vehicle, and performed SC RNA-seq (Chromium 10x) in 4 responsive (R), and one non-responsive (NR) patient samples in early, mid and late timepoints. GEX signatures were used to annotate cells according to function (undifferentiated [U], early and late GM [EGM and LGM]) and CC states. In R samples, ENA yielded more dividing late-GM at mid-late timepoints than DMSO (18% vs 6.5%), and more terminally differentiated neutrophils at late timepoints (46% vs 16%). Using SCENIC (Aibar et al., 2017) to assign highly differentially-expressed genes to TF motifs, we computed regulatory networks (regulons, 'R'). Expression of the SP1 R was strongly correlated with active proliferation and ENA conditions led to generation of more cells that co-expressed CEBPA R or CEBPE R with SP1 R, emphasising simultaneous engagement of CC and GM programmes. SP1 function is associated with CC and GM differentiation, and silencing of its binding to its targets contributes to AML pathogenesis (Maiques-Diaz et al., 2012). Control and NR samples failed to produce neutrophils, had reduced co-expression of CEBPE/SP1 R and yielded more poorly differentiated cells expressing GATA2 R. At the individual gene level, ENA stimulated downregulation of GATA2, GFI1B, IKZF1/2, and RUNX3 together with upregulation of immediate early genes which respond to cytokine and mitogenic stimuli (EGR1, IER2, AP-1) in early-mid phase. Later there is upregulation of CEBP TFs and effector genes FUT4, ELANE, AZU1 and PRTN3. Interestingly, expression of some GM-TFs (RUNX1, SPI1/PU.1, GFI1) was similar between ENA and DMSO, indicating that gene expression alone was insufficient for GM differentiation. Given the effects of 2-HG on JKDM, we assessed chromatin accessibility and TF binding using SC ATAC-seq. Overall, we had 25% of differentially accessible (DA) peaks, from which 75% were more accessible in ENA than in DMSO. ENA DA peaks were highly enriched in promoters. Using ArchR (Granja et al., 2021), we clustered cells and used ELANE expression levels to compute trajectories in parallel with SC RNA-seq data. ENA peaks were sequentially enriched for CBF/RUNX and GATA families, followed by AP-1 (JUN/FOS) and EGR/CEBP/KLF motifs. Footprinting analysis showed sequential decrease and increase of TF binding for GATA2 and CEBPA/E respectively during ENA-induced differentiation. Although it did not cause higher expression of SPI1/PU.1, ENA induced increased accessibility of its target binding sites at promoters, which included CEBPA/E and GM effectors (MPO, FUT4, PRTN3). This provides a novel mechanism by which ENA induces differentiation of L-prog. Regulatory network analysis around active, differentially expressed TFs at different phases of ENA-induced differentiation showed a switch from a repressive transcriptional landscape driven by stem-progenitor TFs, to one where AP-1 and GM-TFs activate expression of GM-effector genes. We postulate a model where MYC, E2F8 and EGR1 upregulate the CEBP family in early-mid differentiation. In addition to stimulation of promoter accessibility of TFBS, we find that ENA increases accessibility of cis-regulatory elements of CEBP TFs, adding another mechanism by which differentiation of L-Prog occurs. Our data on the mechanism of action of ENA suggest that differentiation arrest in IDHm AML involves suppression of CC and GM differentiation programs in a repressive chromatin landscape, likely via inhibition of KDM6A and demethylation of repressive H3K27me3 marks. Disclosures Silveira: Astellas: Speakers Bureau; Abbvie: Speakers Bureau; Servier/Agios: Research Funding; BMS/Celgene: Research Funding. Hasan: Bristol Myers Squibb: Current Employment. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Vyas: Gilead: Honoraria; Astellas: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Takeda: Honoraria; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Daiichi Sankyo: Honoraria; Jazz: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Quek: BMS/Celgene: Research Funding; Servier/Agios: Research Funding.

Purpose: Azacitidine (AZA) is a novel therapeutic option in older patients with acute myeloid leukemia (AML), but its rational utilization is compromised by the fact that neither the determinants of clinical response nor its mechanism of action are defined. Co-administration of histone deacetylase inhibitors, such as vorinostat (VOR), is reported to improve the clinical activity of AZA, but this has not been prospectively studied in patients with AML. Experimental Design: We compared outcomes in 259 adults with AML (n = 217) and MDS (n = 42) randomized to receive either AZA monotherapy (75 mg/m2 × 7 days every 28 days) or AZA combined with VOR 300 mg twice a day on days 3 to 9 orally. Next-generation sequencing was performed in 250 patients on 41 genes commonly mutated in AML. Serial immunophenotyping of progenitor cells was performed in 47 patients. Results: Co-administration of VOR did not increase the overall response rate (P = 0.84) or overall survival (OS; P = 0.32). Specifically, no benefit was identified in either de novo or relapsed AML. Mutations in the genes CDKN2A (P = 0.0001), IDH1 (P = 0.004), and TP53 (P = 0.003) were associated with reduced OS. Lymphoid multipotential progenitor populations were greatly expanded at diagnosis and although reduced in size in responding patients remained detectable throughout treatment. Conclusions: This study demonstrates no benefit of concurrent administration of VOR with AZA but identifies a mutational signature predictive of outcome after AZA-based therapy. The correlation between heterozygous loss of function CDKN2A mutations and decreased OS implicates induction of cell-cycle arrest as a mechanism by which AZA exerts its clinical activity. Clin Cancer Res; 23(21); 6430–40. ©2017 AACR.

Single sitting procedures where the mononuclear cell fraction is extracted from bone marrow and implanted directly into cartilage and bone defects are becoming more popular as novel treatments for cartilage defects which have, until now had few treatment options. This is on the basis that the mesenchymal stem cells (MSCs) contained within will repair the damaged tissue. This study sought to determine if the femur and tibia could provide equivalent amounts of mesenchymal stem cells, with equivalent viability and proliferative capacity, to that obtained from the gold standard of the pelvis in order to potentially reduce the morbidity associated with these procedures. Bone marrow was extracted from the pelvis, femur, and tibia of human subjects. The mononuclear cell fraction was extracted and cultured in the laboratory. Mesenchymal stem cell populations were assessed using a colony forming unit count. Viability was assessed using a PrestoBlue viability assay. Population doubling number was calculated between the end of passage 0 and passage three to determine the proliferative abilities of the different populations. Finally, the cell surface phenotype of the cells was determined by flow cytometry. The results showed that the pelvis was superior to the femur and tibia in terms of the number of stem cells isolated. There was no statistically significant difference in the phenotype of the cells isolated from different locations. This work shows that when undertaking single sitting procedures, the pelvis remains the optimum source for obtaining MSCs, despite the morbidity associated with bone marrow collection from the pelvis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1868-1875, 2017.

Disease relapse is the major cause of treatment failure after allogeneic stem cell transplantation (allo-SCT) in acute myeloid leukemia (AML). To identify AML-associated genes prognostic of AML relapse post–allo-SCT, we resequenced 35 genes in 113 adults at diagnosis, 49 of whom relapsed. Two hundred sixty-two mutations were detected in 102/113 (90%) patients. An increased risk of relapse was observed in patients with mutations in WT1 (P = .018), DNMT3A (P = .045), FLT3 ITD (P = .071), and TP53 (P = .06), whereas mutations in IDH1 were associated with a reduced risk of disease relapse (P = .018). In 29 patients, we additionally compared mutational profiles in bone marrow at diagnosis and relapse to study changes in clonal structure at relapse. In 13/29 patients, mutational profiles altered at relapse. In 9 patients, mutations present at relapse were not detected at diagnosis. In 15 patients, additional available pre–allo-SCT samples demonstrated that mutations identified posttransplant but not at diagnosis were detectable immediately prior to transplant in 2 of 15 patients. Taken together, these observations, if confirmed in larger studies, have the potential to inform the design of novel strategies to reduce posttransplant relapse highlighting the potential importance of post–allo-SCT interventions with a broad antitumor specificity in contrast to targeted therapies based on mutational profile at diagnosis.

The concept of cancer stem cells (CSCs) emerged from our understanding of the way in which normal tissues are generated from multipotent stem cells. Regenerative tissues exhibit a cellular hierarchy of differentiation, which is maintained by stem cells. Evidence from experimental models has indicated that a similar hierarchy is seen in at least some cancers, where CSCs give rise to disordered and dysfunctional tissues, leading to disease. The CSC model proposes that tumours can be divided into at least two distinct populations. The stem cells are a specialized population of cancer cells with the unique property of long-term self-renewal that maintain the growth of the cancerous clone. These stem cells give rise to the second population of cells, which form the bulk of the tumour, and lack indefinite self-renewal. Recently, our understanding of CSCs has been refined through combining genetic, epigenetic, and functional models of tumorigenesis. Malignant transformation occurs as the result of sequential acquisition of genetic mutations. Capacity for self-renewal is essential for a clone to survive and progress to become cancerous. If an oncogenic mutation occurs in a cell that is incapable of self-renewal, the clone will become exhausted through differentiation. CSCs may survive anticancer chemotherapy and increasing evidence indicates their role in mediating treatment resistance and relapse. Therefore, strategies to eradicate cancers must effectively target the stem cells that maintain their growth. CSC-directed therapeutic strategies are currently being explored in experimental studies and clinical trials but reducing toxicity to normal tissue stem cells represents a significant challenge.

Background Aminoacid catabolism has emerged as an important immune escape mechanism in cancer. Arginase, the enzyme that breaks down arginine, is exploited by cancer cells to dampen tumour-specific immune responses. We have shown that arginase-2 is released in plasma of patients with acute myeloid leukaemia to suppress T-cell growth. Here we aimed to elucidate the adaptive mechanism through which cancer cells resist arginine depletion in the tumour microenvironment. Methods We used colorimetric assays to measure arginine concentrations and arginase activity in plasma of patients and mice with acute myeloid leukaemia. T-cell proliferation and activation were assessed with flow cytometry. Differentially expressed genes in THP1 leukaemic cells compared with T cells upon in-vitro arginine depletion were identified in a microarray (Illumina, San Diego, CA, USA) and confirmed by real-time PCR. RNA sequencing (RNA-Seq, Illumina) of human acute myeloid leukaemia cells versus healthy myeloid cells was done to verify results ex vivo. Functional effects were assessed by western blot and proliferation assays. Findings Arginase-2 was released in plasma of patients and mice with acute myeloid leukaemia to cause systemic arginine depletion. Arginine deprivation inhibited T-cell proliferation and function, effects abolished with arginase-specific inhibitors. In contrast to T cells, cancer cells continued to proliferate by converting citrulline to arginine. This dichotomy relies on the ability of cancer cells to upregulate ASS1, the enzyme that controls citrulline conversion to arginine. Knockdown of ASS1 abrogated the ability of cancer cells to proliferate in arginine-free medium. Knockdown of transcription factor ATF4 blocked ASS1 upregulation and citrulline recycling. RNA-sequencing analysis of acute myeloid leukaemia blasts showed ASS1 and ATF4 upregulation in patients compared with healthy controls. Interpretation These results identify arginine metabolism as an important pathway for acute myeloid leukaemia to suppress T-cell proliferation. Furthermore, they prove that cancer cells, but not T cells, continue to proliferate in arginine-deprived environments by upregulating ASS1, a target of transcription factor ATF4. Our findings lead to potential therapeutic opportunities where arginase-2, ASS1, and ATF4 can be targeted as part of cancer immunotherapy. Funding Wellcome Trust clinical research training fellowship (awarded to AVH).

Summary Depleting the microenvironment of important nutrients such as arginine is a key strategy for immune evasion by cancer cells. Many tumors overexpress arginase, but it is unclear how these cancers, but not T cells, tolerate arginine depletion. In this study, we show that tumor cells synthesize arginine from citrulline by upregulating argininosuccinate synthetase 1 (ASS1). Under arginine starvation, ASS1 transcription is induced by ATF4 and CEBPβ binding to an enhancer within ASS1. T cells cannot induce ASS1, despite the presence of active ATF4 and CEBPβ, as the gene is repressed. Arginine starvation drives global chromatin compaction and repressive histone methylation, which disrupts ATF4/CEBPβ binding and target gene transcription. We find that T cell activation is impaired in arginine-depleted conditions, with significant metabolic perturbation linked to incomplete chromatin remodeling and misregulation of key genes. Our results highlight a T cell behavior mediated by nutritional stress, exploited by cancer cells to enable pathological immune evasion., Graphical abstract, Highlights • Arginine starvation induces ASS1 expression in some cancers but not in T cells • ATF4 binds an internal ASS1 enhancer to drive expression in cancer but not T cells • T cell activation is disrupted by arginine starvation, with a loss of reprogramming • Arginine starvation compacts chromatin in T cells, disrupting ATF4 binding, Arginine depletion by many tumors generates an immunosuppressive microenvironment. Crump et al. show that cancer cells can tolerate this by inducing ATF4-dependent upregulation of ASS1, allowing de novo arginine synthesis. T cells are unable to synthesize arginine as ASS1 is repressed, disrupting T cell function and the chromatin remodeling associated with activation.

7501 Background: ENA and AZA each induce overall response rates (ORR) of ~30% and complete remission (CR) rates of ~20% in ND-AML. In vitro, combining ENA + AZA enhances cell differentiation. We report results of the phase II portion of an open-label, randomized phase I/II study of ENA + AZA (“E+A”) vs. AZA monotherapy (“A”) in patients (pts) with m IDH2 ND-AML (NCT02677922). Methods: Pts age ≥ 18 years ineligible for intensive chemotherapy, with ECOG PS ≤ 2 and intermediate- or poor-risk cytogenetics, were randomized 2:1 to E+A or A in 28-day (d) cycles. All pts received SC AZA 75 mg/m2/d x 7 d/cycle; pts randomized to E+A also received ENA 100 mg QD. The primary endpoint was ORR (CR, CR with incomplete recovery, partial remission, morphologic leukemia-free state). Other endpoints include duration of response (DOR), overall and event-free survival (OS, EFS), safety, and m IDH2 VAF. Results: 101 pts received E+A (n = 68) or A (n = 33). Median age was 75 years (57–85); most pts (83%) had intermediate-risk cytogenetics. 21 pts in the E+A arm and 1 in the A arm were ongoing at data cutoff (Aug 2019). Most common reason for discontinuation was disease progression (E+A 31%, A 52%). Median number Tx cycles was 10 (1–26) in the E+A arm and 6 (1–28) in the A arm. 7 pts (21%) in the A arm received subsequent Tx with ENA. ORR, CR rate and DOR were significantly improved with E+A vs. A (Table). Median OS was 22 mo in both arms (HR 0.99 [95%CI 0.52, 1.87]; P = 0.97). Median EFS was 17.2 and 10.8 mo in the E+A and A arms, respectively (HR 0.59 [95%CI 0.30, 1.17]; P = 0.13). Maximal m IDH2 VAF change from BL was –83.4% with E+A vs. –17.7% with A ( P < 0.01). No baseline co-mutation predicted primary resistance. Common Tx-related grade 3–4 AEs in the E+A arm were thrombocytopenia (37%), neutropenia (35%), anemia (19%), and febrile neutropenia (15%); these occurred in 19%, 22%, 22%, and 16% in the A arm. Grade 3–4 infections occurred in 18% of E+A pts and 31% of A pts. IDH differentiation syndrome occurred in 12 pts (18%) in the E+A arm. 5 E+A pts (7%) and 1 A pt (3%) died in the first 60 d. Conclusions: Combining ENA + AZA resulted in significantly improved response rates and durations, and was generally well-tolerated in older patients with m IDH2 ND-AML. The impact of subsequent Tx on OS/EFS and new translational data will be presented at the meeting. Clinical trial information: NCT02677922 . [Table: see text]

International audience; The hierarchy of human hemopoietic progenitor cells that produce lymphoid and granulocytic–monocytic (myeloid) lineages is unclear. Multiple progenitor populations produce lymphoid and myeloid cells, but they remain incompletely characterized. Here we demonstrated that lympho-myeloid progenitor populations in cord blood — lymphoid-primed multi-potential progenitors (LMPPs), granulocyte-macrophage progenitors (GMPs) and multi-lymphoid progenitors (MLPs) — were functionally and transcriptionally distinct and heterogeneous at the clonal level, with progenitors of many different functional potentials present. Although most progenitors had the potential to develop into only one mature cell type (‘uni-lineage potential’), bi- and rarer multi-lineage progenitors were present among LMPPs, GMPs and MLPs. Those findings, coupled with single-cell expression analyses, suggest that a continuum of progenitors execute lymphoid and myeloid differentiation, rather than only uni-lineage progenitors’ being present downstream of stem cells.

Recurrent mutations at R140 and R172 in isocitrate dehydrogenase 2 (IDH2) occur in many cancers, including ∼12% of acute myeloid leukemia (AML). In preclinical models these mutations cause accumulation of the oncogenic metabolite R-2-hydroxyglutarate (2-HG) and induce hematopoietic differentiation block. Single-agent enasidenib (AG-221/CC-90007), a selective mutant IDH2 (mIDH2) inhibitor, produced an overall response rate of 40.3% in relapsed/refractory AML (rrAML) patients with mIDH2 in a phase 1 trial. However, its mechanism of action and biomarkers associated with response remain unclear. Here, we measured 2-HG, mIDH2 allele burden, and co-occurring somatic mutations in sequential patient samples from the clinical trial and correlated these with clinical response. Furthermore, we used flow cytometry to assess inhibition of mIDH2 on hematopoietic differentiation. We observed potent 2-HG suppression in both R140 and R172 mIDH2 AML subtypes, with different kinetics, which preceded clinical response. Suppression of 2-HG alone did not predict response, because most nonresponding patients also exhibited 2-HG suppression. Complete remission (CR) with persistence of mIDH2 and normalization of hematopoietic stem and progenitor compartments with emergence of functional mIDH2 neutrophils were observed. In a subset of CR patients, mIDH2 allele burden was reduced and remained undetectable with response. Co-occurring mutations in NRAS and other MAPK pathway effectors were enriched in nonresponding patients, consistent with RAS signaling contributing to primary therapeutic resistance. Together, these data support differentiation as the main mechanism of enasidenib efficacy in relapsed/refractory AML patients and provide insight into resistance mechanisms to inform future mechanism-based combination treatment studies.

Background: Mutations in IDH2 occur in 8-19% of patients (pts) with AML. Enasidenib (ENA; AG-221) is an oral, small-molecule inhibitor of mutant IDH2 (mIDH2) that promotes myeloid cell differentiation. ENA is approved in the US for use in adult pts with relapsed/refractory mIDH2 AML. Azacitidine (AZA) is a hypomethylating agent that prolongs survival vs. conventional care regimens in older unfit pts with newly diagnosed (ND) AML. AZA promotes DNA hypomethylation by inhibiting DNA methyltransferases. ENA indirectly reduces DNA methylation by suppressing the oncometabolite, 2-hydroxyglutarate (2-HG), thereby restoring function to α-ketoglutarate-dependent TET family enzymes, among other substrates. In vitro, combination ENA + AZA enhances cell differentiation. This is the first report of interim outcomes from the randomized, phase II portion of an ongoing, open-label, phase I/II study of ENA + AZA vs. AZA monotherapy (AZA-only) in pts with mIDH2 ND-AML who are not candidates for intensive chemotherapy (IC) (NCT02677922). Methods: Adult pts with mIDH2 ND-AML who were ineligible to receive IC and had ECOG PS scores ≤2 were randomized in a 2:1 ratio to receive ENA + AZA or AZA-only in repeated 28-day cycles. All pts receive SC AZA 75 mg/m2/day for the first 7 days of each treatment (Tx) cycle; pts randomized to ENA + AZA also receive continuous ENA 100 mg QD. The primary endpoint is overall response rate (ORR), which includes complete remission (CR), CR with incomplete blood or platelet count recovery (CRi/CRp), partial remission (PR), and morphologic leukemia-free state (MLFS), per modified IWG 2003 AML response criteria. P values for response comparisons between Tx arms were derived using chi-square test. Duration of response (DOR) was estimated by Kaplan-Meier method. mIDH2 variant allele frequencies (VAF) in bone marrow mononuclear cells (BMMCs) were assessed by digital PCR. Results: Between Oct. 2016 and Aug. 2018, 101 pts were randomized to receive ENA + AZA (n=68) or AZA-only (n=33). Median ages were 74 years (range 62-85) in the ENA + AZA arm and 75 years (57-85) in the AZA-only arm. Among pts with available data, 78% in the ENA + AZA arm (43/55) and 90% (19/21) in the AZA-only arm had intermediate-risk cytogenetics, respectively, and 18% and 10% had poor-risk cytogenetics. At data cutoff (Feb. 2019), 39 pts were still receiving their randomized Tx. Most common reasons for study discontinuation in the ENA + AZA and AZA-only arms were death (31% and 27%, respectively) and pt decision (4% and 12%). Two pts in the ENA + AZA arm and 1 pt in the AZA-only arm proceeded to transplant. Median number of Tx cycles was 8 (range 1-24) in the ENA + AZA arm and 6 (1-22) in the AZA-only arm; 27% and 19% of pts, respectively, received ≥12 Tx cycles. Response rates were significantly higher with combination treatment vs. AZA alone: ORRs were 68% vs. 42%, respectively (P=0.0155), and CR rates were 50% vs. 12% (P=0.0002) (Table). Median DOR was not reached with ENA + AZA and was 10.2 months in the AZA-only arm (P=0.13). Maximal mIDH2 VAF suppression from baseline was significantly greater with ENA + AZA vs. AZA-only (median -69.3% vs. -14.1%, respectively; P=0.0004). Tx-related grade 3-4 adverse events occurring in ≥10% of pts in the combination arm were neutropenia (34%), thrombocytopenia (34%), anemia (21%), febrile neutropenia (12%), and IDH differentiation syndrome (IDH-DS; 10%); these events occurred in 19%, 19%, 22%, 13%, and 0% of pts in the AZA-only arm. Rate of Tx-related grade 3-4 infections was 16% in the ENA + AZA arm and 31% in the AZA-only arm. In all, 12 pts (18%) in the ENA + AZA arm experienced IDH-DS (any grade) at a median of 37 days. In the first 60 days, 5 deaths (7%) were reported in the ENA + AZA arm (3 due to infectious complications and 2 due to possible IDH-DS) and 1 death (3%) was reported in the AZA-only arm due to progressive disease. Conclusions: ENA + AZA was associated with significantly improved complete remission and overall response rates and significant mIDH2 VAF reductions compared with AZA-only. Combination Tx was generally well tolerated, with a safety profile similar to that reported for either monotherapy. An updated data cutoff that includes at least 1 year of follow-up for all pts will be presented at the conference. Updated data will include overall survival, event-free survival, and comprehensive analyses of 2-HG and co-mutation dynamics. Disclosures DiNardo: syros: Honoraria; jazz: Honoraria; medimmune: Honoraria; agios: Consultancy, Honoraria; abbvie: Consultancy, Honoraria; celgene: Consultancy, Honoraria; daiichi sankyo: Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees. Schuh:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Teva Canada Innovation: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: 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; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria; Teva Canada Innovation: 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; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Stein:Genentech: Membership on an entity's Board of Directors or advisory committees; Novartis: 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; 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; 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; Syros: Membership on an entity's Board of Directors or advisory committees. Fernandez:Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Wei:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Honoraria, Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: AHW is a former employee of the Walter and Eliza Hall Institute and receives a fraction of its royalty stream related to venetoclax, Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Macrogenics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. De Botton:Novartis: Consultancy; Astellas: Consultancy; AbbVie: Consultancy; Pfizer: Consultancy; Pierre Fabre: Consultancy; Daiichi: Consultancy; Celgene Corporation: Consultancy, Speakers Bureau; Servier: Consultancy; Syros: Consultancy; Janssen: Consultancy; Forma: Consultancy, Research Funding; Bayer: Consultancy; Agios: Consultancy, Research Funding. Zeidan:ADC Therapeutics: Research Funding; Jazz: Honoraria; Ariad: Honoraria; Agios: Honoraria; Novartis: Honoraria; Astellas: Honoraria; Daiichi Sankyo: Honoraria; Cardinal Health: Honoraria; Seattle Genetics: Honoraria; BeyondSpring: Honoraria; Medimmune/AstraZeneca: Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Otsuka: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Acceleron Pharma: Consultancy, Honoraria, Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Trovagene: Consultancy, Honoraria, Research Funding. Fathi:Amphivena, Kite, Jazz, NewLink Genetics,: Honoraria; Agios, Astellas, Celgene, Daiichi Sankyo, Novartis, Takeda, Amphivena, Kite, Forty Seven,Trovagene, NewLink genetics, Jazz, Abbvie, and PTC Therapeutics: Consultancy. Quek:Celgene: Research Funding, Speakers Bureau; Agios: Research Funding. Kantarjian:Immunogen: Research Funding; Cyclacel: Research Funding; Agios: Honoraria, Research Funding; Astex: Research Funding; BMS: Research Funding; Novartis: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Research Funding; Ariad: Research Funding; Daiichi-Sankyo: Research Funding; Amgen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding. Frattini:Celgene Corporation: Employment, Equity Ownership. Lersch:Celgene: Employment, Equity Ownership. Gong:Celgene: Employment, Equity Ownership. Franovic:Celgene: Employment, Equity Ownership. MacBeth:Celgene Corporation: Employment, Equity Ownership. Vyas:Celgene: Research Funding, Speakers Bureau; Daiichi Sankyo: Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Forty Seven, Inc.: Research Funding; Abbvie: Speakers Bureau; Pfizer: Speakers Bureau; Astellas: Speakers Bureau. Döhner:Celgene, Novartis, Sunesis: Honoraria, Research Funding; AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria; AROG, Bristol Myers Squibb, Pfizer: Research Funding.

Introduction: Acute myeloid leukemia (AML), a hematologic malignancy characterized by clonal expansion of abnormal myeloid progenitors, is a complex disease exhibiting a dynamic mutational landscape over time. Somatic mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes occur in ~20% of patients with AML, resulting in production of the oncometabolite D-2-hydroxyglutarate (2-HG). Ivosidenib (IVO), a mutant IDH1 (mIDH1) inhibitor, is approved in the US for mIDH1 relapsed or refractory (R/R) AML and newly diagnosed mIDH1 AML in patients ≥75 years old or with comorbidities precluding intensive induction chemotherapy. Durable remissions in mIDH1 R/R AML were achieved with IVO in a phase 1 study (NCT02074839), with a complete remission (CR) plus complete remission with partial hematologic recovery (CRh) rate of >30%, and a median duration of CR+CRh response of >8 months. In these patients, bulk next-generation sequencing (NGS) identified the most frequent baseline co-mutations as DNMT3A (34%), NPM1 (23%), and SRSF2 (20%), with mIDH2 detected in 2 of 101 (~2%) patients (DiNardo et al. N Engl J Med 2018). Though a recent case study (Harding et al. Cancer Discov 2018) described the appearance of mIDH2 in patients who relapsed to IVO (isoform switching), the frequency of this phenomenon is unknown. In addition, it is unclear whether mIDH2 and other co-occurring mutations exist within the same clone as mIDH1 at baseline and relapse, as dynamic clonal architecture cannot be precisely imputed by bulk NGS. Aim: To define clonal architecture heterogeneity and pattern of mechanism of relapse at single-cell resolution in a subset of patients with mIDH2 detectable by bulk NGS following IVO treatment. Methods: Single-cell targeted DNA sequencing (scDNA-seq) was performed on matched patient peripheral blood mononuclear cell (PBMC) samples at baseline and relapse, using a microfluidic platform (Tapestri®) with a 19-gene AML panel (Pellegrino et al. Genome Res 2018) capable of detecting rare subclones to a level of 0.1%. Data were processed and analyzed using Tapestri® Insights software and the timescape R package. Results: Of 129 patients with available longitudinal genomic profiling data, 15 (12%) patients had detectable mIDH2 on treatment. Here we report findings from 9 of 15 patients with available scDNA-seq data. Seven of 9 patients had no detectable mIDH2 at baseline. Six of these 7 acquired mIDH2 at relapse within the same clone as the original mIDH1, whereas mIDH2 was identified in a separate clone to mIDH1 in 1 patient. In the 2 of 9 cases in which mIDH2 was detected at baseline, mIDH2 was present in a separate clone to mIDH1. AML-related gene mutations (e.g. PTPN11, NRAS, ASXL1) were also identified upon relapse following IVO treatment. Figure 1 demonstrates the emergence of mIDH2 in the same cell as mIDH1, concurrent with the expansion of a separate mIDH1 clone harboring a PTPN11 mutation at relapse. Figure 2 demonstrates two distinct mIDH1 clones at baseline, one harboring NPM1/NRAS co-mutations and the other harboring NPM1/FLT3-TKD co-mutations. Following IVO treatment, the IDH1/NPM1/NRAS clone was no longer detected. Reduction in the IDH1/NPM1/FLT3-TKD clone was observed at Cycle 2 Day 1, but it ultimately expanded at relapse with the acquisition of mIDH2. In both cases, plasma 2-HG was first inhibited by >95% but increased at relapse. Furthermore, phylogenetic tree reconstruction from clonotypes indicated patterns of both branching and linear clonal evolution. Conclusions: In a subset of patients with mIDH2 detectable at relapse, mIDH2 was mostly not detectable at baseline but emerged within the same clone as mIDH1, highlighting 2-HG restoration as an important mechanism of resistance to IVO. Moreover, these data provide unique insights into the clonal dynamics in patients with mIDH1 R/R AML harboring mutations in the receptor tyrosine kinase (RTK) pathway, notably that the presence of RTK mutations at baseline does not universally preclude a clinical response. scDNA-seq proved to be a powerful tool in delineating molecular outcomes for patients with mIDH1 R/R AML. These findings, a part of emerging data highlighting the interplay between baseline mutation profiles and response and clonal evolution on treatment, support combinations or sequential treatment modifications at early relapse before overt clinical progression. Disclosures Wang: Agios: Employment, Equity Ownership. Choe:Agios: Employment, Equity Ownership. DiNardo:jazz: Honoraria; celgene: Consultancy, Honoraria; daiichi sankyo: Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees; abbvie: Consultancy, Honoraria; medimmune: Honoraria; agios: Consultancy, Honoraria; syros: Honoraria. Stein:Astellas Pharma US, Inc: 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; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, 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. de Botton:Agios: Consultancy, Research Funding; Celgene: Consultancy, Speakers Bureau; Pierre Fabre: Consultancy; Servier: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Janssen: Consultancy; Forma: Consultancy, Research Funding; Syros: Consultancy; Bayer: Consultancy; AbbVie: Consultancy; Daiichi Sankyo: Consultancy; Astellas: Consultancy. Fathi:Agios, Astellas, Celgene, Daiichi Sankyo, Novartis, Takeda, Amphivena, Kite, Forty Seven,Trovagene, NewLink genetics, Jazz, Abbvie, and PTC Therapeutics: Consultancy; Amphivena, Kite, Jazz, NewLink Genetics,: Honoraria. Tallman:Biosight: Research Funding; 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; Daiichi-Sankyo: 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; BioLineRx: 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; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; UpToDate: Patents & Royalties; ADC Therapeutics: Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; 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; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; Jazz Pharmaceuticals: 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; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Research Funding; Biosight: Research Funding; UpToDate: Patents & Royalties; 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; Biosight: Research Funding; 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; UpToDate: Patents & Royalties; Rigel: 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; 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; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; UpToDate: Patents & Royalties; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellerant: Research Funding; Jazz 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; Tetraphase: 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; Cellerant: Research Funding; Rigel: 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; BioLineRx: 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; Biosight: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BioLineRx: 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; 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; 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; 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; 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; UpToDate: Patents & Royalties; Nohla: 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; Orsenix: 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; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Daiichi-Sankyo: 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; Jazz Pharmaceuticals: 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; 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; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; 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; Biosight: Research Funding; Cellerant: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellerant: Research Funding; Cellerant: Research Funding. Kantarjian:Ariad: Research Funding; Astex: Research Funding; BMS: Research Funding; Cyclacel: Research Funding; Novartis: Research Funding; Agios: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Immunogen: Research Funding; Jazz Pharma: Research Funding; Takeda: Honoraria; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding. Stone:AbbVie, Actinium, Agios, Argenx, Arog, Astellas, AstraZeneca, Biolinerx, Celgene, Cornerstone Biopharma, Fujifilm, Jazz Pharmaceuticals, Amgen, Ono, Orsenix, Otsuka, Merck, Novartis, Pfizer, Sumitomo, Trovagene: Consultancy; Argenx, Celgene, Takeda Oncology: Other: Data and Safety Monitoring Board/Committee: ; Novartis, Agios, Arog: Research Funding. Quek:Agios: Research Funding; Celgene: Research Funding, Speakers Bureau. Zhang:Agios: Employment, Equity Ownership; Agios: Employment, Equity Ownership. Liu:Agios: Employment, Equity Ownership. Attar:Aprea Therapeutics: Employment; Agios: Employment, Equity Ownership. Wu:Agios: Employment, Equity Ownership.

Background: Somatic mutations in isocitrate dehydrogenase 1 (IDH1) are reported in 6-10% of patients (pts) with acute myeloid leukemia (AML). Ivosidenib (IVO; AG-120) is an oral, potent, targeted inhibitor of mutant IDH1 (mIDH1) approved for treatment of mIDH1 relapsed/refractory AML, and newly diagnosed (ND) AML ineligible for intensive chemotherapy (IC). Here we report the genetic mutation and multiparameter flow analyses on longitudinal samples collected from pts receiving IVO + azacitidine (AZA) in the phase 1b portion of the ongoing phase 1b/2 study of mIDH1/2 inhibitors + AZA in pts with IC-ineligible ND AML (NCT02677922). Methods: Pts received oral IVO 500 mg daily continuously and subcutaneous AZA 75 mg/m2 on Days 1-7 in 28-day cycles. The secondary efficacy endpoint of complete remission (CR) plus CR with partial hematologic recovery (CR+CRh) rate was sponsor derived, and CRh was defined as CR with absolute neutrophil count >0.5 × 109/L and platelets >50 × 109/L. Genomic DNA from baseline and longitudinal bone marrow (BMMCs) and peripheral blood mononuclear cells (PBMCs) were used for molecular studies. Co-occurring mutation profiling of 20/23 (14 CR/CRh) pts by targeted next-generation sequencing (NGS) using the ACE Extended Cancer Panel was performed, with 500× average target coverage for the full coding region of 1400 genes (detection limit 2%). mIDH1 variant allele frequency (VAF) for 23/23 (16 CR/CRh) pts was also tested by a highly sensitive BEAMing digital PCR assay (detection limit 0.02-0.04%). Multiparameter flow cytometry was conducted centrally on bone marrow aspirate from 14/23 (12 CR/CRh) pts (detection limit 1%). Fisher's exact test (two-sided) was applied for statistical analysis. Results: As of February 19, 2019, 23 pts received IVO+AZA (11 male; median age 76 years [range 61-88]). Median duration of treatment was 15.1 months (range 0.3-32.2); 10 pts remained on treatment as of the data cutoff. Overall response rate was 78% (18/23 pts): CR 61% (14/23), CR with incomplete hematologic or platelet recovery 9% (2/23), and morphologic leukemia-free state 9% (2/23). CR+CRh rate was 70% (16/23). The top 3 most frequently co-mutated genes at study entry were RUNX1 (7/20 pts, 35%), SRSF2 (7/20, 35%), and DNMT3A (4/20, 20%). Given the small sample size, no gene or pathway identified at baseline was statistically associated with clinical response or resistance. Interestingly, CR/CRh was achieved in ND AML pt populations who typically have a poor prognosis, or did not achieve a CR/CRh response to single-agent IVO therapy: 3/3 pts with poor risk karyotypes (local cytogenetics), 1/2 harboring TP53 mutations, and 3/5 with RTK pathway (KRAS, NRAS, PTPN11) mutations. Longitudinal mutation clearance (MC) of mIDH1 and the most frequent baseline co-mutations in CR/CRh and non-CR/CRh pts are summarized in the Table. IDH1-MC in BMMCs was observed in 13/14 (93%) CR/CRh pts by NGS and in 11/16 (69%) by digital PCR. Utilizing the 2-log more sensitive digital PCR assay specific to mIDH1, there was strong concordance in the mIDH1 VAF observed in BMMCs and PBMCs (Pearson correlation coefficient [r]=0.919) with 12/16 (75%) CR/CRh pts achieving MC in PBMCs, and 11/12 (92%) achieving MC in both BMMCs and PBMCs. These IDH1-MC rates are higher than those previously observed in IC-ineligible ND AML pts treated with single agent IVO (Roboz et al. ASCO 2019; NCT02074839). Similarly, in CR/CRh pts with available baseline co-mutation data by NGS, all mutations were cleared in 11/14 (79%) pts, apart from mutations in the "DTA" (DNMT3A/TET2/ASXL1) genes typically associated with clonal hematopoiesis. In contrast, mutations in the "DTA" genes were cleared in 2/5 (40%) CR/CRh pts. Orthogonal evaluation of the depth of these remissions by flow cytometry found that 10/12 (83%) CR/CRh pts achieved measurable residual disease (MRD) negativity. Conclusion: Combination of IVO+AZA in IC-ineligible ND AML leads to a high rate of clinical response with molecular remissions. The strong association between MC, clinical response, and flow cytometry MRD in this phase 1b study warrants further investigation of single gene mIDH1 VAF as a biomarker for monitoring response in pts with mIDH1 AML treated with IVO+AZA. Furthermore, the high concordance of mIDH1 VAF between BMMCs and PBMCs indicates that peripheral blood could be a surrogate tissue for monitoring mIDH1 VAF in these pts. Disclosures Daigle: Agios: Employment, Equity Ownership. Choe:Agios: Employment, Equity Ownership; Agios: Employment, Equity Ownership. Quek:Celgene: Research Funding, Speakers Bureau; Agios: Research Funding. DiNardo:jazz: Honoraria; syros: Honoraria; celgene: Consultancy, Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees; agios: Consultancy, Honoraria; medimmune: Honoraria; daiichi sankyo: Honoraria; abbvie: Consultancy, Honoraria. Stein:Stemline: Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Speakers Bureau. Stein: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; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; 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; 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. Fathi:Agios, Astellas, Celgene, Daiichi Sankyo, Novartis, Takeda, Amphivena, Kite, Forty Seven,Trovagene, NewLink genetics, Jazz, Abbvie, and PTC Therapeutics: Consultancy; Amphivena, Kite, Jazz, NewLink Genetics,: Honoraria. Schuh:Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Teva Canada Innovation: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kantarjian:Agios: Honoraria, Research Funding; Immunogen: Research Funding; Pfizer: Honoraria, Research Funding; Jazz Pharma: Research Funding; Daiichi-Sankyo: Research Funding; Cyclacel: Research Funding; BMS: Research Funding; Novartis: Research Funding; Amgen: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Astex: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Ariad: Research Funding. Zhang:Agios: Employment, Equity Ownership; Agios: Employment, Equity Ownership. Winkler:Agios: Employment. Vyas:Celgene: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Pfizer: Speakers Bureau; Daiichi Sankyo: Speakers Bureau; Forty Seven, Inc.: Research Funding; Abbvie: Speakers Bureau; Astellas: Speakers Bureau. Wu:Agios: Employment, Equity Ownership. OffLabel Disclosure: Ivosidenib is an IDH1 inhibitor indicated for the treatment of AML with a susceptible IDH1 mutation as detected by an FDA-approved test in: 1) adult patients with newly-diagnosed AML who are more than 75 years old or who have comorbidities that preclude use of intensive induction chemotherapy; 2) Adult patients with relapsed or refractory AML. It is being investigated in clinical trials in combination with azacitidine in patients with IDH1-mutant newly diagnosed AML.

Introduction: Risk stratification in acute myeloid leukemia (AML) is continually being refined as we learn more about the molecular pathophysiology of this heterogeneous disease. European LeukemiaNet (ELN) 2017 used widely to stratify the 'genetic' risk of AML, but there are considerable challenges in its application, particularly in centres where molecular genetic testing either not available, or not sufficiently timely for clinical decision making. Up to a third of the study subjects cannot be stratified using a full cytogenetic-molecular model in real-time, real-life setting. There are few attempts to combine clinical features and genetic factors aiming to find a scoring system to improve AML survival prediction. There is only one to our knowledge (Sorror ML et al. 2017). This study has generated an Adapted Genetic Risk (AGR) assessment, and used it in combination with clinical risk parameters to create a novel scoring system which has now been validated using two independent cohorts. Methods: A training cohort from São Paulo (FMUSP, n = 167) of intensively treated AML patients (18-65 years) was assessed using ELN2017 genetic criteria. A comparative validation with our AGR which permits missing cytogenetic or molecular data (Figure 1) split these patients into favorable-risk (FR), intermediate-risk (IR), and adverse-risk (AR). This cohort was also used for Cox Proportional-Hazard Model (CPHM) univariate and multivariate analysis to find clinical parameters that would inform a novel Survival AML Score (SAMLS). Variables which are included in SAMLS had to be either significant in both CPHM models or significant in univariate and crucial for multivariate fitness as measured for the Akaike Information Criterion (AIC). We then applied the AGR strategy and SAMLS to 2 independent test cohorts of intensively treated adult AML patients : Riberao Preto (FMRP, n=145) and Oxford (OUH, n=157). The study was approved by the institutional review boards of the 3 participating centers. Informed consent was obtained from all patients according to the Declaration of Helsinki. Results: Table 1 shows the clinical characteristics for all the 3 cohorts. The median follow-up (FUP) was 72.3, 44.4, and 70.5 months for FMUSP, FMRP, and OUH, respectively. The median Overall Survival (OS) was 12.4, 12.5, and 56.4 months and the 5-year OS were 29.6%, 29.7%, and 49.7% respectively. Both ELN2017 and AGR correlated with significant differences in OS (p-value Multivariate CPHM model for OS in FMUSP which yielded the smallest AIC were: Age, HR 1.52; Albumin, HR 0.50; WBC, HR 1.37; Haemoglobin, HR 1.48; Platelet count, HR 0.64; and AGR-IR, HR 2.23 and AGR-AR HR, 4.66 (Figure 3). Variables which met SAMLS inclusion criteria are in Table 3. SAMLS stratifies AML as low-risk (LR-AML) (SAMS =2) (Table 3 and Figure 4 C-D), where there is a significant difference in survival in the FMUSP training cohort (p Conclusion: ELN2017 genetic risk score for AML was validated in a cohort from a low-medium income country (LMIC). AGR is a proposed risk evaluation that can be used for AML patients for whom fully genetic testing is not feasible (required for ELN2017) and was as accurate as ELN2017. AGR predicts survival in 3 independent cohorts, from LMIC and developed nation settings. When other biological and clinical factors are added to AGR, we were able to show SAMLS is better to predict overall survival than AGR. Importantly, serum Albumin, a simple and routine test parameter, was an independent predictor of OS for AML in all 3 cohorts. Therefore the use of SAMLS can predict early and late mortality and can impact on treatment decisions. Disclosures Quek: Celgene: Research Funding, Speakers Bureau; Agios: Research Funding. Vyas:Astellas: Speakers Bureau; Pfizer: Speakers Bureau; Abbvie: Speakers Bureau; Daiichi Sankyo: Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Forty Seven, Inc.: Research Funding; Celgene: Research Funding, Speakers Bureau.

Introduction: Somatic mutations in the metabolic enzymes isocitrate dehydrogenase (IDH) 1 and 2 are found in multiple solid and hematologic tumors and drive tumorigenesis via production of the oncometabolite D-2-hydroxyglutarate (2-HG). The mutant IDH1 (mIDH1) inhibitor ivosidenib is approved in the US for the treatment of adults with mIDH1 newly diagnosed acute myeloid leukemia (AML) in patients ≥75 years old or with comorbidities that preclude induction chemotherapy, and mIDH1 relapsed or refractory (R/R) AML. In patients with R/R AML who were enrolled in a phase 1 study (NCT02074839), ivosidenib was associated with an overall response rate of 42%, a complete remission (CR) rate of 24%, and a CR plus CR with partial hematologic response (CR+CRh) rate of 32%, with durable responses and additional clinical benefits observed (Pollyea et al. J Clin Oncol 2018 Abs). Baseline co-occurring mutations in receptor tyrosine kinase (RTK) pathway genes were shown to be associated with primary resistance to ivosidenib (DiNardo et al. N Engl J Med 2018). Case studies of secondary resistance to ivosidenib monotherapy have revealed examples of 2-HG restoring mechanisms at relapse, including IDH2 isoform switching and the emergence of mIDH1-S280F (Harding et al. Cancer Discov 2018; Intlekofer et al. Nature 2018). Aim: To characterize the mechanisms of response and relapse to ivosidenib monotherapy via a comprehensive genomic analysis of samples from a large cohort of patients with mIDH1 R/R AML enrolled in the pivotal phase 1 study whose starting dose was 500 mg daily (approved dose). Methods: Baseline and longitudinal co-occurring mutation profiling was performed on whole bone marrow, bone marrow mononuclear cell, and peripheral blood mononuclear cell samples by next generation sequencing (NGS; detection sensitivity 1-5%). 3D modelling of second-site IDH1 mutations was performed with an ivosidenib analog using Molecular Operating Environment 10.0 software. Enzymatic assays were performed with mIDH1 as previously reported. The clinical response data cut-off was Nov 10, 2018. Results: In 167 patients with baseline NGS data from whole bone marrow, the genes with most frequent co-occurring mutations were DNMT3A (35%), NPM1 (26%), SRSF2 (24%), ASXL1 (18%), RUNX1 (18%), NRAS (14%), and TP53 (13%). RTK pathway mutations, and mutations in the individual genes NRAS and PTPN11, were significantly associated with a lack of CR or CRh response to ivosidenib (p Conclusions: Relapse to ivosidenib monotherapy is mediated by diverse mechanisms that include emergent mutations, most frequently in RTK pathway genes and IDH1/IDH2. The observation of frequent 2-HG restoration at relapse underscores the key role of 2-HG production in mIDH AML disease. These results are important for the rational design of combination treatment strategies in mIDH1 AML, and to understand whether these patterns are replicated in ongoing combination trials. Disclosures Choe: Agios: Employment, Equity Ownership. Wang:Agios: Employment, Equity Ownership. DiNardo:syros: Honoraria; abbvie: Consultancy, Honoraria; medimmune: Honoraria; celgene: Consultancy, Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees; agios: Consultancy, Honoraria; jazz: Honoraria; daiichi sankyo: Honoraria. Stein: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; 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; Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees. De Botton:Agios: Consultancy, Research Funding; Celgene: Consultancy, Speakers Bureau; Pierre Fabre: Consultancy; Servier: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Janssen: Consultancy; Forma: Consultancy, Research Funding; Syros: Consultancy; Bayer: Consultancy; AbbVie: Consultancy; Daiichi Sankyo: Consultancy; Astellas: Consultancy. Fathi:Amphivena, Kite, Jazz, NewLink Genetics,: Honoraria; Agios, Astellas, Celgene, Daiichi Sankyo, Novartis, Takeda, Amphivena, Kite, Forty Seven,Trovagene, NewLink genetics, Jazz, Abbvie, and PTC Therapeutics: Consultancy. Tallman:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Research Funding; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; UpToDate: Patents & Royalties; 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; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; UpToDate: Patents & Royalties; Oncolyze: 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; Biosight: Research Funding; Jazz Pharmaceuticals: 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; Cellerant: Research Funding; Cellerant: Research Funding; Cellerant: Research Funding; 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; 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; 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; 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; Nohla: 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; BioLineRx: 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; ADC Therapeutics: Research Funding; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Biosight: Research Funding; Biosight: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellerant: Research Funding; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; UpToDate: Patents & Royalties; Rigel: 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; 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; 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; Biosight: Research Funding; Cellerant: Research Funding; Delta Fly Pharma: 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; Daiichi-Sankyo: 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; ADC Therapeutics: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; 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; 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; Jazz Pharmaceuticals: 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; 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; Tetraphase: 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; 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; ADC Therapeutics: Research Funding; UpToDate: Patents & Royalties; 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; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: 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; UpToDate: Patents & Royalties; Jazz Pharmaceuticals: 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; Tetraphase: 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; 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; Jazz Pharmaceuticals: 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; UpToDate: Patents & Royalties. Kantarjian:Daiichi-Sankyo: Research Funding; Novartis: Research Funding; Astex: Research Funding; Cyclacel: Research Funding; Jazz Pharma: Research Funding; Takeda: Honoraria; Immunogen: Research Funding; Ariad: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Research Funding; BMS: Research Funding; Amgen: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Agios: Honoraria, Research Funding. Stone:AbbVie, Actinium, Agios, Argenx, Arog, Astellas, AstraZeneca, Biolinerx, Celgene, Cornerstone Biopharma, Fujifilm, Jazz Pharmaceuticals, Amgen, Ono, Orsenix, Otsuka, Merck, Novartis, Pfizer, Sumitomo, Trovagene: Consultancy; Novartis, Agios, Arog: Research Funding; Argenx, Celgene, Takeda Oncology: Other: Data and Safety Monitoring Board/Committee: . Quek:Celgene: Research Funding, Speakers Bureau; Agios: Research Funding. Konteatis:Agios: Employment, Equity Ownership. Dang:Agios: Employment, Equity Ownership. Zhang:Agios: Employment, Equity Ownership; Agios: Employment, Equity Ownership. Liu:Agios: Employment, Equity Ownership. Attar:Aprea Therapeutics: Employment; Agios: Employment, Equity Ownership. Wu:Agios: Employment, Equity Ownership.

Single sitting procedures where the mononuclear cell fraction is extracted from bone marrow and implanted directly into cartilage and bone defects are becoming more popular as novel treatments for cartilage defects which have, until now had few treatment options. This is on the basis that the mesenchymal stem cells (MSCs) contained within will repair the damaged tissue. This study sought to determine if the femur and tibia could provide equivalent amounts of mesenchymal stem cells, with equivalent viability and proliferative capacity, to that obtained from the gold standard of the pelvis in order to potentially reduce the morbidity associated with these procedures. Bone marrow was extracted from the pelvis, femur, and tibia of human subjects. The mononuclear cell fraction was extracted and cultured in the laboratory. Mesenchymal stem cell populations were assessed using a colony forming unit count. Viability was assessed using a PrestoBlue viability assay. Population doubling number was calculated between the end of passage 0 and passage three to determine the proliferative abilities of the different populations. Finally, the cell surface phenotype of the cells was determined by flow cytometry. The results showed that the pelvis was superior to the femur and tibia in terms of the number of stem cells isolated. There was no statistically significant difference in the phenotype of the cells isolated from different locations. This work shows that when undertaking single sitting procedures, the pelvis remains the optimum source for obtaining MSCs, despite the morbidity associated with bone marrow collection from the pelvis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1868-1875, 2017.

To assess factors influencing the success of whole-genome sequencing for mainstream clinical diagnosis, we sequenced 217 individuals from 156 independent cases or families across a broad spectrum of disorders in whom previous screening had identified no pathogenic variants. We quantified the number of candidate variants identified using different strategies for variant calling, filtering, annotation and prioritization. We found that jointly calling variants across samples, filtering against both local and external databases, deploying multiple annotation tools and using familial transmission above biological plausibility contributed to accuracy. Overall, we identified disease-causing variants in 21% of cases, with the proportion increasing to 34% (23/68) for mendelian disorders and 57% (8/14) in family trios. We also discovered 32 potentially clinically actionable variants in 18 genes unrelated to the referral disorder, although only 4 were ultimately considered reportable. Our results demonstrate the value of genome sequencing for routine clinical diagnosis but also highlight many outstanding challenges.

Myelodysplastic syndromes (MDS) are a heterogeneous group of chronic hematological malignancies characterized by dysplasia, ineffective hematopoiesis and a variable risk of progression to acute myeloid leukemia. Sequencing of MDS genomes has identified mutations in genes implicated in RNA splicing, DNA modification, chromatin regulation, and cell signaling. We sequenced 111 genes across 738 patients with MDS or closely related neoplasms (including chronic myelomonocytic leukemia and MDS-myeloproliferative neoplasms) to explore the role of acquired mutations in MDS biology and clinical phenotype. Seventy-eight percent of patients had 1 or more oncogenic mutations. We identify complex patterns of pairwise association between genes, indicative of epistatic interactions involving components of the spliceosome machinery and epigenetic modifiers. Coupled with inferences on subclonal mutations, these data suggest a hypothesis of genetic "predestination," in which early driver mutations, typically affecting genes involved in RNA splicing, dictate future trajectories of disease evolution with distinct clinical phenotypes. Driver mutations had equivalent prognostic significance, whether clonal or subclonal, and leukemia-free survival deteriorated steadily as numbers of driver mutations increased. Thus, analysis of oncogenic mutations in large, well-characterized cohorts of patients illustrates the interconnections between the cancer genome and disease biology, with considerable potential for clinical application.

Quek and colleagues identify human leukemic stem cells (LSCs) present in CD34− AML. In-depth characterization of the functional and clonal aspects of CD34− LSCs indicates that most are similar to myeloid precursors., Our understanding of the perturbation of normal cellular differentiation hierarchies to create tumor-propagating stem cell populations is incomplete. In human acute myeloid leukemia (AML), current models suggest transformation creates leukemic stem cell (LSC) populations arrested at a progenitor-like stage expressing cell surface CD34. We show that in ∼25% of AML, with a distinct genetic mutation pattern where >98% of cells are CD34−, there are multiple, nonhierarchically arranged CD34+ and CD34− LSC populations. Within CD34− and CD34+ LSC–containing populations, LSC frequencies are similar; there are shared clonal structures and near-identical transcriptional signatures. CD34− LSCs have disordered global transcription profiles, but these profiles are enriched for transcriptional signatures of normal CD34− mature granulocyte–macrophage precursors, downstream of progenitors. But unlike mature precursors, LSCs express multiple normal stem cell transcriptional regulators previously implicated in LSC function. This suggests a new refined model of the relationship between LSCs and normal hemopoiesis in which the nature of genetic/epigenetic changes determines the disordered transcriptional program, resulting in LSC differentiation arrest at stages that are most like either progenitor or precursor stages of hemopoiesis.

Introduction Therapy-related myeloid neoplasms (tMN) are an increasing healthcare problem resulting from rising long-term survival from primary cancers. Approximately 80% of cases are associated with an adverse karyotype and have a dismal prognosis; pre-existing clonal haematopoiesis (CH) appears to be a predisposing factor and mutations in genes including TP53, PPM1D and DNMT3A have been detected prior to chemo- or radiotherapy exposure. In contrast ~20% of tMN are characterised by balanced chromosome rearrangements and have a relatively favourable outcome; chemotherapy agents targeting topoisomerase II have been implicated in generating these rearrangements however the involvement of CH has not previously been described. Methods and results We performed whole exome sequencing (WES) of samples taken at diagnosis (D) and molecular complete remission (mCR) followed by targeted capture and error-corrected deep sequencing (ECS) on ≥2 mCR samples for 11 patients with therapy-related acute promyelocytic leukemia (tAPL) and 20 patients with de novo APL (dnAPL). All patients had t(15;17) / PML-RARA at diagnosis with no additional cytogenetic abnormality. The median age of patients with tAPL was 46.7y (range 30-78); primary cancer types were breast (4), colorectal (2), lymphoma (3), CNS (1) and testicular (1). Ten patients had received chemotherapy and 4 radiotherapy. The median latency between primary cancer treatment and tAPL diagnosis was 3.9y (range 2.2-6.1). Patients received a mixture of chemotherapy- and arsenic-based treatments. The median age of patients with dnAPL was 40.3y (range 18-69); all patients were treated with the AIDA regimen and none developed tMN subsequently. There were no significant differences between the number or type of mutations between dnAPL and tAPL however disease associated somatic mutations were detectable in mCR samples by ECS in 4/11 tAPL samples compared to 0/20 dnAPL samples (p=0.04). Mutations detected in mCR were UPN1: PPM1D exon (e) 6 1bp deletion (del, variant allele fraction, VAF, D 32% mCR 11.3%); UPN2: DNMT3A e8 1bp del (VAF D 40.2% mCR 0.48%); UPN3: DNMT3A e10 1bp del (VAF D 35.4% mCR 2.9%); UPN4: MYCN e3 6bp insertion (ins, VAF D 38% mCR 0.37%). We screened samples from these patients and a further 39 dnAPL patients for a panel of genes with known CH associated mutations (CH-M) using ECS and detected additional mutations in 2 tAPL patients (UPN4, DNMT3A G104R, VAF D 0% mCR 3% and UPN5 DNMT3A R693H VAF D 2% mCR 25%, who subsequently developed tMN with complex karyotype). We did not detect CH-M in diagnostic samples from any patient with dnAPL and publicly available NGS datasets encompassing 220 patients only showed 1 APL case with a CH-M (DNMT3A e8 ins, prior cytotoxic exposure unknown). We detected treatment-emergent CH clones in mCR by ECS in 6/59 dnAPL patients treated with AIDA (DNMT3A n=3, PPM1D n=2, TP53 n=1, SF3B1 n=1). Applying the same techniques to six patients with therapy-related core-binding factor AML, we identified a persistent CH-M in mCR in one (DNMT3A e15 del, VAF D 38%, mCR 11.8%). DNA samples taken at the time of primary cancer diagnosis were available from UPN 1-3. In UPN1 we detected the PPM1D mutation in a lymph node (LN) involved with T-cell lymphoma (LN) (VAF 2.1%) and an uninvolved staging bone marrow (VAF 3.3%). In UPN3 the DNMT3A mutation was detected in a breast biopsy (VAF 0.7%) and LN involved with carcinoma (VAF 0.9%). In UPN2 the DNMT3A mutation was not detected in the LN biopsy diagnostic for Hodgkin lymphoma. We used FACS to isolate T, B, monocyte, granulocyte and CD34+ cells from complete remission samples from UPN1-4 with >99% purity and detected the persistent mutations in each cell compartment e.g. UPN3 VAF: T cell 1.3%, B cell 8.6%, monocyte 4%, granulocyte 3.7%, CD34+ 3.9%. Diagnostic material from UPN1 and 4 was injected into irradiated NSG mice. After 12 weeks we detected multilineage human engraftment in both samples by FACS in 1/3 mice from UPN1 and 2/3 mice from UPN4. We detected the PPM1D and MYCN mutations respectively in bone marrow samples from each engrafting mouse. Conclusions Together these findings indicate that tMN with balanced chromosome rearrangements can occur on a background of non-malignant CH. Using ECS we observed this phenomenon in 5/17 (29%) patients with therapy-related APL or CBF AML. This has important implications for planning curative therapy, notably for tAPL where effective cytotoxic-free regimens are available. Disclosures Russell: Pfizer: Consultancy, Honoraria, Speakers Bureau; Jazz Pharma: Speakers Bureau; Daiichi Sankyo: Consultancy. Hills:Daiichi Sankyo: Consultancy, Honoraria.

Background Mutations in Isocitrate Dehydrogenase 2 (IDH2) occur in many cancers including Acute Myeloid Leukemia (AML). In preclinical models mutant IDH2 (mIDH2) causes partial hemopoietic differentiation block1. Recently, we showed that single agent enasidenib, a first-in-class, selective mIDH2 inhibitor, produces a 40% response in relapsed/refractory AML patients by promoting differentiation2. Here, we studied response and acquired resistance to enasidenib, in sequential samples treated in the Phase 1 study of Enasidenib in relapsed/ refractory AML patients. Results We studied a cytogenetically and genetically representative subset of 25 patients enriched for enasidenib responders, genotyped by whole exome sequencing (WES) or cancer gene panel targeted re-sequencing. Pre-enasidenib, differentiation arrest in these AML patients resulted in abnormally expanded leukaemic progenitors or precursors and diminished mature haematopoietic populations. Complete remission (CR) post-enasidenib was associated with in increased mature populations, near-normalisation of haematopoietic progenitor profiles, and restoration of in vitro progenitor function. In most patients, mature blood cells (of erythroid and granulocyte-monocyte lineages) post-enasidenib are IDH2 mutant, consistent with enasidenib inducing differentiation of IDH2 mutant leukaemic progenitors/ precursors. Each mIDH2 patient studied had on average 13 somatic, non-synonymous exonic or splice site mutations in addition to IDH2 . We used single cell genotyping (SCG) to reveal linear or branching clonal structures in mIDH2 AML. We combined clonal structure data and immunophenotyping of haematopoietic progenitor, precursor and mature populations to track functional behaviour of mIDH2 clones before, and during enasidenib treatment. We demonstrate, for the first time, that mIDH2 subclones within the same patient are functionally heterogeneous: both in their ability to differentiate pre-enasidenib, and in their sensitivity to Enasidenib-induced differentiation. This suggests that different combinations of co-operating mutations result in functional heterogeneity of mIDH2 clones. When we studied the contribution of mIDH2 clones to functional haematopoiesis at CR, we found that this was supported by either ancestral or leukaemic terminal mIDH2 clones. Despite a median survival of 18-21 months in patients who respond to enasidenib, most patients eventually relapse3. In contrast to targeted therapies such as tyrosine kinase inhibitors, in all 12 relapse samples studied, none had second site mutations in IDH2 . Furthermore, 2-hydroxyglurate (2HG) levels remain suppressed in most patients suggesting enasidenib remains effective in inhibiting mIDH2 enzyme. Instead, mIDH2 clones, which had persisted at CR or partial remission (PR) acquired additional mutations or aneuploidy, highlighting bypass pathways which re-impose differentiation arrest. We found 4 patterns: i) acquisition of IDH1 codon R132 mutations which resulted in a rise in 2HG (n=2), ii) deletion of chromosome 7q (n=4), iii) gain of function mutations in genes implicated in cell proliferation (FLT3, CSF3R) (n=3) and iv) mutation in hematopoietic transcription factors (GATA2, RUNX1) (n=2). We also found mutations in 4 genes (DHX15 and DEAF1 (n=1) ; NFKB1 (n=1) and MTUS1 (n=1)) not previously implicated in haematopoietic differentiation arrest which were selected for, or evolved in mIDH2 subclones at relapse. Conclusion This study provides a paradigm of how deep clonal single cell analysis in purified hemopoietic compartments in sequential samples through therapy reveals clonal complexity and the impact of the selective pressure of therapy on clonal architecture. Furthermore, we gain insights into the functional heterogeneity of mIDH2 subclones in their ability to differentiate pre-and post-Enasidenib. Further analysis of this kind in a larger cohort of IDH2 -inhibitor-treated patients would also provide insight to improve efficacy of this novel class of therapeutics, and design of combination therapies in AML and other cancers. Finally, this provides a platform for further study of the pathways mediating enasidenib resistance. References 1. Kats, L.M. , et al. Cell Stem Cell14, 329-341 (2014). 2. Amatangelo, M.D. , et al. Blood (2017). 3. Stein, E.M. , et al. Blood (2017). Disclosures Quek: Celgene Corporation: Research Funding. Amatangelo: Celgene Corporation: Employment. Agresta: Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Yen: Agios: Employment, Equity Ownership. Stein: Pfizer: Consultancy, Other: Travel expenses; Agios Pharmaceuticals, Inc.: Consultancy, Research Funding; Constellation Pharma: Research Funding; Novartis: Consultancy, Research Funding; GSK: Other: Advisory Board, Research Funding; Celgene Corporation: Consultancy, Other: Travel expenses, Research Funding; Seattle Genetics: Research Funding. De Botton: Agios: Honoraria, Research Funding; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Servier: Honoraria. Thakurta: Celgene Corporation: Employment, Equity Ownership. Levine: Qiagen: Equity Ownership; Qiagen: Equity Ownership; Celgene: Research Funding; Roche: Research Funding; Celgene: Research Funding; Roche: Research Funding. Vyas: Jazz Pharmaceuticals: Speakers Bureau; Celgene Corporation: Speakers Bureau.

The beta-thalassaemias have a major global impact on health and mortality. Allogeneic haemopoietic stem cell transplantation is the only approach that may lead to a cure but this approach is not available to most patients. The mainstay treatment for the majority remains life-long blood transfusion in combination with a rigorous regime of iron chelation. Improved understanding of the pathophysiology and molecular basis of the disease has provided clues for more effective strategies that aim to correct the defect in beta-globin chain synthesis at the primary level or redress the alpha/beta-globin chain imbalance at the secondary level. Improved understanding of the molecular basis of the disease complications, such as iron overloading, has also provided clues for potential molecular targets at the tertiary level.