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Supplementary Figure 1. Synthesis and pharmacokinetic characterization of AG-221. Supplementary Figure 2. Biochemical attributes of AG-221. Supplementary Figure 3. Biochemistry of AG-221 with respect to substrate and cofactor. Supplementary Figure 4. TF-1 IDH2R140Q cells treated with AG-221. Supplementary Figure 5. AG-221 can reverse the block in EPO-induced differentiation caused by the expression of IDH2R140Q in the TF-1 erythroleukemia cell line. Supplementary Figure 6. Pharmacokinetics/pharmacodynamics of AG-221 in IDH2R140Qmutant U87MG xenograft tumor-bearing mice. Supplementary Figure 7. AG-221 does not affect intrinsic hematological parameters or body weight. Supplementary Figure 8. AG-221 strongly reduces the number of human IDH2R140Q blasts in the liver and spleen in AML-1, AML-2, and AML-3. Supplementary Figure 9. Flow cytometry analyses of bone marrow-derived hCD45+ cells in primary human AML xenograft models. Supplementary Figure 10. Affinity of the IDH2R140Q homodimer for NADPH. Supplementary Figure 11. Electron density map diagrams for bound ligands for IDH2R140Q co-complex structures determined by X-ray crystallography.

Supplementary Table 1. Drug metabolism and pharmacokinetic attributes of AG-221. Supplementary Table 2. Selectivity of AG-221 confirmed by testing against a panel of kinases. Supplementary Table 3. Clinical characteristics of patients with IDH2R140Q-mutated AML. Supplementary Table 4. Treated NSG mice (AML-1, AML-2, AML-3) engrafted with human IDH2R140Q mononuclear cells display stable levels of AG-221 in serum. Supplementary Table 5. AG-221 inhibits 2HG production in models AML-1, AML-2, and AML-3. Supplementary Table 6. Summary of pharmacokinetics/pharmacodynamics in primary human acute myeloid leukemia xenograft model (AML-4). Supplementary Table 7. Summary of data collection and refinement statistics. Supplementary Table 8. Percentage of human chimerism in peripheral blood in models AML-1 and AML-2.

Supplementary Methods

Background: Despite that cytogenetic and molecular analysis of tumor cells can rapidly identify recurring molecular abnormalities, no personalized therapy is currently available in the setting of relapsed/refractory multiple myeloma (r/r MM). Methods: MM-EP1 is a retrospective study aimed at comparing a personalized molecular-oriented (MO) versus a non-molecular-oriented (no-MO) approach in r/r MM. Actionable molecular targets and their associated therapies were the BRAF V600E mutation and BRAF inhibitors; t(11;14)(q13;q32) and BCL2 inhibitors; and t(4;14)(p16;q32) with FGFR3 fusion/rearrangements and FGFR3 inhibitors. Results: One hundred three highly pretreated r/r MM patients with a median age of 67 years (range 44–85) were included. Seventeen (17%) patients were treated using an MO approach with BRAF inhibitors (vemurafenib or dabrafenib, n = 6), BCL2 inhibitor (venetoclax, n = 9), or FGFR3 inhibitor (erdafitinib, n = 2). Eighty-six (86%) patients received non-MO therapies. Overall response rate was 65% in MO patients versus 58% in the non-MO group (p = 0.053). Median PFS and OS were 9 and 6 months (HR = 0.96; CI95 = 0.51–1.78; p = 0.88) and 26 and 28 months (HR = 0.98; CI95 = 0.46–2.12; p = 0.98), respectively, in MO and no-MO patients. Conclusion: Despite the low number of patients treated with an MO approach, this study highlights the strengths and weakness of a molecular-targeted approach for the treatment of multiple myeloma. Widespread biomolecular techniques and improvement of precision medicine treatment algorithms could improve selection for precision medicine in myeloma.

Advances in molecular profiling of newly diagnosed diffuse large cell B lymphoma (DLBCL) have recently refine genetic subgroups. Genetic subgroups remain undetermined at the time of relapse or refractory (RR) disease. This study aims to decipher genetic subgroups and search for prognostic molecular biomarkers in patients with RR-DLBCL. From 2015 to 2021, targeted next-generation sequencing analyses of germline-matched tumor samples and fresh tissue from RR-DLBCL patients were performed. Unsupervised clustering of somatic mutations was performed and correlations with patient outcome were sought. 120 patients with RR-DLBCL were included in LNH-EP1 study and a molecular tumor landscape was successfully analyzed in 87% of patients (104/120 tumor samples). The median age was 67.5 years (range 27.4-87.4), median number of previous treatments was 2 (range 1-9). The most frequently mutated genes were TP53 (n=53 mutations; 42% of samples), CREBBP (n=39; 32%), BCL2 (n=86; 31%), KMT2D (n=39; 28%) and PIM1 (n=54; 22%). Unsupervised clustering separated three genetic subgroups entitled BST (enriched in BCL2, SOCS1 and TNFRSF14 mutations); TKS (enriched in TP53, KMT2D and STAT6 mutations); and PCM (enriched in PIM1, CD79B and MYD88 mutations). Median overall survival was 11.0 (95%CI: 8.1-12.6) months. Overall survival was not significantly different between the three genetic subgroups. GNA13 mutant was significantly associated with an increased risk of death (HR: 6.6 [95%CI: 2.1-20.6]; p=0.0011) and shorter overall survival (p=0.0340). At the time of relapse or refractory disease, three genetic subgroups of DLBCL patients were delineated, which could help advance precision molecular medicine programs. This article is protected by copyright. All rights reserved.

Isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) mutations in Myeloid Neoplams (MNs) exhibit DNA hypermethylation via 2-hydroxyglutarate (2HG) over-production. Clinical impact of azacitidine (AZA) re...

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.

Somatic point mutations at a key arginine residue (R132) within the active site of the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) confer a novel gain of function in cancer cells, resulting in the production of d-2-hydroxyglutarate (2-HG), an oncometabolite. Elevated 2-HG levels are implicated in epigenetic alterations and impaired cellular differentiation. IDH1 mutations have been described in an array of hematologic malignancies and solid tumors. Here, we report the discovery of AG-120 (ivosidenib), an inhibitor of the IDH1 mutant enzyme that exhibits profound 2-HG lowering in tumor models and the ability to effect differentiation of primary patient AML samples ex vivo. Preliminary data from phase 1 clinical trials enrolling patients with cancers harboring an IDH1 mutation indicate that AG-120 has an acceptable safety profile and clinical activity.

International audience; The TET2 gene encodes an α-ketoglutarate-dependent dioxygenase able to oxidize 5-methylcytosine into 5-hydroxymethylcytosine, which is a step toward active DNA demethylation. TET2 is frequently mutated in myeloid malignancies but also in B- and T-cell malignancies. TET2 somatic mutations are also identified in healthy elderly individuals with clonal hematopoiesis. Tet2-deficient mouse models showed widespread hematological differentiation abnormalities, including myeloid, T-cell, and B-cell malignancies. We show here that, similar to what is observed with constitutive Tet2-deficient mice, B-cell-specific Tet2 knockout leads to abnormalities in the B1-cell subset and a development of B-cell malignancies after long latency. Aging Tet2-deficient mice accumulate clonal CD19+ B220low immunoglobulin M+ B-cell populations with transplantable ability showing similarities to human chronic lymphocytic leukemia, including CD5 expression and sensitivity to ibrutinib-mediated B-cell receptor (BCR) signaling inhibition. Exome sequencing of Tet2-/- malignant B cells reveals C-to-T and G-to-A mutations that lie within single-stranded DNA-specific activation-induced deaminase (AID)/APOBEC (apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like) cytidine deaminases targeted motif, as confirmed by the lack of a B-cell tumor in compound Tet2-Aicda-deficient mice. Finally, we show that Tet2 deficiency accelerates and exacerbates T-cell leukemia/lymphoma 1A-induced leukemogenesis. Together, our data establish that Tet2 deficiency predisposes to mature B-cell malignancies, which development might be attributed in part to AID-mediated accumulating mutations and BCR-mediated signaling.

Background: Despite recent advances in the treatment of multiple myeloma (MM) patients, cure remains rare. MM is in an era of intense clinical research and the molecular abnormalities landscape of refractory and relapsed (R/R) patients is of major interest for drug development. Furthermore, innovative molecular-oriented treatments for these R/R patients could be oriented by mutational characterization of myeloma plasma cells (PC). At relapse but also for clonal minimal residual disease monitoring on therapy, bone marrow aspiration is an invasive procedure that can give limited information since PC infiltration is heterogeneous and can be modest. Mutational profiling on circulating cell-free tumoral DNA (ctDNA) could be a simple and appropriate alternative. Method: We compared molecular landscape in myeloma PC versus in ctDNA in a cohort of 45 R/R MM patients screened at Gustave Roussy for a salvage therapy, most of them for a Phase I trial in the DITEP department, with a median age at time of molecular analysis of 69 years. All patients had received ≥ 1 prior lines of myeloma therapy (median: 2, range: 1-8), 33/45 (73%) had previously undergone autologous stem cell transplant and 14/45 (31%) had received prior daratumumab; 6/45 (13%) were double-refractory (to at least a proteasome inhibitor (PI) and an immunomodulatory imide drug (IMiD), 4/45 (9%) were triple-refractory and 1/45 (2%) was penta-refractory. Four patients (9%) had prior daratumumab and were also double-refractory. Paired samples, as well as normal sorted CD3 + T cells were sequenced using an Ion Torrent custom panel covering 30 myeloma-related genes previously reported as the most frequent mutated ones. The human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents under an IRB/EC approved protocol. Results: One hundred and two variants were found in magnetic-sorted CD138 + myeloma PC, and 99 variants were detected in ctDNA; more than half of the variants detected in myeloma PC were also found in ctDNA (55/102, 54%). Variant allelic frequencies (VAF) in PC and in ctDNA were significantly correlated (p10% of R/R MM). Considering these five key driver genes, the kappa coefficient of concordance per gene was medium/good between both samples, as defined by the Landis-Koch scale. The mean and median sensitivity of ctDNA detection per gene was 55% and 58% respectively (range: 38-67), and specificity 94% and 97% (range: 80-100); positive predictive value of TP53 mutations detection was poor as theses mutations were more frequently detected in ctDNA (12/45, 27%) than in myeloma PC (6/45, 13%). At the patient level, the similarity between myeloma PC and ctDNA (level defined as the ratio between SNV number in PC and SNV number in ctDNA) was greater than a threshold of 80% in 20/39 (51%) cases (median level: 100%). Importantly, key driver gene mutations were reported in ctDNA for 13/28 (48%) patients without cytological evidence of infiltrated plasmocytosis (less than 10% PC) in the bone marrow aspiration. Conclusions: ctDNA profiling may complete molecular description of R/R MM patients thanks to a less-invasive procedure, allowing to fully characterize mutational profile prior to molecular-oriented treatment decision. ctDNA can give information on the clonal architecture in patients without bone marrow infiltration even after CD138 + cells magnetic-sorted isolation. Disclosures Michot: BMS: Consultancy, Honoraria; GSK: Consultancy, Honoraria. Lazarovici: Mundipharma: Other: Travel grant. Ribrag: Infinity Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Nanostring: Membership on an entity's Board of Directors or advisory committees; Astex Pharmaceuticals: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Argen-X: Research Funding; GSK: Research Funding; Epizyme: Honoraria, Research Funding; MSD Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; PharmaMar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

Somatic gain-of-function mutations in isocitrate dehydrogenases (IDH) 1 and 2 are found in multiple hematologic and solid tumors, leading to accumulation of the oncometabolite (R)-2-hydroxyglutarate (2HG). 2HG competitively inhibits α-ketoglutarate–dependent dioxygenases, including histone demethylases and methylcytosine dioxygenases of the TET family, causing epigenetic dysregulation and a block in cellular differentiation. In vitro studies have provided proof of concept for mutant IDH inhibition as a therapeutic approach. We report the discovery and characterization of AG-221, an orally available, selective, potent inhibitor of the mutant IDH2 enzyme. AG-221 suppressed 2HG production and induced cellular differentiation in primary human IDH2 mutation–positive acute myeloid leukemia (AML) cells ex vivo and in xenograft mouse models. AG-221 also provided a statistically significant survival benefit in an aggressive IDH2R140Q-mutant AML xenograft mouse model. These findings supported initiation of the ongoing clinical trials of AG-221 in patients with IDH2 mutation–positive advanced hematologic malignancies. Significance: Mutations in IDH1/2 are identified in approximately 20% of patients with AML and contribute to leukemia via a block in hematopoietic cell differentiation. We have shown that the targeted inhibitor AG-221 suppresses the mutant IDH2 enzyme in multiple preclinical models and induces differentiation of malignant blasts, supporting its clinical development. Cancer Discov; 7(5); 478–93. ©2017 AACR. See related commentary by Thomas and Majeti, p. 459. See related article by Shih et al., p. 494. This article is highlighted in the In This Issue feature, p. 443

Background. Diffuse large B cell lymphoma (DLBCL) is the most common adult non-Hodgkin lymphoma. Despite therapeutic advances, 40% of the patients (pts) and especially pts older than 65 years old (y/o), will experience a relapsed or refractory (R/R) disease. Novel therapies as well as predictive biomarkers are required for these patients with a poor prognosis after standard salvage regimens. We aimed at establishing whether tumor molecular characterization by a customized target panel in pts with R/R DLBLC prior to enrollment in a phase I clinical trials might impact therapeutic decision making and outcome prediction. Methods. Paired targeted next generation sequencing for all consecutive pts with R/R DLBCL from germline sample and fresh tumor biopsy were performed at the time of enrollment in phase I clinical trials within the early phase clinical department. Tumor and germline samples of enrolled patients were sequenced using Ion Torrent technology on an in-house customized panel of 44 genes. Molecular and cluster mapping of recurrent molecular abnormalities was determined by integrated completed likelihood (ICL) in patients with relapsed diffuse large B-cell lymphoma. Predictive and prognostic impact of molecular profile was assessed on overall survival (OS). Results. Between 2013 and 2020, 89 pts with R/R DLBCL were included in the study. At time of inclusion, mean age was 62 (range 23-83) y/o, 50 pts (56%) were male, median prior lines of systemic treatment was 2 (range 1-9). Age adjusted international prognostic index (aaIPI) score was 0-1 in 42 pts (47%) and 2-3 in 47 pts (53%). Among the 82 cases with a cell of origin (COO) status assessed by immunohistochemistry, 55 (67%) pts had a germinal center (GC) and 27 pts (33%) a non-GC. The sequencing panel was informative in 86 pts (97%) and 3 pts (3%) had no variants identified. The most recurrently altered genes by mutations (m) were TP53m (n=38; 43%), CREBBPm (n=29; 33%), KMT2Dm (n=24; 27%), PIM1m (n=23; 26%) and BCL2m (n=20; 22%). Mutual exclusivity and co-occurrence analysis underlined that KMT2Dm were fully exclusive from MEF2Bm (p Conclusion. A molecular tumor characterization of patients with R/R DLBCL emphasizes high incidence of TP53 and epigenetic modifying oncogenes CREBBP and KMT2D mutations. Four distinct genetic clusters were identified that could serve as the basis for a molecular-matched therapeutic approach. Disclosures Michot: Medimmune: Research Funding; Lytix Biopharma: Research Funding; Lysarc: Research Funding; Janssen: Other, Research Funding; Celgene: Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Agios: Research Funding; AZD: Honoraria, Membership on an entity's Board of Directors or advisory committees; Xencor: Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Other; Sanofi: Research Funding; Roche: Research Funding; Argen-x: Research Funding; Abbvie: Research Funding; Gustave Roussy: Honoraria, Other: Abbvie, Aduro, Agios, Amgen, Argen-x, Astex, AstraZeneca, Aveo pharmaceuticals, Bayer, Beigene, Blueprint, BMS, Boeringer Ingelheim, Celgene, Chugai, Clovis, Daiichi Sankyo, Debiopharm, Eisai, Eos, Exelixis, Forma, Gamamabs, Genentech, Gortec, GSK, H3 bio, Research Funding; Astex: Membership on an entity's Board of Directors or advisory committees, Research Funding; Exelixis: Research Funding; Eos: Research Funding; Eisai: Research Funding; Lilly: Research Funding; Mundi Pharma: Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Amgen: Research Funding; AstraZeneca: Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; Debiopharm: Research Funding; Daiichi Sankyo: Research Funding; Forma: Research Funding; Genentech: Research Funding; Kyowa: Research Funding. Varga:Astra Zeneca: Current Employment. Ribrag:Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; arGEN-X-BVBA: Research Funding; BAY1000394 studies on MCL: Patents & Royalties; Nanostring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; Immune Design: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; Eisai: Honoraria; AZD: Honoraria, Other; Institut Gustave Roussy: Current Employment; Pharmamar: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; argenX: Current equity holder in publicly-traded company, Research Funding; Epizyme: Consultancy, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Infinity: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Background: Early drug development is associated with a high rate of failure in patients with relapsed or refractory diffuse large B-cell lymphoma (R/R DLBLC), partly due to genomic heterogeneity. Precision medicine aims at identifying patients with cancer who are likely to respond to targeted therapies. Here we assessed the clinical impact of characterizing actionable and molecular targets and matching targeted therapy in pts with R/R DLBCL prior enrollment in early phase clinical trials (epCT). Methods: Pts with R/R DLBCL enrolled in epCT, provided paired samples including germline and fresh tumor tissue sample. Paired samples were analyzed by targeted next generation sequencing (NGS) using a customized panel of 44 genes (Ion Torrent). Clinical actionability of genomic alterations and orientation to targeted therapy were prospectively assessed at the Hematological Tumor Board from our Institution according to the ESMO Scale for Clinical Actionability of molecular Targets (ESCAT) tiers (ESCAT scale; J. Mateo, Annals of Oncology, 2018[1]). Pts were classified as molecularly matched (MM) i.e. treated based on the presence of an ESCAT actionable molecular abnormality, or non-molecularly matched (no-MM). Tumor and clinical characteristics and outcome of pts were collected. Results: From 2013 to 2020, 90 pts with R/R DLBCL screened for early clinical trial had tumor & germline samples sequenced by NGS. The mean age was 62 years old (range, 23-83) and median prior lines of systemic therapy was 2 (range 1-9). Sixteen (18%) pts were enrolled in epCT and treated with MO drugs, 43 pts (48%) were enrolled in epCT and treated with no-MM drugs, and 31 (34%) pts were not enrolled in epCT. The main clinical characteristics of the pts were similar in both MM and no-MM groups (table 1). Based on ESCAT scale, targeted therapies of the 16 pts MM were distributed as "investigational-level" tier II in 6 pts (3 pts EZH2 mutant [m] treated with EZH2 inhibitors; 3 patients with a CD79Bm treated with BTK inhibitors); "hypothetical-level" tier III in 1 pt BRAFm treated with a BRAF inhibitor; and "hypothetical-level" tier IV in 9 pts (6 pts MYD88m treated with cereblon modulator drugs; 2 pts with BCL7Am/ARID1Am treated with investigational epigenetic modifier drugs; 1 pt with MYCm treated with a PI3K inhibitor). Median progression-free survival in MM and no-MM pts, were 3.3 and 1.7 months, respectively (p=0.0092; HR=0.47 [CI95:0.27-0.83]). Median overall survival in MM and no-MM pts were 20.0 and 6.7 months, respectively (p=0.0238; HR=0.49 [CI95: 0.26-0.91]) (figure 1). Conclusions: Molecular orientation before inclusion in early phase clinical trials was associated with better outcomes in patients with R/R DLBCL. A molecular tumor profile could be useful to manage patients with R/R DLBCL with precision medicine helping to identify patients likely to respond to targeted therapies. Reference. 1- Mateo J, Chakravarty D, Dienstmann R et al. A framework to rank genomic alterations as targets for cancer precision medicine: the ESMO Scale for Clinical Actionability of molecular Targets (ESCAT). Annals of Oncology 2018; 29(9):1895-1902. Table 1.Patient's characteristics at time of R/R DLBCL relapse and tumor sample in molecularly matched patients (MM group) and non-molecularly matched patients (no-MM group). Data presented are n (%) unless otherwise stated. MM= Molecularly matched. no-MM= Non-molecularly matched. R/R DLBCL= relapsed or refractory diffuse large B-cell lymphoma. y.o. = years old. mo.= months. ECOG PS= Eastern Cooperative Oncology Group performance status. NHL=non-Hodgkin lymphoma. ASCT= Autostem cell transplantation. IHC= Immunohistochemistry. COO= Cell of origin. GC= Germinal center. ABC= Activated B-cell. NA= Not available. LDH= Lactate deshydrogenase. *the p-value indicates the comparison between the proportions of the groups from a contingency table, and was calculated by the exact Fisher test. All tests were non-parametric. The p values were calculated by two-tailed and a 95% confidence interval was used, with values less than 0.005 considered being significantly different. Disclosures Michot: Argen-x: Research Funding; Eos: Research Funding; Exelixis: Research Funding; Amgen: Research Funding; Astex: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai: Research Funding; Daiichi Sankyo: Research Funding; AstraZeneca: Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; Lysarc: Research Funding; Lilly: Research Funding; Kyowa: Research Funding; Genentech: Research Funding; Forma: Research Funding; Agios: Research Funding; AZD: Honoraria, Membership on an entity's Board of Directors or advisory committees; Mundi Pharma: Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Celgene: Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Other; Xencor: Research Funding; Sanofi: Research Funding; Roche: Research Funding; Medimmune: Research Funding; Lytix Biopharma: Research Funding; Janssen: Other, Research Funding; Debiopharm: Research Funding; Gustave Roussy: Honoraria, Other: Abbvie, Aduro, Agios, Amgen, Argen-x, Astex, AstraZeneca, Aveo pharmaceuticals, Bayer, Beigene, Blueprint, BMS, Boeringer Ingelheim, Celgene, Chugai, Clovis, Daiichi Sankyo, Debiopharm, Eisai, Eos, Exelixis, Forma, Gamamabs, Genentech, Gortec, GSK, H3 bio, Research Funding; Abbvie: Research Funding. Varga:Astra Zeneca: Current Employment. Ribrag:argenX: Current equity holder in publicly-traded company, Research Funding; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; arGEN-X-BVBA: Research Funding; BAY1000394 studies on MCL: Patents & Royalties; Institut Gustave Roussy: Current Employment; Epizyme: Consultancy, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AZD: Honoraria, Other; Pharmamar: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Eisai: Honoraria; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nanostring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; Immune Design: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; Infinity: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Introduction Aggressive B-cell lymphomas are heterogeneous in their clinical course and biological characteristics. They include diffuse large B-cell lymphoma not otherwise specified (DLBCL-NOS), high grade-B-cell lymphoma with double/triple-hit or NOS (HGBL), primary mediastinal B-cell lymphoma (PMBL). In order to better differentiate these entities, the WHO classification recommends using immunohistochemistry (IHC), FISH, targeted sequencing and gene expression profiles (GEP). However, these techniques are most often performed retrospectively in clinical trials, which is not representative of real life. In order to use these information to improve the current standard of treatment with targeted therapies adapted to lymphoma biology, we have set up a national network on behalf of the LYSA called RT3 (for Real-time tailored therapy) to demonstrate that we are able to comprehensively characterize aggressive B cell lymphomas in a clinically relevant timeline. Materials and methods Patients > 18 years of age with untreated aggressive B-cell lymphoma were included prospectively from 21 LYSA centers. FFPE specimens were analyzed and classified according to WHO classification, benefiting from an IHC profile (Hans algorithm, BCL2, MYC), FISH analysis (BCL6, BCL2, MYC break apart and MYC-IGH/IGL/IGK fusion probes), targeted NGS analysis and a targeted GEP using RT-MLPA (Bobée et al. J Mol diagn 2017). A first part of the study phase was carried out in an oligocentric manner with only 2 referral platforms for pathological analysis and molecular characterization. The second phase was the implementation of 7 RT3 platforms spread over France. The main objective was to evaluate the capacity of the network to provide an exhaustive histopathological and molecular characterization 4 days before theoretical R-CHOP21 C3 administration (within 38 days after D1 cycle 1). Results The oligocentric cohort 1 prospectively included 72 patients in 6 months: 19 had insufficient material or inappropriate diagnosis to be qualified and 53 benefited from the complete analysis on referral platforms. A complete characterization 4 days before RCHOP-C3 was provided to the clinician in 47 cases (88.7%), allowing to further implement the network with 7 platforms and 23 clinical investigation centers. 183 patients were included in the second phase in 9 months, 35 were excluded for inadequate diagnosis/material. On this population, 143 (96.6%) complete patient-reports were provided with a median time of 32 days (1-50). Finally, 201 cases were retained in the Full Analysis Set for which tumor samples fulfilled all prerequisites and diagnosis of DLBCL was confirmed by RT3 platforms. The clinical characteristics were as follows: median age of 61 y (20-92), with 45.2% of pts with aaIPI 2-3and 67.1% with Ann Arbor stage III-IV; 1L treatment consisted mainly of RCHOP (RCHOP14/21 = 136, 72.3%). 7% of patients were treated with experimental drugs. 76% presented with extranodal involvement. After pathological review, 139 (69%) patients were classified as DLBCL-NOS (41% GCB; 58% nGCB), 11 (5%) as HGBL-NOS, 8 (4%) as HGBL-DH and 18 (9%) as PMBL, 3 (1%) as EBV+ DLBCL-NOS, 9 (4%) as FL-3B, 7 (3%) transformed DLBCL, 2 (1%) plasmablastic, 1 (0.5%) unclassified, 1 (0.5%) DLBCL with IRF4 rearrangement. By immunohistochemistry, 22 cases were CD5+, 74% were BCL2+, and 53% MYC+ and 49% as double expressors. BCL2/18q21, BCL6/3q27 and MYC/8q24 breaks were observed in 14% (29/201), 25% (50/201) and 12% (25/201) of the cases respectively. MYC partner gene was available in 10/29 cases (5 MYC-IG, 5 non-IG). At the molecular level, PIM1 was the most frequently mutated gene in the entire cohort (36%). In the ABC subtype, MYD88 was mutated in 48% of cases. In the GCB cohort, SOCS1 was the most frequently mutated gene (30%). In the HGBL cohort, MYC was the most mutated gene (43%). Conclusion This study demonstrates that this RT3 LYSA network allowed a real time pathological and molecular characterization of aggressive B-cell lymphomas according to the WHO recommendations. The RT3 network provides relevant informations to the clinician before R-CHOP21 C3, that might contribute to modify the treatment eventually adding targeted therapies or tailor a second line treatment. This network should be used as a basis for future clinical trials. Inter platforms reproducibility and prognostic relevance of the RT3 project will be presented. Disclosures Haioun: Gilead: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Miltenyi: Honoraria; Servier: Honoraria; Roche: Honoraria; Celgene: Honoraria. Le Gouill:Roche Genentech, Janssen-Cilag and Abbvie, Celgene, Jazz pharmaceutical, Gilead-kite, Loxo, Daiichi-Sankyo and Servier: Honoraria; Loxo Oncology at Lilly: Consultancy. Ribrag:arGEN-X-BVBA: Research Funding; BAY1000394 studies on MCL: Patents & Royalties; Institut Gustave Roussy: Current Employment; argenX: Current equity holder in publicly-traded company, Research Funding; Epizyme: Consultancy, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Infinity: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; Nanostring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria; Pharmamar: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AZD: Honoraria, Other; Eisai: Honoraria; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Immune Design: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Salles:Takeda: Honoraria; Karyopharm: Honoraria; Genmab: Honoraria, Other; Debiopharm: Consultancy, Honoraria, Other: consultancy or advisory role; Autolos: Other: consultancy or advisory role; Abbvie: Other: consultancy or advisory role; Roche: Honoraria, Other: consultancy or advisory role; Novartis: Honoraria, Other: consultancy or advisory role; MorphoSys: Honoraria, Other: consultancy or advisory role; Janssen: Honoraria, Other: consultancy or advisory role; Epizyme: Honoraria, Other: consultancy or advisory role; Kite, a Gilead Company: Honoraria, Other: consultancy or advisory role ; BMS/Celgene: Honoraria, Other: consultancy or advisory role. Sujobert:Sunesis: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead/Kyte: Membership on an entity's Board of Directors or advisory committees. Bouabdallah:Gilead Sciences: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Sibon:takeda france: Consultancy.

D-2-hydroxyglutaric aciduria (D2HGA) type II is a rare neurometabolic disorder caused by germline gain-of-function mutations in isocitrate dehydrogenase 2 (IDH2), resulting in accumulation of D-2-hydroxyglutarate (D2HG). Patients exhibit a wide spectrum of symptoms including cardiomyopathy, epilepsy, developmental delay and limited life span. Currently, there are no effective therapeutic interventions. We generated a D2HGA type II mouse model by introducing the Idh2R140Q mutation at the native chromosomal locus. Idh2R140Q mice displayed significantly elevated 2HG levels and recapitulated multiple defects seen in patients. AGI-026, a potent, selective inhibitor of the human IDH2R140Q-mutant enzyme, suppressed 2HG production, rescued cardiomyopathy, and provided a survival benefit in Idh2R140Q mice; treatment withdrawal resulted in deterioration of cardiac function. We observed differential expression of multiple genes and metabolites that are associated with cardiomyopathy, which were largely reversed by AGI-026. These findings demonstrate the potential therapeutic benefit of an IDH2R140Q inhibitor in patients with D2HGA type II. Electronic supplementary material The online version of this article (doi:10.1007/s10545-016-9960-y) contains supplementary material, which is available to authorized users.

IDHology Among the most exciting drug targets to emerge from cancer genome sequencing projects are two related metabolic enzymes, isocitrate dehydrogenases 1 and 2 (IDH1, IDH2). Mutations in the IDH1 and IDH2 genes are common in certain types of human cancer. Whether inhibition of mutant IDH activity might offer therapeutic benefits is unclear (see the Perspective by Kim and DeBerardinis ). F. Wang et al. (p. 622 , published online 4 April) isolated a small molecule that selectively inhibits mutant IDH2, describe the structural details of its binding to the mutant enzyme, and show that this compound suppresses the growth of patient-derived leukemia cells harboring the IDH2 mutation. Rohle et al. (p. 626 , published online 4 April) show that a small molecule inhibitor of IDH1 selectively slows the growth of patient-derived brain tumor cells with the IDH1 mutation.

AG-221 or enasidenib is a first-in-class selective inhibitor of mutated isocitrate dehydrogenase 2 (IDH2) with early demonstrated clinical efficacy in acute myeloid leukemia as a single agent, yet with persistence of mutant IDH2 clones. Two papers in this issue of Cancer Discovery provide further insight into the biological activity of AG-221 in promoting differentiation of IDH2 mutant cells and reversing aberrant DNA methylation over time, and demonstrating pre-clinical activity in combination with a targeted FLT3 kinase inhibitor to eliminate IDH2 mutant clones.

The TET family of proteins has been described a few years ago. Only recently, their roles in DNA modification, through the oxidation of methyl-cytosine, and in normal and malignant development, through the description of TET2 as a tumor suppressor have been documented. The conjunction of these findings has prompted large efforts to understand the biology of these novel entities. Here, we summarize the recent results implicating TET2 in hematological malignancies suggesting that further studies are required to fully understand the role of DNA methylation alterations during transformation.

SummaryLoss-of-function mutations affecting one or both copies of the Ten-Eleven-translocation (TET)2 gene have been described in various human myeloid malignancies. We report that inactivation of Tet2 in mouse perturbs both early and late steps of hematopoiesis including myeloid and lymphoid differentiation in a cell-autonomous manner, endows the cells with competitive advantage, and eventually leads to the development of malignancies. We subsequently observed TET2 mutations in human lymphoid disorders. TET2 mutations could be detected in immature progenitors endowed with myeloid colony-forming potential. Our results show that the mutations present in lymphoid tumor cells may occur at both early and later steps of lymphoid development and indicate that impairment of TET2 function or/and expression predisposes to the development of hematological malignancies.

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.

Mutations in the isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) genes are present in ∼20% of acute myeloid leukemia, and cause a neomorphic enzyme activity that results in the production of 2-hydroxyglutarate (2HG). Mutational and epigenetic profiling of a large patient cohort of acute myeloid leukemia (AML) has revealed that IDH1/2-mutant AMLs display global DNA hypermethylation and impaired hematopoietic differentiation. To further investigate the intrinsic effect of 2HG on hematopoietic proliferation and differentiation, we transfected an erythroleukemia cell line (TF-1) with either IDH1 or IDH2 mutant alleles. These cells overexpress the mutant enzyme, have high levels of 2HG, and exhibit GM-CSF independent growth. Consistent with clinical observations, overexpression of the IDH mutant proteins led to hypermethylation of both histones and DNA. These results suggest that mutations in IDH1/2 could lead to epigenetic rewiring of cells that could facilitate the gain of function phenotype. We are currently studying the global and specific effects of IDH1/2 mutant overexpression to gain a broader understanding of the biological consequence of the IDH1/2 gain of function mutations. We have also generated mutation selective molecules that are capable of inhibiting IDHm enzymes. Upon compound treatment in vitro, we are able to reverse hypermethylation of both histones and DNA and induce cellular differentiation in IDHm cell lines and primary human IDHm AML patient samples(1, 2). These data suggest that an inhibitor of IDH1/2 mutations could correct the altered gene expression patterns seen in IDH1/2 mutant AML tumors leading to a profound effect on hematopoietic differentiation, proliferation and tumor growth. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PL02-04. Citation Format: F. Wang, J. Travins, B. DeLaBarre, V. Penard-Lacronique, S. Schalm, E. Hansen, K. Straley, A. Kernytsky, W. Liu, C. Gliser, H. Yang, S. Gross, E. Artin, V Saada, E. Mylonas, C. Quivoron, J. Popovici-Muller, J. O. Saunders, F. G. Salituro, S. Yan, S. Murray, W. Wei, Y. Gao, L. Dang, M. Dorsch, S. Agresta, D. P. Schenkein, S. A. Biller, S. M. Su, S. de Botton, Katharine E. Yen. IDH mutations and tumorigenicity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PL02-04.

Isocitrate dehydrogenase ( IDH ) 2 R140Q mutation induces myeloid and lymphoid neoplasms in mice

Purpose Mutated isocitrate dehydrogenases (IDHs) 1 and 2 produce high levels of 2-hydroxyglutarate (2-HG). We investigated whether, in acute myeloid leukemia (AML), serum 2-HG would predict the presence of IDH1/2 mutations at diagnosis and provide a marker of minimal residual disease (MRD). Patients and Methods Serum samples from 82 patients at diagnosis of de novo AML (IDH1/2 mutated, n = 53) and 68 patients without AML were analyzed for total 2-HG and its ratio of D to L stereoisomers by mass spectrometry. We measured 2-HG levels and molecular markers of MRD (WT1 and NPM1) in serial samples of 36 patients with IDH1/2 mutations after induction therapy. Results In patients with AML with IDH1/2 mutations, 2-HG serum levels were significantly higher than in patients with IDH1/2 wild type (P < .001). Area under the receiver operating characteristic curve was 99%. The optimum diagnostic cutoff between IDH1/2 mutated and normal was 2 μmol/L (sensitivity, 100%; specificity, 79%). Quantification of the D/L stereoisomers increased specificity (100%; 95% CI, 83% to 100%) compared with total 2-HG (P = .031). In patients with IDH2 R172 mutations, 2-HG levels were higher relative to those with other IDH1/2 mutations (P < .05). During follow-up, serum 2-HG levels showed strong positive correlation with WT1 and NPM1 (P < .001). After induction therapy, total 2-HG serum levels < 2 μmol/L were associated with better overall (P = .008) and disease-free survival (P = .005). Conclusion Serum 2-HG is a predictor of the presence of IDH1/2 mutations and outcome in these patients. Discrimination between D/L stereoisomers improved specificity.

Myeloid sarcoma (MS) is defined as a tumor mass consisting of myeloid blasts with or without maturation occurring at an anatomical site other than bone marrow (BM). MS may occur before, concurrently or after a characterized acute myeloid leukemia (AML). Cytogenetic abnormalities are found in 50% of the cases but molecular alterations are less well described and involved FLT3 and/or NPM1 mutations. Mutations in IDH1 and IDH2 genes are found in 15% to 20% of patients with AML but have never been described in MS. Mutated IDH enzymes produce in vast excess D-2-hydroxyglutarate (2-HG) in leukemic cells, which can act as a biomarker predictive of the presence of IDH1 and IDH2 mutations. As availability of DNA sequencing techniques on paraffin samples are limited, molecular characterization of MS remained difficult. We asked whether in MS, serum 2-HG would predict the presence of IDH1/2 mutations at diagnosis, and could provide a biomarker for follow up. Tissue samples and serum samples from 8 patients with a MS diagnosis were analyzed. High quality genomic DNA was extracted from frozen MS samples using conventional phenol/chloroforme extraction procedures. Exon 4 of IDH1 and IDH2 genes (IDH1/R132 and IDH2/R140 and /R172 codons) was amplified by PCR using HotStar Taq polymeraze (Qiagen) and primers. Direct sequencing was performed using the Sanger method as previously described. In case of MS relapse or AML evolution, IDH1 and IDH2 genes were analyzed in the same way from frozen tissue sample or bone marrow sample. Serum samples at MS diagnosis were analyzed for total 2-HG, D-2-HG and L-2-HG by reverse-phase liquid chromatography coupled to mass spectrometry. In case of myeloid sarcoma with IDH1/2 mutation, 2-HG values were compared to 18F-FDG-PET results when available during remission phase and at relapse. Three patients (3/8; 37.5%) had an IDH2 R140Q mutation at diagnosis of MS localized to lymph node, soft tissue, skin or pharynx. At MS diagnosis, serum total 2-HG, D-2-HG and ratio D/L-2-HG were significantly higher in case of myeloid sarcoma with IDH2 R140Q mutation compared to patients with no IDH mutation (Table 1). Serum total 2-HG level ≥2µM or D-2-HG level ≥1.8µM or ratio D/L 2-HG >2.5 were significantly associated with the presence of IDH2 mutation (Fisher's exact test P≤0.02). Table 1. Myeloid sarcoma with IDH2 R140Q mutation (N=3) Myeloid sarcoma without IDH2 R140Q mutation (N=5) Median total 2-HG (µM) 4.1 (range: 3.1-30.1) 1.4 (range: 1-1.6) Median D-2-HG (µM) 3.7 (range: 2.3-28) 0.6 (range: 0.5-0.8) Median L-2-HG (µM) 0.8 (range: 0.4-2.1) 0.8 (range: 0.4-0.8) Median ratio D/L 2-HG 8.3 (range: 2.9-18.8) 1 (range: 0.7-1.7) All 3 patients with IDH2 R140Q mutated MS received intensive chemotherapy treatment and achieved complete remission (CR). Two patients relapsed: one experienced isolated extramedullary relapse (thigh muscle); one had a bone marrow relapse. IDH2 R140Q mutation was found at the site of relapse in both cases. When available, serum 2-HG values and 18F-fluorodeoxyglucose-positron-emission tomography (FDG-PET) were compared at different time points (at diagnosis, remission and relapse; Table 2). Table 2. Patient #1 Patient #2 Patient #3 FDG-PET at diagnosis (SUVmax) - 17 5.25 Serum 2-HG at diagnosis (µM) 4.1 3.1 30.1 FDG-PET in remission (SUVmax) 0 - 9.6 (N=4) 0 (N=4) - Serum 2-HG in remission (µM) 0.5 - 1.1 (N=4) 0.6 - 3.1 (N=4) 3.4 - 17.9 (N=3) FDG-PET at MS relapse/evolution to AML (SUVmax) 6.1 - 0 Serum 2-HG at MS relapse/evolution to AML (µM) 2.3 - 16.7 Time between diagnosis and MS relapse/evolution to AML (months) 30 - 9 Serum 2-HG values were in accordance with FDG-PET interpretations except in patient #1 who presented a transient hypermetabolic splenic nodule (SUVmax 9.6) without serum 2-HG increase. Patient #2 remained in CR but had recently increased 2-HG values without overt relapse. Patient #3 presented relapse as a refractory anemia with excess of blast without extra-medullary localization. FDG-PET didn't find any abnormality contrary to the persistent increased value of serum 2-HG (total 2-HG: 16.7µM). These data show that myeloid sarcoma can be associated with IDH2 R140Q mutation and suggest that 2-HG measurement in the serum predicts the presence of IDH1/2 mutations at diagnosis. During follow-up, serum 2-HG values could be representative of the disease status. Because of IDH inhibitors promising results in AML, 2-HG screening at MS diagnosis could be useful. Disclosures Ribrag: Celgene: Research Funding; Esai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmamar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: 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:Agios pharmaceuticals: Research Funding.

Introduction: Mutations in IDH1 and IDH2 genes are found in 15% to 20% of patients with acute myeloid leukemia (AML). Mutated IDH enzymes produce in excess the D stereoisomer 2-hydroxyglutarate (2-HG) in leukemic cells, which can act as a biomarker predictive of the presence of IDH1 and IDH2 mutations. Total serum 2-HG (D and L stereoisomers) >2µM and ratio D/L 2-HG >2.5 are strong predictors of the presence of IDH1/2 mutations at diagnosis (Janin et al; JCO 2014). We measured 2-HG levels in serial samples of 47 patients with IDH1/2 mutations during induction or first salvage therapy to determine whether 2HG can serve as a surrogate marker of treatment efficacy. Methods: Between 2007 and 2015, 47 AML patients with IDH1 or IDH2 mutation received intensive chemotherapy as induction therapy (n=42) or as first salvage regimen (n=5) in our center (Table 1). Genomic DNA was extracted from BM samples using conventional procedures. IDH1-(codon R132) and IDH2- targeted (codons R140 and R172) regions were amplified by PCR with primers containing Ion Torrent adapters and unique barcodes to generate libraries. Pooled amplicons libraries were clonally amplified on Ion Spheres using the Ion Xpress Template 200 Kit. All available serum samples (n=394) prior and during induction/salvage chemotherapy were analyzed for total 2-HG, D-2-HG and L-2-HG by reverse-phase liquid chromatography coupled to mass spectrometry. We studied the serum 2-HG values (D-2-HG and ratio D/L 2-HG) evolution during IC according to complete remission (CR) evaluated at the end of IC. When a patient had a missing value for 2-HG, the missing value was replaced by the last available observation (up to 7 days before the missing value). At each day we tested the difference between the dosage values of the responders and the non-responders with non-parametric Wilcoxon 2-sided tests. The analyses were done with SAS software (version 9.3; SAS Institute, Cary, NC). Table 1. Patients and treatment characteristics: AML at diagnosis / RR AML (N) 42 / 5 Median age (years) 58.2 (23.1 - 75.7) Sex ratio (M/F) 23/24 Conventional cytogenetic: - Normal karyotype - Trisomy 8 - Monosomy 7 39 (83%) 3 (6%) 2 (4%) Associated gene mutations: - NPM1- FLT3-ITD- FLT3-TKD 24 (51%) 10 (21%) 5 (11%) IDH mutation: - IDH1 R132 (N) - IDH2 R140 (N) - IDH2 R172 (N) 8 (17%) 32 (65%) 7 (15%) Intensive treatment: - "3+7" regimen - Intensified induction chemotherapy - High-dose cytarabine based chemotherapy 24 (51%) 19 (40%) 4 (8%) Results: Complete remission (CR) 1 was obtained in 35/42 patients (83%) and CR2 in 3/5 (60%) after IC. All (8/8) IDH1 -R132 mutated patients achieved CR1, 25/28 (89%) IDH2 -R140 mutated patients and 3/4 achieved CR1 and CR2 respectively; 2/6 (33%) IDH2 -R172 mutated patients and 0/1 achieved CR1 and CR2, respectively. Median serum 2-HG values before IC were as follow: total 2-HG 10.9 µM (range: 2.1-135.5); D-2-HG 10.7 µM (range: 1.7-132.6) and ratio D/L 36.9 (range: 3.9-131.2). Median number of serum 2-HG values per patient was 7 (range: 5-10). Serum D-2-HG and ratio D/L 2-HG evolution according to CR status after IC began to differ significantly at day 5 after IC (Wilcoxon test P=0.02, Figure 1). The difference between responders and non-responders increased overtime (Wilcoxon test P Conclusion: The oncometabolite 2-HG can act as a biomarker to monitor early response to conventional IC in AML patients with IDH1/2 mutations. D-2-HG and ratio D/L 2-HG measurements are rapid, robust, non-invasive and can predict IC failure in these patients, allowing selection of patients who could potentially benefit from early salvage treatment or IDH inhibitors. Figure 1. D-2-HG and ratio D/L 2-HG evolution according to response to IC (CR) and p-value Wilcoxon tests. Figure 1. D-2-HG and ratio D/L 2-HG evolution according to response to IC (CR) and p-value Wilcoxon tests. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Ribrag: Celgene: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Esai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmamar: 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:Agios pharmaceuticals: Research Funding.

According to the World Health Organization classification, myeloproliferative neoplasms (MPN) include chronic myelogenous leukemia, also known as BCR-ABL1–positive MPN, classic BCR-ABL1-negative MPN including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), and non-classic forms (i.e. systemic mastocytosis, chronic eosinophilic leukemia not otherwise specified, chronic neutrophilic leukemia and unclassifiable MPN). All these subtypes are stem cell-derived clonal myeloproliferation, associated with the overproduction of mature blood elements and variable rates of transformation to acute myeloid leukemia (AML).1 JAK2V617F activating mutation is the most prevalent abnormality observed in BCR-ABL1-negative MPN, found in virtually all cases of PV and in about half of ET and PMF (96%, 55% and 65%, respectively). This mutation lies in the pseudokinase-domain of JAK2 and disrupts its regulatory activity. Another mutation affecting JAK2 exon 12 is observed in 3% of all PV cases. Mutations affecting W515 of the thrombopoietin receptor MPL are detected in PMF and ET patients. Additional mutations have been identified in MPN (reviewed in 1). Defects in the control of intracellular signaling involve mutations in LNK and CBL genes. Genetic abnormalities affecting epigenetic regulation, and possibly responsible for disease initiation, concern the ASXL1, EZH2 and TET2 genes. Finally, mutation in IKZF1 and IDH1/2 may be implicated in MPN transformation. In PV, among these additional mutations to JAK2V617F, TET2 mutations are those most frequently reported (16%); the others are only described in small subsets of patients.1 TET2 belongs to a family of three conserved genes in mammals: TET1, TET2 and TET3. The founding member of the family, TET1, has been identified as a fusion partner of MLL in the t(10;11)(q22;q23) translocation of acute leukemia.2,3 The involvement of TET3 in hematologic disorder has not yet been described. The TET proteins are members of the 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenase that are able to convert 5-methyl-cytosine (5-mC) to 5-hydroxymethylcytosine (hmC).4,5 Recent reports indicate an important role for TET1 and TET2 (and, therefore, hmC) in the control of ES cell self-renewal and differentiation.6 TET3 might be involved in genome reprogramming following fecundation.7

Point mutations in isocitrate dehydrogenase (IDH) define distinct subsets of acute myelogenous leukemia (AML). IDH is a metabolic enzyme that interconverts isocitrate and α-ketoglutarate (α-KG), but cancer-associated point mutations in IDH1 and IDH2 confer a neomorphic activity that allows reduction of α-KG to the oncometabolite R-2-hydroxyglutarate (2-HG). High levels of 2-HG have been shown to inhibit α-KG-dependent dioxygenases including histone and DNA demethylases, which play a key role in regulating the epigenetic state of cells, but the relationship between 2-HG and oncogenesis is not completely understood. Consistent with 2-HG promoting cancer via an effect on chromatin structure, patients harboring IDH mutations display a CpG island methylator phenotype (CIMP) and several studies have shown that overexpression of IDH mutant enzymes can induce histone and DNA hypermethylation as well as block cellular differentiation. In addition, mice engineered to express IDH1-R132H in hematopoietic tissue have increased early hematopoietic progenitors, splenomegaly, anemia, hypermethylated histones and altered DNA methylation patterns similar to those found in AML patients harboring IDH1/2 mutations.[i] Taken together, these data suggest that cancer-associated IDH mutations may induce a block in cellular differentiation to promote tumorigenesis. To investigate whether selective pharmacological inhibition of the mutant IDH1 enzyme could provide an effective way to lower intracellular 2-HG levels and restore normal differentiation, we treated TF-1 cells or primary human AML patient samples expressing mutant IDH1 with AG-120, an oral, selective, first-in-class, potent IDH1 mutant inhibitor currently in phase I clinical trials. Treatment with AG-120 decreased intracellular 2-HG levels, inhibited growth factor independent proliferation and restored erythropoietin (EPO)-induced differentiation in TF-1 IDH1-R132H cells. Similarly, pharmacological inhibition of mutant IDH1 enzyme with AG-120 in primary human blast cells cultured ex vivo provided an effective way to lower intracellular 2-HG levels and induced myeloid differentiation. Taken together, these data demonstrate that AG-120 is effective at lowering 2-HG levels and restoring cellular differentiation, and support further clinical development of this compound. Figure 1: Diagnosis and karyotypes of primary AML patient samples used in ex vivo studies Figure 1:. Diagnosis and karyotypes of primary AML patient samples used in ex vivo studies PB = peripheral blood, BM = bone marrow Figure 2: Percent 2-HG remaining relative to DMSO control after 6-day treatment with AG-120 in IDH1 R132H or IDH1 R132C patient samples Figure 2:. Percent 2-HG remaining relative to DMSO control after 6-day treatment with AG-120 in IDH1 R132H or IDH1 R132C patient samples or following 6 days of treatment with control (DMSO) or AG-120 (0.5, 1.0, and 5.0 μM) Figure 3: Relative proportion of cell types in human AML bone marrow samples untreated Figure 3:. Relative proportion of cell types in human AML bone marrow samples untreated [i] M. Sasaki et al., IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics. Nature 488(7413):656-9, 2012. Disclosures Hansen: Agios Pharmaceuticals: Employment, Stockholder Other. Quivoron:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la recherche contre le Cancer (ARC): Grant, Grant Other; AGIOS: Grant Other. Straley:Agios Pharmaceuticals: Employment, Stockholder Other. Lemieux:Agios Pharmaceuticals: Employment, Stockholder Other, US20130190249 (pending) Patents & Royalties. Popovici-Muller:Agios Pharmaceuticals: Employment, Stockholder Other. Fathi:Agios Pharmaceuticals: Advisory board participation Other. Gliser:Agios Pharmaceuticals: Employment, Stockholder Other. David:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la Recherche contre le Cancer (ARC): Grant, Grant Other; Association Laurette Fugain: Grant, Grant Other; AGIOS: Grant Other. Bernard:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Ligue Nationale contre le cancer (LNCC): Grant, Grant Other; AGIOS: Grant Other. Dorsch:Agios Pharmaceuticals: Employment, Stockholder Other. Yang:Agios Pharmaceuticals: Employment, Stockholder Other. Su:Agios Pharmaceuticals: Employment, Stockholder Other. Agresta:Agios Pharmaceuticals: Employment, Stockholder Other. de Botton:AGIOS: Grant Other. Penard-Lacronique:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la recherche contre le Cancer (ARC): Grant, Grant Other; AGIOS: Grant Other. Yen:Agios Pharmaceuticals: Employment, Stockholder Other.

Somatic point mutations in the active site of IDH (isocitrate dehydrogenase) 1 and IDH2 genes are observed in acute myeloid leukemia (AML). These mutations lead to the production and accumulation of R-2-hydroxyglutarate (2-HG) in the tumor blast cells as well as in the plasma of patients. High levels of 2-HG have been shown to inhibit alpha-ketoglutarate-dependent dioxygenases, including epigenetic regulators (i.e. histone or DNA demethylases), resulting in altered cellular differentiation. Ex vivo experiments in primary cells from AML IDH2 mutant patients have demonstrated that IDH2 mutant inhibitors are able to revert this phenotype. In order to test the biological activity of AG-221, an oral, reversible and selective inhibitor of mutated IDH2 currently in phase I trials, we developed primary human AML xenograft models. Primary blast cells from 3 AML patients with normal karyotype (n=3), NPM1 mutation (n=3) and FLT3ITD (n=1) were injected into immunodeficient NOD SCID IL2R gamma null (NSG) mice by intrafemoral injection after sub-lethal irradiation. Medullar and peripheral tumoral engraftment was monitored by flow cytometry (using species-specific antibodies) and serum 2-HG measurements. Engraftment of IDH2-R140Q human blast cells was not lethal, but these AML cells persisted over time. Twelve months after injection, IDH2-R140Q blast cell-engrafted mice were randomized into two groups, for treatment with AG-221 (30 mpk) or vehicle (0.5% methylcellulose / 0.2% Tween 80, in water) (n=10 mice; 5 mice per condition). Treatments were administered by oral gavage for 38 days twice per day (BID). Peripheral blood was collected at multiple time points for the determination of pharmacokinetics (PK) and pharmacodynamics (PD), and the assessment of the differentiation effects by AG-221 on human tumor cells. PK/PD analyses showed good plasma exposure of AG-221 and reduction of 2-HG. Furthermore, AG-221 administration also induced a burst of proliferation of human blasts followed by myeloid differentiation starting at day 20 in peripheral blood as measured by the expression of CD11b, CD14, CD15, CD24 and cell morphology. No effects on proliferation or differentiation were seen in the absence of AG-221 administration. Histological analyses of hematopoietic organs in treated animals showed a decrease of infiltrating human cells, as well as obvious morphological changes in the human cell population in AG-221-treated animals compared with vehicle-treated animals. Flow cytometry confirmed the differentiation of the human cells in the spleen and bone marrow of the AG-221-treated mice. Furthermore, cells undergoing differentiation retained the R140Q mutation, demonstrating the differentiating effect of the compound. In conclusion, AG-221 reduced serum 2-HG levels and triggered differentiation of leukemic blast cells engrafted in NSG mice. These results are consistent with what has been observed clinically in IDH2 mutant AML patients treated with AG-221 in a phase I dose escalation trial. Disclosures Quivoron: Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la recherche contre le Cancer (ARC): Grant, Grant Other; AGIOS: Grant Other. David:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la Recherche contre le Cancer (ARC): Grant, Grant Other; Association Laurette Fugain: Grant, Grant Other; AGIOS: Grant Other. Straley:Agios Pharmaceuticals: Employment, Stockholder Other. Travins:Agios Pharmaceuticals: Employment, Stockholder Other. Kim:Agios Pharmaceuticals: Employment, Stockholder Other. Chen:Agios Pharmaceuticals: Employment, Stockholder Other. Zhu:Agios Pharmaceuticals: Employment, Stockholder Other. Bawa:AGIOS: Grant Other. Bernard:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Ligue Nationale contre le cancer (LNCC): Grant, Grant Other; AGIOS: Grant Other. Yang:Agios Pharmaceuticals: Employment, Stockholder Other. Agresta:Agios Pharmaceuticals: Employment, Stockholder Other. de Botton:AGIOS: Grant Other. Yen:Agios: Employment. Penard-Lacronique:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la recherche contre le Cancer (ARC): Grant, Grant Other; AGIOS: Grant Other.