IDH Inhibitors Research Articles

Outcomes for Patients with Late-Stage Mutant-IDH2 (m IDH2) Relapsed/Refractory Acute Myeloid Leukemia (R/R AML) Treated with Enasidenib Vs Other Lower-Intensity Therapies in the Randomized, Phase 3 IDHentify Trial

Outcomes for Patients with Late-Stage Mutant-IDH2 (m IDH2) Relapsed/Refractory Acute Myeloid Leukemia (R/R AML) Treated with Enasidenib Vs Other Lower-Intensity Therapies in the Randomized, Phase 3 IDHentify Trial

IDH Mutations Are Enriched in Myelodysplastic Syndromes Patients with Severe Neutropenia: A Potential Targeted Therapy

IDH Mutations Are Enriched in Myelodysplastic Syndromes Patients with Severe Neutropenia: A Potential Targeted Therapy

Roles of metal ions in the selective inhibition of oncogenic variants of isocitrate dehydrogenase 1

Cancer linked isocitrate dehydrogenase (IDH) 1 variants, notably R132H IDH1, manifest a ‘gain-of-function’ to reduce 2-oxoglutarate to 2-hydroxyglutarate. High-throughput screens have enabled clinically useful R132H IDH1 inhibitors, mostly allosteric binders at the dimer interface. We report investigations on roles of divalent metal ions in IDH substrate and inhibitor binding that rationalise this observation. Mg2+/Mn2+ ions enhance substrate binding to wt IDH1 and R132H IDH1, but with the former manifesting lower Mg2+/Mn2+KMs. The isocitrate-Mg2+ complex is the preferred wt IDH1 substrate; with R132H IDH1, separate and weaker binding of 2-oxoglutarate and Mg2+ is preferred. Binding of R132H IDH1 inhibitors at the dimer interface weakens binding of active site Mg2+ complexes; their potency is affected by the Mg2+ concentration. Inhibitor selectivity for R132H IDH1 over wt IDH1 substantially arises from different stabilities of wt and R132H IDH1 substrate-Mg2+ complexes. The results reveal the importance of substrate-metal ion complexes in wt and R132H IDH1 catalysis and the basis for selective R132H IDH1 inhibition. Further studies on roles of metal ion complexes in TCA cycle and related metabolism, including from an evolutionary perspective, are of interest.

Nonmyeloablative Allogeneic Transplantation With Post-Transplant Cyclophosphamide for Acute Myeloid Leukemia With IDH Mutations: A Single Center Experience

IntroductionMutations in the IDH1 or IDH2 genes are detected in approximately 20% of cases of acute myeloid leukemia (AML). Few studies have examined the impact of IDH mutations in AML on allogeneic bone marrow transplant (alloBMT) outcomes. Patients and MethodsIn this single center study, alloBMT outcomes for 61 patients with IDH-mutated (mIDH) AML were compared to those for 146 patients with IDH-wildtype (wtIDH) AML. ResultsPatients with mIDH AML had a 2-year overall survival (OS) of 85% (95% CI 76%-95%), 2-year relapse free survival (RFS) of 71% (95% CI 59%-85%), 1-year cumulative incidence of relapse (CIR) of 14% (95% CI 5%-23%) and a 1-year cumulative incidence of transplant related mortality (CITRM) of 3% (95% CI 0%-8%). Patients with wtIDH had a 2-year OS of 61% (95% CI 53%-70%), 2-year RFS of 58% (95% CI 50%-67%), 1-year CIR of 27% (95% CI 20%-35%), and a 1-year CITRM of 9% (95% CI 5%-14%). In a univariate analysis cox-proportional hazard model, mIDH was associated with significantly better OS (HR 0.52, 95% CI 0.29-0.96) and a trend toward better RFS (HR 0.60, 95% CI 0.35-1.01). After controlling for donor age, diagnosis, and ELN risk category, mIDH was associated with a nonsignificantly improved OS (HR 0.54, 95% CI 0.29-1.01) and RFS (HR 0.67, 95% CI 0.39-1.15). ConclusionAmong patients with mIDH AML, patients who received a peritransplant IDH inhibitor had improved OS (P = .03) compared to those who did not, but there was no detectable difference for RFS (P = .29).

The cure of leukemia through the optimist's prism.

Progress is occurring at a dizzying rate across all leukemias. Since the authors' review of the topic in Cancer in 2018, numerous discoveries have been made that have improved the therapy and outcomes of several leukemia subsets. Hairy cell leukemia is potentially curable with a single course of cladribine followed by rituximab (10-year survival, ≥90%). Acute promyelocytic leukemia is curable at a rate of 80% to 90% with a nonchemotherapy regimen of all-trans retinoic acid and arsenic trioxide. The cure rate for core-binding factor acute myeloid leukemia (AML) is ≥75% with fludarabine, high-dose cytarabine, and gemtuzumab ozogamicin. Survival for patients with chronic myeloid leukemia is close to that for an age-matched normal population with BCR-ABL1 tyrosine kinase inhibitors (TKIs). Chronic lymphocytic leukemia, a previously incurable disease, may now be potentially curable with a finite duration of therapy with Bruton tyrosine kinase inhibitors and venetoclax. The estimated 5-year survival rate for patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL) exceeds 70% with intensive chemotherapy and ponatinib, a third-generation BCR-ABL1 TKI, and more recent nonchemotherapy regimens using dasatinib or ponatinib with blinatumomab are producing outstanding results. Survival in both younger and older patients with ALL has improved with the addition of antibodies targeting CD20, CD19 (blinatumomab), and CD22 (inotuzumab) to chemotherapy. Several recent drug discoveries (venetoclax, FLT3 and IDH inhibitors, and oral hypomethylating agents) are also improving outcomes for younger and older patients with AML and for those with higher risk myelodysplastic syndrome.

Biomimetic mesoporous vectors enabling the efficient inhibition of wild-type isocitrate dehydrogenase in multiple myeloma cells

The discovery of isocitrate dehydrogenases (IDHs) mutations in several malignancies has brought to the approval of drugs targeting IDH1/2 mutants in cancers. More recently it has been suggested that the enzymatic inhibition of IDHs may have therapeutic potentials also for wild-type IDH cancers. Specifically, IDH2 inhibition can sensitize multiple myeloma cells to proteasome inhibitors. However, inhibitors directed against native IDHs are not present on the market. Here, we exploited an allosteric inhibitor of mutant IDH2 (AGI-6780), known to also decrease the activity of wild-type IDH2. Since AGI-6780 effectiveness in vivo is limited by its high hydrophobicity and very low bioavailability, the drug was loaded into mesoporous silica nanoparticles (MSNs) with the aim to enhance its efficacy. Furthermore, to enable high drug retention into the silica pores, improve biocompatibility, and reduce the off-target delivery of the drug, a Supported phosphoLipidic Bilayer (SLB) was self-assembled on the outer MSN surface. The silica nanoparticles were thus coated with three different lipid formulations and characterized in terms of structure, size, and morphology. We demonstrated that MSN@SLB nanoparticles have improved colloidal stability and hemocompatibility with respect to pristine MSN. We showed that MSN@SLB formulation displays an excellent loading and retention of the IDH2 inhibitor AGI-6780, with a limited drug leakage depending on the lipid formulation. Finally, we proved that AGI-6780-loaded MSN@SLB nanoparticles efficaciously inhibited the IDH2 enzymatic activity of multiple myeloma cells. Overall, this study provides a proof of concept of drug delivery to multiple myeloma cells by repurposing a neglected/dismissed drug (AGI-6780) with the use of smart nanoparticles and enabling the sensitization of multiple myeloma cells towards other possible treatments. • Mesoporous silica nanoparticles are self-assembled with 3 different phospholipids bilayers at increasing complexities. • Nanoconstructs are loaded by an enzymatic inhibitor, showing effective decrease of IDH2 activity in multiple myeloma cells. • These hybrid nanoconstructs enable high drug retention, improved water stability and haemocopatibility. • This nanotech approach, exploiting mesoporous biocompatible nanocarriers, allow to reposition an abandoned drug.

Somatic IDH1 variant (p.R132C) in an adult male with Maffucci syndrome

Maffucci syndrome is a rare, highly variable somatic mosaic condition, and well-known cancer-related gain-of-function variants in either the IDH1 or IDH2 genes have been found in the affected tissues of most reported individuals. Features include benign enchondroma and spindle-cell hemangioma, with a recognized increased risk of various malignancies. Fewer than 200 affected individuals have been reported; therefore, accurate estimates of malignancy risk are difficult to quantify and recommended surveillance guidelines are not available. The same gain-of-function IDH1 and IDH2 variants are also implicated in a variety of other benign and malignant tumors. An adult male presented with several soft palpable lesions on the right upper limb. Imaging and histopathology raised the possibility of Maffucci syndrome. DNA was extracted from peripheral blood lymphocytes and tissue surgically resected from a spindle-cell hemangioma. Sanger sequencing and droplet digital polymerase chain reaction (PCR) analysis of the IDH1 gene were performed. We identified a somatic mosaic c.394C > T (p.R132C) variant in exon 5 of IDH1, in DNA derived from hemangioma tissue at ∼17% variant allele fraction. This variant was absent in DNA derived from blood. This variant has been identified in the affected tissue of most reported individuals with Maffucci syndrome. Although this individual has a potentially targetable variant, and there is a recognized risk of malignant transformation in this condition, a decision was made not to intervene with an IDH1 inhibitor. The reasons and prospects for therapy in this condition are discussed.

Targeted Therapies for the Evolving Molecular Landscape of Acute Myeloid Leukemia.

Simple SummaryAcute myeloid leukemia (AML) is a predominately fatal blood cancer. For a period of forty years, treatment options for AML remained relatively stagnant. Recently, multiple new agents have been approved. In this review, we discuss considerations surrounding the use of these newly approved therapies. We outline the molecular profiles of AML disease status and highlight subsets of patients for whom therapies are best suited based on available data.Despite considerable growth in our understanding of the heterogeneous biology and pathogenesis of acute myeloid leukemia (AML) in recent decades, for nearly forty years, little progress was gained in the realm of novel therapeutics. Since 2017, however, nine agents have been FDA-approved for patients with AML in both the upfront and relapsed/refractory (R/R) settings. Most of these compounds function as inhibitors of key cell cycle enzymatic pathways or mediators of leukemic proliferation and survival. They have been approved both as single agents and in combination with conventional or reduced-intensity conventional chemotherapeutics. In this article, we review the molecular landscape of de novo vs. R/R AML and highlight the potential translational impact of defined molecular disease subsets. We also highlight several recent agents that have entered the therapeutic armamentarium and where they fit in the AML treatment landscape, with a focus on FLT3 inhibitors, IDH1 and IDH2 inhibitors, and venetoclax. Finally, we close with a survey of two promising novel agents under investigation that are poised to enter the mainstream clinical arena in the near future.

Accelerated Phase of MPN: What Is It and What to Do About It

Introduction The classic Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) are hematopoietic stem cell diseases characterized by activated JAK/STAT signaling pathways and a variable propensity to evolve toward accelerated or blast phase disease (MPN AP/BP). Outcomes are poor in MPN AP/BP and have not changed substantially in the past decade. Our increasing understanding of the molecular-genomic factors and pathways that drive MPN progression have the potential to lead to meaningful targeted therapeutic approaches. This argues for consideration of a personalized approach that takes the mutational spectrum of the individual patient into account, in addition to clinical factors, when fashioning a therapeutic strategy.1 For example, emerging retrospective data with IDH inhibitors in MPN AP/BP lend credence to this approach and are now informing prospective early phase clinical investigation.2,3

IDH Mutated AML: Beyond Enasidenib and Ivosidenib Monotherapy

Introduction Mutations in the isocitrate dehydrogenase (IDH) genes, IDH1 or IDH2, occur in approximately 20% of patients with acute myeloid leukemia (AML) and promote leukemogenesis through a block in normal myeloid differentiation and through the production of the oncometabolite 2-hydroxyglutarate (2HG).1 Selective oral inhibitors of mutant IDH1 and IDH2, leading to restoration of normal myeloid differentiation and reduction of 2HG have been developed, and are now approved as single-agent therapy, based on clinical efficacy observed within the original first-in-human trials.

Updates on targeted therapies for acute myeloid leukaemia.

In the past few years research in the underlying pathogenic mechanisms of acute myeloid leukaemia (AML) has led to remarkable advances in our understanding of the disease. Cytogenetic and molecular aberrations are the most important factors in determining response to chemotherapy as well as long-term outcome, but beyond prognostication are potential therapeutic targets. Our increased understanding of the pathogenesis of AML facilitated by next-generation sequencing has spurred the development of new compounds in the treatment of AML, particularly the creation of small molecules that target the disease on a molecular level. Many of the hopeful predictions outlined in our AML review of 2018 are now therapeutic realities: gemtuzumab ozogamicin, venetoclax, FLT3 inhibitors (midostaurin, gilteritinib), IDH inhibitors (ivosidenib, enasidenib), CPX-351, glasdegib, oral decitabine, and oral azacitidine. Others may soon be (quizartinib, APR246 magrolimab, menin inhibitors). The wealth of positive data allows reconsideration of what might soon be new standards of care in younger and older patients with AML. In this review we give an overview of recently approved therapies in AML and address present and future research directions.

Mutant Idh2 Cooperates with a NUP98-HOXD13 Fusion to Induce Early Immature Thymocyte Precursor ALL.

Mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes are frequently observed in a wide variety of hematologic malignancies, including myeloid and T-cell leukemias. In this study, we generated Idh2R140Q transgenic mice to examine the role of the Idh2R140Q mutation in leukemia. No leukemia developed in Idh2R140Q transgenic mice, suggesting a need for additional genetic events for leukemia development. Because myeloid cells from NUP98-HOXD13 fusion (NHD13) transgenic mice frequently acquire somatic Idh mutations when they transform to acute myeloid leukemia, we generated Idh2R140Q/NHD13 double transgenic mice. Idh2R140Q/NHD13 transgenic mice developed an immature T-cell leukemia with an immunophenotype similar to double-negative 1 (DN1) or DN2 thymocytes. Idh2R140Q/NHD13 leukemic cells were enriched for an early thymic precursor transcriptional signature, and the gene expression profile for Idh2R140Q/NHD13 DN1/DN2 T-ALL closely matched that of human early/immature T-cell precursor (EITP) acute lymphoblastic leukemia (ALL). Moreover, recurrent mutations found in patients with EITP ALL, including KRAS, PTPN11, JAK3, SH2B3, and EZH2 were also found in Idh2R140Q/NHD13 DN1/DN2 T-ALL. In vitro treatment of Idh2R140Q/NHD13 thymocytes with enasidenib, a selective inhibitor of mutant IDH2, led to a marked decrease in leukemic cell proliferation. These findings demonstrate that Idh2R140Q/NHD13 mice can serve as a useful in vivo model for the study of early/immature thymocyte precursor acute lymphoblastic leukemia development and therapy. SIGNIFICANCE: T-cell leukemia induced in Idh2R140Q/NUP98-HOXD13 mice is immunophenotypically, transcriptionally, and genetically similar to human EITP ALL, providing a model for studying disease development and treatment.

Mutant IDH1 inhibitors activate pSTAT3-Y705 leading to an increase in BCAT1 and YKL-40 levels in mutant IDH1-expressing cells

IDH1 mutations are frequent and early events in gliomas. Mutant IDH1 produces D-2HG that causes epigenetic changes by increasing histone and DNA methylations, thereby contributing to tumor growth. Mutant IDH1 rewires metabolism and endows a few therapeutic vulnerabilities in cells. But, mutant IDH1 inhibitor(s) treatments reverse these therapeutic vulnerabilities by increasing cell growth. Nevertheless, it is unclear how mutant IDH1 inhibitor(s) increases cell growth. As mutant IDH1 inhibitor(s) increase cell growth, therefore we asked whether mutant IDH1 inhibitor(s) activate oncogenes in mutant IDH1-expressing cells. To answer this question, we used allosteric mutant IDH1 inhibitors to treat mutant IDH1-expressing HT1080 cells, and examined for activation of oncogenes by assessing the levels of our read-outs: BCAT1 and YKL-40. We found that mutant IDH1 inhibitors' treatments increased BCAT1 and YKL-40 levels in HT1080 cells. Next, we observed that mutant IDH1 inhibitors activated STAT3 by phosphorylation at Tyr-705 position (pSTAT3-Y705) and its nuclear translocation. Upon examining the molecular mechanism of pSTAT3-Y705 activation in mutant IDH1 inhibitor-treated cells, we found that mutant IDH1 strongly bound STAT3, but mutant IDH1 inhibitor treatment decreased mutant IDH1-STAT3 binding. Furthermore, we observed that STAT3-knockdown and pharmacological inhibition of STAT3 attenuated the mutant IDH1 inhibitor-mediated increase in BCAT1 and YKL-40 levels, whereas STAT3 overexpression and Interleukin-6 (STAT3 activator) treatments increased BCAT1 and YKL-40 levels. We conclude that mutant IDH1 inhibitors activate the oncogenic transcription factor-STAT3 leading to an increase in BCAT1 and YKL-40 levels in mutant IDH1-expressing cells.

Predicting Response to IDH1/IDH2 Inhibitors Beyond IDH Mutations Using a Computational Model and Its Validation: A Beat AML Project Study

Predicting Response to IDH1/IDH2 Inhibitors Beyond IDH Mutations Using a Computational Model and Its Validation: A Beat AML Project Study

Synergistic Activity of IDH1 Inhibitor Bay-1436032 with Azacitidine in IDH1 Mutant Acute Myeloid Leukemia

Background: Only about one third of AML patients with mutant IDH1 respond to IDH1 inhibitor monotherapy with a median response duration of 7 months, indicating the strong need for combination treatments. We have previously shown that BAY-1436032 is a highly effective oral pan-mutant IDH1 inhibitor, which has strong anti-leukemic activity in patient derived xenograft (PDX) models of IDH1 mutant leukemia in vivo. In the present study, we assessed the combination of azacitidine with IDH1 inhibitor BAY-1436032 in a preclinical PDX model of IDH1 mutated AML.Methods: We tested the activity of BAY-1436032 alone or in combination with escalating doses of azacitidine in IDH1 mutated and IDH1 wildtype AML patient cells in vitro evaluating the effects on colony formation, apoptosis, cell cycle, differentiation and global gene expression changes. Leukemic cells from an AML patient with mutated IDH1, NPM1, FLT3-TKD and NRAS were xenografted in immunocompromised mice and treatment was started 28 days after transplantation. The control groups were treated with either vehicle, BAY-1436032 150 mg/kg once daily p.o. continuously, or azacitidine 1 mg/kg once daily s.c. days 1-5, repeated once after 28 days. The test groups were treated with BAY-1436032 and azacitidine in the doses mentioned above either starting both drugs on day 1 (parallel group) or starting azacitidine on day 1 but BAY-1436032 on day 6 (sequential group). The treatment was stopped after 84 days.Results: Our IDH1 inhibitor reduced colony formation specifically in human IDH1 mutated AML cells, while IDH1 wildtype cells were not affected (IC50 100 nM). Combination of BAY-1436032 (100 nM) with azacitidine 100 nM further reduced colony formation by 50% in IDH1 mutated AML cells, while the IC50 for IDH1 wildtype AML cells was not reached even at 1000 nM azacitidine in the presence of 100 nM BAY-1436032. For in vivo experiments human AML cells from an IDH1 mutated AML patient were transplanted into sublethally irradiated NSG mice and treatment was started four weeks after transplantation. Leukemic cells in peripheral blood constantly increased in vehicle and azacitidine treated mice, while leukemic cells declined from week 4 until the end of treatment at week 12 in mice treated with BAY-1436032 alone as well as in the parallel and sequential combination groups treated with BAY-1436032 and azacitidine. However, in week 20 (8 weeks after stopping the BAY-1436032 treatment) leukemia relapsed in the BAY-1436032 alone as well as the sequential combination groups. Interestingly, the frequency of leukemic cells remained low in peripheral blood in mice treated in parallel with the combination of BAY-1436032 and azacitidine even at 24 weeks after starting the treatment (Figure 1). To obtain mechanistic insights into the efficacy of the combination treatment we treated human IDH1 mutant and IDH1 wildtype AML cells in vitro with either 100 nm BAY-1436032 or 100 nm azacitidine alone or in combination (in parallel). Vehicle treatment served as control. There were no significant differences in percentage of apoptotic cells between BAY-1436032 or azacitidine treatment as single agents or in combination. However, the proportion of cells in S phase of the cell cycle was synergistically decreased by the combination treatment compared with either monotherapy or vehicle. Gene expression profiling showed that the target genes of EGR1-3 and GFI1 transcription factors were upregulated by the combination treatment over BAY-1436032 treatment alone, while target genes of NFkB and its targets ELF1 and STAT1/3 were down regulated, suggesting synergy in induction of differentiation and inhibition of self-renewal of the combination treatment through distinct pathways.Conclusion: Our study provides the first evidence of synergistic activity of an IDH1 inhibitor with hypomethylating agents through inhibition of the cell cycle by dysregulating EGR, GFI1 and NFkB signaling, and strongly argues for simultaneous application of the IDH1 inhibitor BAY-1436032 with azacitidine in future clinical trials. Clinical development is ongoing with phase 1 studies using BAY-1436032 in IDH1 mutant solid tumors and AML.Figure 1: Development of human IDH1 mutated AML in peripheral blood of NSG mice treated with BAY-1436032 and azacitidine alone or in combination (PRL, in parallel; SEQ, sequential, i.e. first azacitidine days 1-5, then BAY-1436032 from day 6 onwards). [Display omitted] DisclosuresChaturvedi:Bayer Pharma AG, Berlin, Germany: Research Funding. Kaulfuss:Bayer Pharma AG, Berlin, Germany: Employment. Panknin:Bayer Pharma AG, Berlin, Germany: Employment. Wagner:Bayer Pharma AG, Berlin, Germany: Employment, Equity Ownership. Jeffers:Bayer Pharma AG, Whippany, NJ, USA: Employment. Haegebarth:Bayer Pharma AG, Berlin, Germany: Employment, Equity Ownership.

Impact of Next-Generation Sequencing on Treatment Choices for Patients with Acute Myeloid Leukemia

INTRODUCTION: Until recently, therapy options for patients with acute myeloid leukemia (AML) were limited. This is changing due to advent of CLIA-approved next generation sequencing (NGS) panels and development of multiple new anti-leukemia agents. Examples are the recent approval of a FLT3 and IDH2 inhibitors. Multiple other targeted agents are currently being studied in clinical trials for AML. The aim of our study was to evaluate the impact of a NGS panel on the clinical practice at a major clinical trial research center. We report the experience in 1171 patients with AML who had a NGS panel evaluation as part of their clinical care during 2012-2015. METHODS : We included all AML patients for whom an NGS-based analysis for the detection of somatic mutations in the coding sequence of a total of 28 or 53 genes was performed between October 2012 and October 2015. Clinical and demographic data were obtained from clinical records. 17 genes ( ALK, CSF1R, FGFR1, FGFR2, FGFR3, FLT3, IDH1, IDH2, JAK2, KDR, KRAS, NPM1, NRAS, PDGFRA, PRPN11, RET and TP53) were considered potentially actionable due to the possibility to be targeted with established or investigational therapies, either directly or indirectly. Statistical analyses were performed with the IBM SPSS Statistics 23.0 software. RESULTS: A total of 732 patients (62.5%) were evaluated using the 28 gene panel and 439 patients (37.5%) using the 53 gene panel. 36% of patients were evaluated frontline (n=421), 27.6% during treatment (n=313) and 37.4% (n=437) at relapse. The median time to inform the results were 9 days (range 2-90). Median age at AML diagnosis was 62 years (range 10-92). Baseline characteristics are shown in Table 1. 881 patients (75%) had at least one detectable mutation: 669 (57.1%) on a potential actionable gene and 502 (42.9%) on non-actionable genes. The most frequently potentially actionable mutation was IDH2 (n=196; 16.7%), followed by TP53 (N=174; 14.9%), NPM1 (n=163; 13.9%), NRAS (n=129; 11.0%), IDH1 (n=116; 9.9%) and KRAS (n=66; 5.6%), present on more than 5% of the patients. The frequency of all identified mutations is shown in Fig. 1A. A total of 421 patients (36.0%) were evaluated at initial diagnosis, with 404 patients subsequently starting therapy for their AML. 277 (65.8%) of these had potentially targetable alterations, and only 5 (1.8%) of these patients were treated with a targeted agent. 437 patients (37.4%) patients were evaluated at the time of relapse/refractory with 388 patients (88.6%) starting new therapy. 281 (72.4%) of these treated patients had a targetable mutation and 46 (16.4%) of them were treated with a target agent. Therefore, considering only patients with an actionable mutation, patients with relapse/refractory AML were more likely to receive targeted therapy than those at diagnosis (16.4% vs 1.8%, p The 614 remaining patients (91.8%) never received a targeted therapy. 9.5% of these patients (n=58) never return to the institution after the mutation analysis and 16.6% (n=102) responded to another therapy. For the remaining 434 patients, documentation of discussion of enrollment in a clinical trial was present on 82.9% (n=358): 85.8% (n=307) enrolled in a clinical trial not involving actionable mutations and 10.1% (n=36) were not eligible owing to poor status or other causes. CONCLUSION: NGS technologies can be used to identify a significant proportion of patients (57%) with potentially actionable mutations. However, until 2015, only a small proportion of patients (8.2%) with AML and actionable alterations were treated with targeted therapy. Relapse/refractory patients were more likely to receive targeted agents than those at initial diagnosis. Possible reasons are: 1) delays in NGS results and physicians not willing to wait to start treatment; 2) presumption that standard therapies may be better than investigational target agents. Personalized medicine based on mutation profiles in AML remains an area under development, but a dramatic increase in the proportion of patients treated with a target agent were observed in 4 years. The results of current clinical trials may change the way we treat these patients. Disclosures Takahashi: Symbio Pharmaceuticals: Consultancy. Jabbour: Bristol-Myers Squibb: Consultancy. Cortes: ARIAD: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Teva: Research Funding; Sun Pharma: Research Funding; BMS: Consultancy, Research Funding. DiNardo: AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding. Daver: Otsuka America Pharmaceutical, Inc.: Consultancy; Bristol-Myers Squibb Company: Consultancy, Research Funding; Kiromic: Research Funding; Jazz: Consultancy; Pfizer Inc.: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Incyte Corporation: Honoraria, Research Funding; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Immunogen: Research Funding; Karyopharm: Consultancy, Research Funding; Daiichi-Sankyo: Research Funding. Pemmaraju: Cellectis: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria; Stemline: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria, Research Funding. Kantarjian: Amgen: Research Funding; ARIAD: Research Funding; Novartis: Research Funding; Bristol-Meyers Squibb: Research Funding; Delta-Fly Pharma: Research Funding; Pfizer: Research Funding.

Barriers to Successful Implementation of Precision Medicine to the Treatment of Refractory AML: Lessons Learned

Tremendous genomic heterogeneity has been reported for patients with acute myeloid leukemia (AML), as well as variability in gene expression. We have developed a custom high throughput drug sensitivity (HTS) assay enabling testing of ~150 drugs and combinations, both FDA approved and investigational, including chemotherapy agents and targeted inhibitors. We enrolled advanced relapsed/refractory AML patients (pts) on two successive trials, NCT01872819 “Treatment for Relapsed / Refractory AML Based on a High Throughput Drug Sensitivity Assay” and NCT NCT02551718 “Individualized Treatment for Relapsed/Refractory Acute Leukemia Based on Chemosensitivity and Genomics/Gene Expression Data.” The first trial enrolled 16 AML pts (3-5 prior regimens) of whom 14 received single agent study directed treatment, and the second trial (2-6 prior regimens) enrolled 25 AML pts of whom 14 received study directed therapy including drug combinations. We were able to choose chemotherapy drugs for all pts that had IC50's of ≤ 0.1 µM, or regimens that demonstrated synergy for drugs in combination. We have established feasibility as defined by the study, in that HTS assay results were reported at an average of 5.6 days (range 4-10), mutation analysis for 194 genes by MyAML™ reported in 12.5 days (range 9-17) with turnaround from receipt median 8 days (range 7-12) and treatment started at 7.8 (mean), 8 (median) days (range 4-11).In the first trial, 1 pt (5prior regimens), achieved complete remission with incomplete count recovery (CRi) with the single chosen drug, and 1 achieved CR, 1 CRi with subsequent regimens of additional drugs based on screening and published combinations. Median overall survival (OS) was 88 days for all patients, range 7 to 411. For the 2nd trial, where combinations were used, there were 2 CRs, 1 CRi and 3 partial responses. Median OS was 122 days, range 19-379. Of the patients in the 1st trial who had therapy that could have been directed by mutation analysis, there were 4 with FLT3, 1 KIT, 1 IDH2, 1 TP53, 1 FLT3+KIT, 2 FLT3+ IDH2 mutations (10/12 tested). For the 2nd trial there were 2 pts with FLT3D835, 2 TP53, 2 translocations involving FLT3, 1 IDH2 + FLT3-ITD, 1 FLT3-ITD + IDH1, 1 IDH1, 1 FLT3D839, 1 FLT3-ITD + TP53, 1 IDH2, 1 BCR-ABL (13/17 tested), and one patient received imatinib for BCR-ABL, 2 sorafenib for Flt3 mutations, and 1 with a TP53 mutation received decitabine. We had a number of logistical difficulties, including very advanced disease , poor performance status, inability to obtain the drugs with the lowest IC50's or as directed by mutations due to investigational status or lack of insurance approval, and inability to treat pts who returned home to their local physicians. We observed a higher response rate in pts who received drug combinations (8) vs. single agents (1). Although these were just feasibility trials, we also tried to compare their outcomes to a historical control group. We selected 2 matches for each of our pts, total of 50. They were matched to age, length of CR1, prior HCT, prior intense salvage and prior number of total salvages. One limitation was to find controls that had received ≥3 salvages like some of our studied population. Most of the patients in our clinical trial received single drug therapy (64 vs 24%) and non-intense therapy (4 vs 44%), when compared to our historical control group. Although these were very important treatment differences, the median OS did not differ statistically between both groups (88 vs 115 days, p: 0.327).We have gathered clinical data, and data on mutation analysis, gene expression, and drug sensitivity testing, that have been analyzed to determine correlations of specific features with drug resistance. Moreover, we have the ability to confirm functional drug sensitivity to the targeted inhibitors based on mutation data. All these data can contribute to future algorithms to assist in optimizing drug choices.In conclusion, our future iterations of this approach will include 1) only drug combinations, 2) study of pts with only 2 prior regimens, and 3) earlier incorporation of targeted inhibitors such as midostaurin and the new IDH2 inhibitor, enasidenib, into combination regimens. The current salvage options for relapsed/refractory AML have dismal results, as exhibited by our historical control group; only 10% were alive at 1 year. We will therefore pursue alternative approaches for this population, including the optimization of precision medicine methodology. DisclosuresWalter:ADC Therapeutics: Research Funding; Aptevo Therapeutics: Research Funding. Patay:Invivoscribe: Employment. Carson:Invivoscribe, Inc.: Employment. Radich:Amgen: Consultancy; Gilliad: Consultancy; Novartis: Consultancy, Other: lab contracts for bid and service assays; BMS: Consultancy; Ariad: Consultancy; Pfizer: Consultancy. Becker:GlycoMimetics, Inc.: Research Funding.

Morphologic Assessment of Bone Marrow Samples in AML Patients Treated with Inhibitors to IDH1 and IDH2—a Single Center Experience

INTRODUCTION:Mutations in isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) are reported in about 8% and 13% of patients with acute myeloid leukemia (AML), respectively, and are less common in patients with myelodysplastic syndromes. These recurrent mutations in AML lead to neomorphic enzymatic activity resulting in accumulation of 2-hydroxyglutarate, DNA and histone hypermethylation, blockage of cellular differentiation and, ultimately, leukemogenesis. Inhibitors to both IDH1 and IDH2 are being developed and have shown clinically significant responses. Perhaps unsurprisingly, these IDH inhibitors appear to promote the differentiation of myeloblasts.Serial bone marrow biopsies are considered the standard of care for the evaluation of the effectiveness of AML-directed therapies. However, due to the unique mechanism of action of IDH1 and IDH2 inhibitors, the morphologic assessment of response to these drugs may be challenging to pathologists and hematologists who may not be familiar with their use. Here we describe our single-center experience with IDH1 and IDH2 inhibitors with the goal of improving our understanding of the bone marrow topography in treated patients.METHODS:This retrospective study reports on all patients at our institution with refractory or relapsed AML who have been treated with either an IDH1 inhibitor (FT-2102) on a clinical protocol (NCT02719574) or an IDH2 inhibitor (AG-221, Enasidenib) on a compassionate use basis. A bone marrow biopsy and aspiration was performed on all patients after each 4-week cycle of therapy. All baseline and on-treatment bone marrow specimens were reviewed by a dedicated hematopathologist. Due to the small sample size, which is reflective of the low prevalence of this particular patient population, reported results are primarily descriptive in nature.RESULTS:A total of three patients were treated with an IDH1 inhibitor (cases 1,2,3), one of whom was treated in combination with azacitidine (case 3). Two patients were treated with an IDH2 inhibitor (cases 4,5). One patient (case 5) is still undergoing early treatment and therefore a full assessment of response and bone marrow morphology is not included at the time of this writing. Ages ranged from 53 to 71 years. All patients had relapsed and/or refractory AML with a median of three prior lines of therapy. Complete response (CR) to IDH1 or IDH2 inhibitor therapy was seen in 3 out of the 4 evaluable patients (cases 2,3,4). Two of the CRs were associated with incomplete platelet count recovery (CRp).In 3 of 4 patients, the end of cycle 1 bone marrow was markedly hypercellular (>90%) with sheets or large aggregates of myeloblasts suggestive of refractory disease. In case 1, the IDH inhibitor therapy was discontinued. In the other two cases, therapy was continued and CRp was eventually achieved after 2 cycles in case 2 and after 3 cycles in case 4. In cases 2 and 3 the morphology in the bone marrow at the time of remission was considered markedly abnormal by the pathologist. In case 2, the marrow was markedly hypercellular (>95%) with a profound myeloid expansion and increased reticulin fibrosis reminiscent of a myeloproliferative disorder. In case 3, the marrow was again markedly hypercellular (>95%) with marked myeloid and megakaryocytic hyperplasia and significant dysplastic features in all three lineages.CONCLUSION:Bone marrow assessments in patients on IDH inhibitor therapy can be challenging. Clinicians should be aware that responses may be delayed and early discontinuation of therapy may be premature particularly in patients who otherwise appear to be deriving clinical benefit. Pathologists should be made aware of this new class of drugs with a novel mechanism of action which can produce unusual bone marrow morphologies, presumably due to a differentiating effect on leukemic myeloblasts. This may mimic other myeloid malignancies such as myelodysplasia and myeloproliferative neoplasms. Thus, communication between the treating hematologist and interpreting pathologist is important for accurate response evaluations and optimal patient outcomes. Results will be updated at the ASH Annual Meeting including results from case 5 and photomicrographs of representative marrow specimens. DisclosuresNo relevant conflicts of interest to declare.

Characterization of Acute Myeloid Leukaemia (AML) Patients with Elevated Peripheral Blood Plasma D-2-Hydroxyglutarate (D-2HG) and/or Isocitrate Dehydrogenase (IDH) Mutational Status

Isocitrate dehydrogenase (IDH) gene mutations are identifiable in AML preleukemic stems cells that persist during post-induction clinical remission, justifying their targeted therapy. Recent monotherapy trials with mutant IDH1 and IDH2 oral inhibitors have shown a manageable safety profile, durable responses of greater than 6 months, and evidence of induced differentiation of malignant cells. IDH mutations commonly lead to an increased oncogenic metabolite, D-hydroxyglutarate (D-2HG). This is a single institution, multi-site study to examine pre-treatment plasma 2HG levels and corresponding IDH mutational status in patients diagnosed with de novo or recurrent AML (n=50) or high grade myelodysplastic syndromes (n=1). D- and L-2HG were quantified by Chiral chromatography coupled mass-spectrometry (2D GCxGC-TOP-MS). Elevated D-2HG in the peripheral blood with increased D/L 2HG ratio was detected in 17% (9 of 51) of patients having 25% to <1% peripheral blood blasts. DNA sequence analyses were performed by Sanger chain-terminating dideoxynucleotide labelling and the droplet digital PCR approach. SNP-specific digital PCR was markedly more sensitive, effectively identifying 10 IDH mutations in the peripheral blood, including one AML patient with <1 % blast load. These include 9 patients with IDH1R132 point mutations and one localized at IDH2R140. Overall prevalence of detectable IDH mutations (19.6%) was consistent with previously described findings, although detectable IDH2 mutation was under-represented in this limited study. D-2HG positivity correlated with IDH mutational status (p=0.0005, Fisher exact test) particularly among older patients (p=0.048, linear regression). The mean clinical follow-up for all patients was 292.6 ± 292.2 days (median 189 days, range 4 - 1201 days). Median time to progression was 248 days and median survival was 461 days. No significant association was observed between overall survival and plasma D-2HG level (p=0.7896) nor IDH mutational status (p=0.7964, log rank test). Progression-free survival also did not differ significantly among patients with or without an elevated plasma D-2 HG level (p=0.9227) nor IDH mutational status (p=0.5522, log rank test). For patients without the benefit of bone marrow archiving, plasma 2HG quantification represents a rapid and cost-effective surrogate indicator of IDH mutational status (sensitivity 0.8, specificity 0.93). Similar to reports by others, we found that not all patients with IDH1/2 mutations displayed elevated D-2HG. Nonetheless, for patients with baseline elevation of this oncometabolite, longitudinal, plasma-based determination of its trajectory may be effective for post-induction clinical prognostication, and/or as a trigger for second line IDH-targeted intervention following mutational status confirmation. DisclosuresNo relevant conflicts of interest to declare.

Genetically-Defined AML Subgroups Represent Diverse Epigenetic Heterogeneity Landscape

Epigenetic allele (epiallele) complexity and clonal evolution are linked to inferior clinical outcome in acute myeloid leukemia (AML). However, the source of epiallelic heterogeneity in AML is not known. Here we investigated whether the abundance and genomic distribution of epialleles might be linked to canonical recurrent somatic mutations. We performed mutational and methylome sequencing in a cohort of 119 clinically annotated AML patients curated to reflect some of the most common genetic and epigenetic lesions. Specifically, patients were selected based on presence of twelve highly recurrent genetic lesions: t(8;21), t(15;17), INV(16), t(8;21), Del(5/7q), EVI1, t(v;11q23), CEBPA double mutation, mutations in DNMT3A, IDH1/2, NPM1, and FLT3. Also included were biphenotypic patients with CEBPA silencing. We used 14 normal bone marrow CD34+ cells samples (NBM) as controls. Epiallele complexity was measured using orthogonal approaches, including “proportion of discordant reads (PDR)”, epipolymorphism, and Shannon entropy. We also measured the degree of global epiallele shift compared to NBM using Methclone.Strikingly, we found that AMLs with certain specific genetic lesions manifested characteristic and distinct genomic distribution and abundance of epialleles (p = 2.51-10, one-way ANOVA test). Overall, epiallele diversity was linked to inferior clinical outcome (p = 0.048, log-rank test of event-free survival). Moreover, epiallele complexity was significantly associated with high-risk cytogenetic AMLs (p = 0.002, Wilcoxon rank sum test). However, certain favorable risk genetic lesions - t(15;17) and CEBPA double mutation - were linked to high levels of epiallele diversity, while others manifested low burdens, including t(8;21), INV(16), and NPM1 mutations. Similarly, certain high-risk genetic lesions - Del(5/7q), CEBPA silencing, EVI1 - manifested high levels of epigenetic diversity whereas other high-risk alleles including t(v;11q23) and DNMT3A mutations had relatively low burdens. We used tSNE and various clustering metrics to determine the relative similarity in epiallele variance and location among genetically defined AMLs. These data indicated striking differences. For example, patients with t(15;17), INV(16), t(8;21), CEBPA silencing, CEPBA double mutation and t(v;11q23) formed unique and distinct clusters, as well as patients with IDH1/2 mutations but not DNMT3A mutations. Patients with Del(5/7q) and mutations in NPM1 did not form discrete clusters suggesting that these lesions do not drive formation or diversity of specific epiallele sets.We next wondered if cooperation between somatic mutations might increase epiallele diversity. We address this question using genetically engineered mice with uniform genetic backgrounds. We tested: i) Tet2-/-, Flt3ITD and Tet2-/-+ Flt3ITD (T2F3) and ii) Idh2R140Q, Flt3ITD and Idh2R140Q+ Flt3ITD (I2F3). In all cases, we obtained purified LSK cells at similar timepoints before development of leukemia, and normal controls. In all cases, the single mutants (Tet2-/-, Idh2R140Q or Flt3ITD) manifested modest if any increase in epiallele diversity. In contrast, both the T2F3 and I2F3 mice had significantly higher levels of epiallele complexity (p=0.01 and p = 0.006, t-test, respectively compared to the single mutants). Therefore, epiallele diversity can precede transformation to acute leukemia and is enhanced by cooperation between somatic mutations.Given that epiallele diversity is linked to inferior outcome, we wondered if it could be potentially reversed using epigenetic targeted therapy. Therefore, we treated T2F3 leukemic mice with 5'Aza, and I2F3 mice with the IDH2 inhibitor AG221. In T2F3, 5'Aza profoundly reduced epiallelic diversity (p = 0.002, t-test), whereas AG-221 was effective in I2F3 but not as strong as 5'Aza in the Tet2 leukemias (p = 0.03, t-test). Both mouse models were also treated with AC220 (FLT3 inhibitor). However, AC220 - while having some anti-leukemia activity - did not reduce epiallele diversity in either model. Our data show that epiallele complexity is potentially reversible by epigenetic therapy. Perhaps part of the benefit of epigenetic therapy is due to this effect, by reducing population fitness of AMLs. Moreover, we show that genetic lesions are a critical source of epigenetic heterogeneity driving the genomic location and abundance of epialleles. DisclosuresLi:The Jackson Laboratory for Genomic Medicine: Employment. Levine:Qiagen: Equity Ownership; Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership; Roche: Research Funding; Celgene: Research Funding.