EZH2 Mutations Research Articles

Mutational patterns and their correlation to CHIP-related mutations and age in hematological malignancies

AbstractAcquired somatic mutations are crucial for the development of most cancers. We performed a comprehensive comparative analysis of the mutational landscapes and their correlation with CHIP-related (clonal hematopoiesis of indeterminate potential) mutations and patient age of 122 genes in 3096 cases of 28 different hematological malignancies. Differences were observed regarding (1) the median number of mutations (highest, median n = 4; lowest, n = 0); (2) specificity of certain mutations (high frequencies in atypical chronic myeloid leukemia [aCML; ASXL1, 86%], follicular lymphoma [FL; KMT2D, 87%; CREBBP, 73%], hairy cell lymphoma [BRAF, 100%], lymphoplasmacytic lymphoma [MYD88, 98%; CXCR4, 51%], myeloproliferative neoplasm [MPN; AK2, 68%]); (3) distribution of mutations (broad distribution within/across the myeloid/lymphoid lineage for TET2, ASXL1, DNMT3A, TP53, BCOR, and ETV6); (4) correlation of mutations with patient's age (correlated with older age across entities: TET2, DNMT3A, ASXL1, TP53, EZH2, BCOR, GATA2, and IDH2; younger age: KIT, POT1, RAD21, U2AF2, and WT1); (5) correlation of mutation number per patient with age. Moreover, we observed high frequencies of mutations in RUNX1, SRSF2, IDH2, NRAS, and EZH2 in cases comprising at least 1 DTA (DNMT3A, TET2, ASXL1) mutation, whereas in cases without DTA mutations, TP53, KRAS, WT1, and SF3B1 were more frequent across entities, suggesting differences in pathophysiology. These results give further insight into the complex genetic landscape and the role of DTA mutations in hematological neoplasms and define mutation-driven entities (myelodysplastic syndrome/MPN overlap; secondary acute myeloid) in comparison with entities defined by chromosomal fusions (chronic myeloid leukemia; myeloid/lymphoid neoplasm with eosinophilia).

Co-occurrence of immature T-lymphoblastic lymphoma and acute myeloid leukemia\u2014microenvironment-dependent lineage differentiation derived from a common progenitor?

Mixed phenotype acute leukemia (MPAL) is an uncommon disease characterized by currently only limited knowledge concerning biology, clinical presentation, and treatment outcome. We here describe a most unusual case of simultaneous occurrence of T-lymphoblastic lymphoma in cervical and mediastinal lymph nodes and acute myeloid leukemia in the bone marrow (BM) successfully treated with allogeneic stem cell transplantation (SCT). Although the blasts in both locations showed additional aberrant expression of other lineage markers (even B-cell markers), diagnostic criteria of MPAL were not fulfilled either in the LN or in the BM. We performed next generation sequencing (NGS) with the objective to look for common genetic aberrations in both tissues. Histology, immunohistochemistry, flow cytometry, AML-associated genetic alterations (FLT3, NPM1, KIT D816V, CEPBA), and clonal T-cell receptor β and γ gene rearrangements were performed according to routine diagnostic workflows. Next generation sequencing and Sanger sequencing were additionally performed in BM and LN. Somatic mutation in the EZH2 gene (p.(Arg684Cys)) was detected in the BM by NGS, and the same mutation was found in the LN. Since an identical genetic aberration (EZH2 mutation) was detected in both locations, a common progenitor with regional dependent differentiation may be involved.

IBCL-419: Non-Invasive Quantitative Analysis of EZH2 Mutations in Follicular Lymphoma

Context Follicular lymphoma (FL) is the most common type of indolent non-Hodgkin lymphoma, with an average overall survival rate of 15 years. The prolonged disease course raises the need for an effective disease monitoring system, which is hampered by the limited specificity and sensitivity of the most widely used, imaging-based PET-CT method. Molecular analysis of tumor-derived circulating DNA (ctDNA), frequently referred to as liquid biopsy, is a promising minimally invasive disease monitoring tool for patients suffering from malignancies. Recent studies interrogating the genomic background of FL have revealed activating mutations in the epigenetic regulator EZH2 gene in 25% of patients. Objective In our study, we tested the potential of liquid biopsy to detect and monitor EZH2 mutations in plasma samples of patients with EZH2-mutant FL treated with immunochemotherapy. Patients and Methods Eighty-five blood plasma samples were collected from 41 EZH2 mutant patients. Cell-free plasma DNA was extracted using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Germany). The EZH2 mutation status was assessed using a multiplex digital droplet PCR (ddPCR, Bio-Rad Laboratories, USA) approach, detecting seven different EZH2 hotspot mutations using only two PCR reactions. The molecular results were correlated with PET-CT images. Results Among the 41 patients, 36 responded to treatment (88%); meanwhile, 5 patients did not (12%). We detected an EZH2 mutation in 89% of the samples collected prior to treatment (17/19). Similarly, 86% of the samples (6/7) collected during therapy from patients not responding to treatment proved to be EZH2 mutated; meanwhile, only 22% of the samples were positive from responding patients (6/33). As expected, all samples collected during remission were EZH2 wild-type. EZH2 variant allele frequencies (VAF) correlated with the total metabolic tumor volume detected on the PET-CT scans. We also demonstrated spatial heterogeneity of EZH2 mutations in two cases, where different EZH2 mutations deriving from distinct anatomical sites could simultaneously be detected in the plasma. The median sensitivity of our ddPCR approach was 0.15%, with EZH2 VAFs ranging between 0.2 and 74%. Conclusions Our results demonstrate that liquid biopsy offers a sensitive, radiation-free, minimally invasive monitoring method for EZH2-mutant FL patients.

Poster: IBCL-419: Non-Invasive Quantitative Analysis of EZH2 Mutations in Follicular Lymphoma

Poster: IBCL-419: Non-Invasive Quantitative Analysis of EZH2 Mutations in Follicular Lymphoma

Abstract 2132: Ezh2-Y641F mutation interacts with Stat3 to regulate the melanoma tumor immune response via disruption of IFN signaling

Abstract Cancer genetic sequencing studies have identified systematic alterations of epigenetic and chromatin modifiers in most cancers, consistent with the fact that chromatin modifiers, such as the Polycomb Repressive Complex 2 (PRC2), regulate the expression of tumor suppressors. In melanoma, 10% of patients exhibit amplifications or point mutations in EZH2, the enzymatic subunit of PRC2; however, the oncogenic mechanisms of EZH2 mutations in melanoma remain elusive. Previously, we observed that expression of Ezh2-Y641F in melanoma cells resulted in upregulation of IFN-related genes. We thus sought to better understand this mutation and the consequences of IFN signaling changes on tumor growth and the tumor immune response. We discovered that Ezh2-Y641F directly interacts with and methylates Stat3, a known regulator of interferon signaling. To understand the effects of this interaction, we performed ChIP-seq for Ezh2 and Stat3 in melanoma cells and found that the binding profiles of Stat3 and Ezh2 are greatly changed in Ezh2-Y641F cells. Furthermore, Ezh2 and Stat3 binding sites co-localized at several genomic loci in Ezh2-Y641F cells, including a region that regulates Ezh2. Knockdown of Stat3 in melanoma cells decreased Ezh2 expression and partially reduced expression of the IFN-related genes that were initially upregulated by Ezh2-Y641F. Analysis of the tumor-infiltrating immune cells by flow cytometry revealed increased CD8+ T cells and decreased myeloid cells in Ezh2-Y641F melanomas, consistent with a more favorable immune response, although tumor growth was unchanged. Genetic inhibition of Stat3 expression via shRNA rescued this phenotype. Our data suggest a novel interaction between the PRC2 complex and Stat3 in melanoma, which has significant physiological consequences on the tumor immune response. Molecular and genomic analysis suggest that Ezh2 and Stat3 together regulate gene expression, including Ezh2 itself. Overall, these data highlight the significant role of chromatin regulators on the tumor immune response. Citation Format: Sarah M. Zimmerman, Samantha Nixon, George P. Souroullas. Ezh2-Y641F mutation interacts with Stat3 to regulate the melanoma tumor immune response via disruption of IFN signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2132.

Genetic Characterization and Clinical Impact of Loss of MHC Class I and II Expression in De Novo Diffuse Large B-Cell Lymphoma

Introduction: Immune recognition by T cells requires the presentation of processed peptides in association with major histocompatibility complex class I (MHC-I) and class II (MHC-II) molecules. Among the tumor immune escape mechanisms described to date, alterations in the expression of MHC molecules play a crucial role in the development of diffuse large B-cell lymphoma (DLBCL). Loss of MHC-I or -II expression in DLBCL can occur through multiple mechanisms, including deletion of the MHC-I and -II locus, loss of function mutations of B2M, and genetic alterations of CIITA ; however, the role of additional genetic and epigenetic mechanisms remains largely unknown. With regard to clinical significance, a number of studies have reported that low MHC-II expression on tumor cells is associated with shorter survival in DLBCL, while the prognostic effect of MHC-I expression has not been systematically evaluated in a large, homogeneously treated cohort. In addition, although the frequency of loss of MHC-II expression differs between ABC- and GCB-DLBCL cell of origin (COO) subtypes, the prognostic impact remains ill-defined. Here, we conducted a comprehensive phenotypic and genetic analysis in a population-based DLBCL cohort to investigate the genetic background and clinical relevance of loss of MHC-I and -II expression.Method: We analyzed biopsies from 347 patients newly diagnosed with de novo DLBCL and uniformly treated with R-CHOP at the BC Cancer Agency. High-resolution copy number analysis was performed using Affymetrix SNP 6.0 arrays. Mutation status was determined using deep targeted re-sequencing of the coding exons of 57 genes with a Truseq Custom Amplicon assay (Illumina) and/or Fluidigm Access Array chips, and RNAseq. Immunohistochemical staining of MHC-I and -II was performed on tissue microarrays (n=332). COO was assigned by gene expression (Lymph2Cx assay) in 323 cases (183 GCB, 104 ABC and 36 unclassifiable).Results: We found the following MHC-I and -II staining patterns:membrane-positive (n=188 (57%) and n=233 (72%), respectively), cytoplasmic only-positive / membrane-negative (n=86 (26%) and 31 (10%), respectively) and negative staining for both membrane and cytoplasmic (n=55 (17%) and n=59 (18%), respectively). Overall, membranous MHC-I and -II expression was not detected in 141/329 (43%) and 90/323 (28%) cases, respectively. While the frequency of loss of MHC-I expression was not different between COO subtypes, loss of MHC-II expression occurred more often in ABC-DLBCL (36% vs 22%, p=0.026). This is consistent with previous observations showing that MHC-II expression is reduced during plasmacytic differentiation and in ABC-DLBCL. Loss of MHC-I expression was associated with B2M mutation and deletion as well as with HLA-C deletions (all p <0.001, Fisher's exact test). Interestingly, mutations of EZH2 and GNA13 were detected more frequently in cases that had loss of MHC-I expression (p <0.001 and p=0.004, respectively). Moreover, mutations of CIITA and RFXAP (components of the MHC-II enhanceosome), and mutations of PRDM1 and TNFAIP3, ABC-DLBCL-specific mutations, were significantly enriched in MHC-II negative cases. Of note, mutations of B2M and EZH2 were significantly associated with loss of MHC-II expression, as well (p=0.001 and p=0.016, respectively). The associations between EZH2 mutation and MHC-I and -II expression were also observed when the analysis was restricted to GCB-DLBCL (both p <0.0001).With a median follow up of 6.5 years for living patients, the loss of MHC-I expression was not associated with survival in the entire DLBCL cohort, as well as within each COO subtype. Although there was also no significant survival difference according to the MHC-II expression status in the entire DLBCL cohort, loss of MHC-II expression was associated with an inferior 5y-time to progression (TTP, 59% vs 79%, p=0.007) in the GCB subtype. Interestingly, in the ABC subtype, loss of MHC-II expression was associated with a superior 5y-TTP (64% vs 40%, p=0.03).Conclusion: Our approach using next generation sequencing and high resolution SNP arrays uncovered the genetic background, as well as the clinical impact of loss of MHC-I and-II expression in a uniformly R-CHOP-treated large population-based cohort of DLBCL patients. Our results warrant further studies investigating a potential mechanism of EZH2 mutations regulating MHC-I and -II expression in DLBCL. DisclosuresSavage:Bristol-Myers Squibb: Honoraria; Seattle Genetics: Consultancy, Honoraria; Roche: Research Funding; Celgene: Consultancy; Merck: Honoraria. Sehn:Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Morin:Epizyme, Inc: Consultancy. Connors:Genentech: Research Funding; Bayer Healthcare: Research Funding; Lilly: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Amgen: Research Funding; Janssen: Research Funding; F Hoffmann-La Roche: Research Funding; NanoString Technologies: Research Funding; Bristol-Myers Squibb: Research Funding; Cephalon: Research Funding; Takeda: Research Funding; NanoString Technologies, Amgen, Bayer, BMS, Cephalon, Roche, Genentech, Janssen, Lilly, Merck, Seattle Genetics, Takeda,: Research Funding. Scott:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; BCCA: Patents & Royalties: Patent describing molecular subtyping of DLBCL licensed to NanoString Technologies. Patent describing measurement of the proliferation signature in MCL.. Steidl:Affimed Therapeutics: Consultancy; Juno Therapeutics: Consultancy.

Discovery of Candidate Predictors of Response to Tazemetostat in Diffuse Large B-Cell Lymphoma and Follicular Lymphoma Using NGS Technology on ctDNA Samples Collected Pre-Treatment

Discovery of Candidate Predictors of Response to Tazemetostat in Diffuse Large B-Cell Lymphoma and Follicular Lymphoma Using NGS Technology on ctDNA Samples Collected Pre-Treatment

Chromatin Regulator Mutations Are Drivers of Clonal Evolution in the Early Multipotent HSC Compartment of Primary Myelofibrosis

The definition of initiating genetic events remains up to date the main research quest in MPN disease biology. Identification of characteristic mutational clusters has shed more light on the complexity of various MPN genotypes, even though no specific genetic lesion or mutational order can be exclusively linked to the different MPN subtypes. Our aim is to determine the sequence of mutational clusters that orchestrate the expansion of aberrant clones in MPN with respect to the prime hematopoietic stem cell compartment.Genotyping for 54 MPN related gene mutations (100 MPN Patient cohort with myelofibrosis) indicated high incidence of mutations in chromatin regulator genes ASXL1 (40%) and EZH2 (14%). In 11% of the patients both ASXL1 and EZH2 gene mutations were detected, which correlated with significantly lower hemoglobin levels, higher leukocyte counts, higher numbers of additional mutations and higher risk score.In our former work we reported the identification of an expanded Lin-CD133+CD34+ neoplastic HSC population in patient peripheral blood that reproduces PMF parameters and fibrosis in vivo. Analysis of this aberrant HSC in the single cell level revealed the heterogeneity of the malignant clones and the mutational hierarchies that propagate myeloproliferation. From this analysis we report that ASXL1 and/or EZH2 mutations precede JAK2 signalling related genetic lesions (JAK2, CALR, MPL) or other epigenetic modifier mutational groups (DMT3A, TET2). Neoplastic stem cells bearing both ASXL1/EZH2 mutations exhibit higher levels of engraftment in vivo and limited erythroid colony output in vitro .In order to determine the exact contribution of chromatin regulator mutations with regard to clonal hematopoiesis determined by changes in prime HSC dynamics we established a sorting strategy for the isolation of early LT-HSC circulating in MF patient peripheral blood. By usage of 27 lineage specific and 6 additional HSC specific markers we sorted HSC on the apex of the hierarchy with the phenotype; 27xLin-CD34+CD38-CD90+CD93+CD49f+CD133+ and all classes of early and more committed progenitors from 11 patients with ASXL1 and/or EZH2 mutations. Our data indicate that both mutations emerge in the LT-HSC fraction (10/11 patients) dominate aberrant myelopoiesis and can be also detected in mature T cells (3/11 Patients). JAK2-V617F mutation was not detected in the early HSC fraction.The incidence of EZH2 mutations with or without the synergistic effect of ASXL1 mutations, is correlated with the expansion of morphologically aberrant erythroid progenitors in patient peripheral blood, which are found to be arrested in G0 phase of the cell cycle.Our results suggest that ASXL1 and EZH2 mutations are the drivers of clonal hematopoiesis in MPN and occur early at the multipotent HSC compartment. EZH2 mutations in PMF represent the first genetic cluster that correlates with aberrant erythropoiesis in these patients. DisclosuresNo relevant conflicts of interest to declare.

RUNX1 Mutations in MDS, s-AML and De Novo AML: Differences in Accompanying Cytogenetic and Molecular Genetic Alterations, Clonal Architecture and Outcome

Background: RUNX1 mutations are frequent in de novo AML, s-AML and MDS cases. AML with mutated RUNX1 constitutes a new provisional entity, was reported to be associated with a distinct pattern of cytogenetic and molecular genetic abnormalities and with an adverse prognosis. However, differences in accompanying aberrations and prognosis between the respective entities have not been analyzed to date. Aims: Evaluation and comparison of associated genetic alterations, prognosis and presence of RUNX1 in the main clone in (1) de novo AML, (2) s-AML and (3) MDS cases with RUNX1 mutations. Patient cohorts and methods: The total cohort comprised 546 cases, 379 with de novo AML, 73 with s-AML and 94 MDS cases with RUNX1 mutations. The total cohort included 348 male and 198 female patients, median age was 72 years (range: 18-91 years). All patients were investigated using chromosome banding analysis (CBA) and amplicon sequencing of RUNX1 . All cases with s-AML and MDS and 141 cases with de novo AML were subject to further mutation analyses of ASXL1, BCOR, CBL, DNMT3A, ETV6, EZH2, FLT3 -ITD , GATA2, IDH1, IDH2, JAK2, KRAS, MPL, SF3B1, SRSF2, TET2, TP53, U2AF1 and ZRSR2 . Variants of unknown significance were excluded from statistical analysis. Results: In the total cohort, CBA analysis revealed a normal karyotype (NK) in 56% of cases, 18% harbored trisomies, 2% a complex karyotype (>3 abnormalities), 23% other aberrations. 56% of cases with trisomies harbored +8, in 26% of patients +13 was found. In the subgroups, the proportion of cases with NK was largest in cases with MDS (73%), followed by s-AML (58%) and de novo AML (52%) (p Mutation analyses of 308 cases (141 de novo AML, 73 s-AML, 94 MDS) revealed SRSF2 (43%), ASXL1 (40%), TET2 (26%), SF3B1 (16%), DNMT3A (14%), EZH2 (12%) and IDH2 (12%) as most frequently mutated genes. In MDS, a higher frequency of ASXL1, TET2 and EZH2 mutations was observed compared to the other cases (ASXL1 : 57%, 44% and 27% in MDS, s-AML and de novo AML, respectively, p=0.001; TET2 : 38%, 33% and 14%, p=0.011; EZH2 : 22%, 7%, 7%, p To analyze if the RUNX1 mutation was present in the main clone or in a subclone of the respective patient, the RUNX1 mutation load (ML) was compared to the ML of accompanying mutations. RUNX1 mutation was considered to constitute the main clone, if the difference between the MLs was ±10%. In the majority of cases with de novo AML and s-AML, RUNX1 was present in the main clone (80% and 82%, respectively), whereas this was the case in merely 64% of MDS patients. Median overall survival (OS) in the total cohort was 14 months. OS was found to be significantly shorter in s-AML (8 months) cases compared to de novo AML (14 months, p=0.035) and MDS (15 months, p=0.017). Mutations in EZH2 and SF3B1 negatively impacted OS in the total cohort (8 vs. 14 months, p=0.024; 8 vs. 14 months, p=0.036), while SRSF2 mutations showed a trend towards a better outcome (16 vs. 13 months), which was especially pronounced in s-AML (16 vs. 6 months). ASXL1 and DNMT3A mutations depicted a negative impact on OS in cases with de novo AML only (9 vs. 20 months, p= 0.033, 11 vs. 16 months, p=0.049), whereas EZH2 and TET2 mutations caused a shorter OS in MDS (14 vs. 17 months, p=0.049; 9 vs. 18 months, p=0.039). Conclusion: 1) MDS cases with RUNX1 mutations differ from RUNX1 -mutated AML by accompanying cytogenetic aberrations (high frequency of NK, lack of +13) and additional mutations (high frequency of ASXL1, TET2 and EZH2 mutations). 2) In the majority of de novo AML and s-AML cases, RUNX1 mutations constitute the main clone. 3) Patients with RUNX1 mutated s-AML had a poorer OS than patients with de novo AML and MDS, although the presence of SRSF2 mutations counterbalances this effect. Disclosures Stengel: MLL Munich Leukemia Laboratory: Employment. Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Mutations in DNMT3A, U2AF1, and EZH2 Identify Intermediate-Risk Acute Myeloid Leukemia Patients with Poor Outcome after CR1

Introduction: Intermediate-risk acute myeloid leukemia (IR-AML) is a clinically heterogeneous disease, for which optimal post-remission therapy has been debated. Several studies using next-generation sequencing have identified important prognostic information and led to modifications in risk stratification. However, the utility of this genetic information in decision making for IR-AML has yet to be elucidated. We investigated the clinical relevance of recurrent driver gene mutations in a well-characterized cohort of IR-AML patients in first complete remission (CR1) after induction chemotherapy.Methods: A total of 1,589 AML patients treated at the Cleveland Clinic (CC) between 2002 and 2016 were screened for eligibility, of whom 825 were in European Leukemia Net (ELN) IR category. Of them, 100 IR-AML patients, who had mutational information at diagnosis and achieved CR after ≥ 1 round of intensive induction were included. CR was defined as <5% bone marrow blasts, absolute neutrophil counts ≥ 1x109/L, and platelet counts ≥ 100x109/L. CR with inadequate count recovery (CRi) was same as CR, except for residual neutropenia or thrombocytopenia. The induction regimen was 7+3, and patients with persistent disease at day 14 marrow were re-induced with 7+3 or 5+2. Patients who did not receive intensive therapy or achieve CR/CRi were excluded. A TruSeq Custom Amplicon panel targeting exons of 62 genes was used for deep sequencing at diagnosis. We used The Cancer Genome Atlas (TCGA) data for validation of our findings.Results: In the CC cohort, median age was 58.5 (24-75) years, 64% had normal cytogenetics, and 31% required >1 induction cycles to achieve CR1. DNMT3A and FLT3-ITD mutations were the most common (19%), followed by RUNX1 (15%), TET2 (12%), IDH2 (12%), NPM1 (12%), ASXL1 (11%), and U2AF1 (11%). The frequency of persistent disease after initial induction was higher in patients with mutations in DNMT3A (47% vs 27%, p= .03) and U2AF1 (55% vs 28%, p= .01), while CRi was more common in patients with BCOR mutation (50% vs 8.5%, p= .02). In univariable analysis, patients with mutations in DNMT3A, U2AF1, and EZH2, and patients who achieved CRi after induction had worse overall (OS) and relapse-free survival (RFS). Patients who had hematopoietic cell transplant (HCT) in CR1 achieved better OS (HR, 0.4; 95% CI, 0.2-0.7; p=0.003) and RFS (HR, 0.3; 95% CI, 0.1-0.6; p< 0.001) than those who did not. Other clinical factors analyzed, such as age, sex, cytogenetics, and the number of induction courses to achieve CR1 were not significant predictors for survival after CR1. To evaluate the impact of these three mutations on risk stratification, we compared patients whose AML harbored any of the mutations with those who did not. The median OS and RFS for patients with mutations were 13 and 9 months, respectively, compared to 41 and 16 months in patients without (p< 0.0001 and p= .003). Similarly, TCGA patients with mutations had significantly poorer OS (median, 9.8 vs 46.8 months, p= .0002) and RFS (median, 9.3 vs 17 months, p= .02). In multivariable analysis, mutations maintained an independent effect on survival (Table 1).Since mutations and HCT were the major predictors of outcome after CR1, we analyzed the outcomes of patients harboring mutations in relation to HCT (Figure 1). In both cohorts, patients who did not have the mutations and underwent HCT had the best outcomes. In the CC cohort, median OS and RFS for patients who had mutations and HCT were 14 and 11 months, respectively, compared to 7.5 and 5 months in patients who did not undergo HCT (p= .04 for OS and p= .002 for RFS). In the TCGA cohort, patients who harbored mutations and did not undergo HCT had significantly worse OS (median, 6.3 vs 24.6 months, p= .001) and RFS (median, 7.8 vs 12.5 months, p= .003). In view of the recently proposed ELN 2017 criteria that place IR patients with RUNX1 or ASXL1 mutations into the adverse risk, we explored outcomes of these patients at the time of CR1. Presence of these mutations did not predict OS and RFS in our cohort, and performance of HCT did not confer a significant survival benefit in patients with these mutations.Conclusions: In two independent cohorts, DNMT3A, U2AF1, and EZH2 mutations were predictors of relapse and OS in IR-AML at CR1, and performance of HCT translated into survival benefit. Our results provide evidence of clinical utility in considering mutation screening to stratify IR-AML patients to guide therapeutic decisions. [Display omitted] DisclosuresSekeres:Celgene: Membership on an entity's Board of Directors or advisory committees. Gerds:CTI BioPharma: Consultancy; Incyte: Consultancy. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fees; Ra Pharma: Consultancy; Apellis Pharmaceuticals: Consultancy. Advani:Pfizer: Consultancy; Takeda/ Millenium: Research Funding.

Aberrant FLT3 Transcription Arised from Polycomb Repressive Complex 2 Inactivation Indiactes a Potential Therapy Target for Pediatric T Cell Acute Lymphoblastic Leukemia

Pediatric T cell acute lymphoblastic leukemia (T-ALL) is an aggressive subtype of pediatric ALL, and is characterized by recurring genetic and epigenetic alterations. Loss-of-function mutations and deletions of Polycomb repressive complex 2 (PRC2) core components, including EED, EZH2 and SUZ12 ,have been identified in around 25% of T-ALL and are associated with diseases progression and poor prognosis. One presented study has demonstrated the specific loss of the repressive mark Lys27 trimethylation of histone 3 (H3K27me3) by abrogating the activity of PRC2 leads to Notch pathway activation; In a mouse model of early T cell precursor ALL (ETP-ALL), inactivation of PRC2 has been revealed to be associated with increasing transcription of early hematopoietic programs and contribute to hyperactive JAK/STAT signaling pathway. However, the detailed mechanism of pediatric T-ALL tumorigenesis driven by PRC2 remains to be further clarified.Through targeted sequencing of PRC2 components of 62 diagnostic pediatric T-ALL samples, we discovered mutations in EZH2 (8/62), SUZ12 (4/62), and EED (2/62) in 12 patients. RNA sequence profiling of the sorted primary samples from these 12 patients revealed a surprisingly upregulated expression of FLT3 and this upregulation was confirmed to be specifically presented in PRC2 mutated patients by qRT-PCR analysis in all the 62 T-ALL primary samples.To explore the interplay between the inactivation of PRC2 and upregulation of FLT3, we constructed EZH2 disrupted Jurkat cell lines by CRISPR-Cas9. QRT-PCR reflected eight-to-twenty-fold increasing of FLT3 mRNA expressions in homozygousknockout clones; ChIP-qPCR showed that EZH2 deletions led to remarkably attenuated H3K27me3 histone mark and prominent enrichment of POLII binding in FLT3 promoter region, which were highly consistent with gene expression levels; Besides, elevation in phosphorylation of STAT5, AKT and ERK also showed high correlation with increased FLT3 expressions, which was reversed by the treatment of FLT3 inhibitors, Sorafeinib and Quizartinib, respectively. This, in turn, suggested that the loss of H3K27me3 caused by PRC2 disruption closely coupled to the activation of FLT3 downstream signaling pathway in human T-ALL.To examine the regulation of FLT3 transcription by PRC2 in hematologic malignances, we exposed cell lines of T-lineage (Jurkat, Molt4, CEM), B-Lineage (Ramos, Raji), and other myeloid malignancies (K562, HL-60, THP-1) to EZH2 inhibitors and observed only T-lineage cell lines specifically presented response to EZH2 inhibitors with increased expression of FLT3, and therefore the activation of downstream pathway. Rather, inhibitions of PRC2 seemed not to uprgulate FLT3 transcriptions in other cell lines.In a model of T-lineage differentiation from immature hematopoietic cells on OP9-DL1 cells exposed to EZH2 inhibitors, lineage-negative, Sca1-positive, and C-kit-positive (LSK) cells, and ETP cells derived from C57BL/6 mice displayed markedly differentiation arrest via flow cytometric analysis, accompanied by upregulation of Flt3 expression and accentuation of down stream signaling pathway.To further test whether the hyperactive signaling pathway mediated by EZH2 -inactivation was a potential therapy target, pediatric T-ALL cells from EZH2 -mutated primary samples were transplanted into NSG mouse and treated with FLT3 inhibitors. Surprisingly, FLT3 inhibitors treated group conferred hypersensitivity to the drugs, and the median survival span from treatment to death in FLT3 inhibitors group was significantly longer than control group treated with PBS (P < 0.0001).In conclusion, our study provides strong evidence that loss of H3K27me3 arised from PRC2 inactivation leads to high level of FLT3 transcription and downstream signaling pathway upregulation in human T-ALL, indicating both a possible mechanism and a potential therapy target for pediatric T-ALL with PRC2 mutations in future clinical practice. DisclosuresNo relevant conflicts of interest to declare.

Gaining a Better “Sense” for ASXL1 Mutations in Myelofibrosis: Assessing the Variable Impact of Frameshift, Nonsense, and Missense Mutations

Introduction: ASXL1 gene mutations commonly occur in myeloid diseases and are associated with inferior prognoses. They can occur as frameshift, nonsense or missense mutations. In myelofibrosis (MF) specifically, ASXL1 mutations are associated with worse overall survival (OS) and leukemia-free survival (LFS). While the general impact of ASXL1 mutations has been well-studied, the variable impact based on type of mutation is less clear. In CMML and MDS, frameshift and nonsense mutations correlate with shorter survival and increased risk of leukemic transformation. In this study, we aimed to analyze the role of ASXL1 mutations in MF while stratifying for type of mutation.Methods: We retrospectively analyzed MF patients seen at our institution who had next-generation sequencing (NGS) performed as part of routine clinical care between 9/1/2013 and 5/1/2017. Laboratory and clinical parameters calculated from first presentation. OS defined from time of NGS assessment.Results: We evaluated 182 MF patients with available molecular profiling. ASXL1 mutations were present in 71 (39%) patients. Among ASXL1 mutations, 44 were frameshift (FS) mutations (62%), 14 were nonsense (NS) mutations (20%) and 13 were missense (MS) mutations (18%). Demographic and clinical features of these groups can be seen in figure 1. Grouped together, ASXL1 mutations were associated with high-risk clinical features such as decreased hemoglobin (p = 0.003), decreased platelet count (p = 0.01), increased bone marrow fibrosis (p = 0.02), transfusion dependence (p = 0.005), increased LDH (p = 0.02), and higher MF-specific prognostic scores (IPSS, DIPSS, and DIPSS+) (p < 0.001, p < 0.001, p = 0.003, respectively). Excluding MS mutations, the correlation with high-risk features was strengthened and new correlations between ASXL1 mutations and leukocytosis (p = 0.006), monocytosis (p = 0.02), and the presence of EZH2 (p = 0.04), NRAS (p = 0.008), and SRSF2 (p = 0.01) mutations emerged. ASXL1 NS mutations were associated with presence of splenomegaly (p = 0.01), increased immature myeloid cells (p = 0.03), and high IPSS (p = 0.04) and DIPSS (p = 0.04) scores when compared to ASXL1 wild-type (WT) patients. ASXL1 MS mutations correlated with decreased hemoglobin (p = 0.03) and decreased bone marrow cellularity (p = 0.007), but did not correlate with prognostic scores.High molecular risk (HMR) mutations (SRSF2, EZH2, IDH1/2) occurred more commonly with ASXL1 FS mutations than with ASXL1 MS mutations (p = 0.003) or ASXL1 WT (p = 0.002). SRSF2 mutations occurred more commonly in patients with ASXL1 FS mutations compared to ASXL1 MS mutations (p = 0.02) and ASXL1 WT (p = 0.03). EZH2 mutations occurred more commonly with ASXL1 FS mutations than with ASXL1 WT (p = 0.02).Including all ASXL1 mutations, there was a trend towards inferior LFS (p = 0.12) when compared to ASXL1 WT. Excluding NS and MS mutations, ASXL1 FS mutations were associated with decreased LFS (p = 0.02). The rate of transformation to blast-crisis was 14% with FS mutations, 0% with NS and MS mutations, and 4% in ASXL1 WT patients. ASXL1 mutations were associated with inferior OS (p = 0.005). When assessed by type of mutation, only FS mutations were associated with inferior OS (p = 0.003). NS mutations were not associated with OS (p = 0.39).Conclusion: The impact of ASXL1 mutations on clinical phenotype and outcome is most clearly demonstrated with frameshift mutations. Nonsense mutations appear clinically significant given their correlation with phenotype and higher-risk disease, but their impact on survival is less clear. Missense mutations do not have an apparent impact in MF. Increased presence of additional high-risk mutations in patients with ASXL1 frameshift mutations suggests a more unstable genome and likely contribute to inferior outcomes. [Display omitted] DisclosuresSallman:Celgene: Research Funding. Padron:Incyte: Honoraria, Research Funding. Lancet:Pfizer: Other: Institutional research funding; Biopath, Biosight, Boehringer Ingelheim, Celator/Jazz, Celgene, Janssen, Karyopharm Therapeutics, and Novartis: Consultancy. Komrokji:Celgene: Honoraria; Novartis: Honoraria, Speakers Bureau.

Age Is Not a Predictive Marker in Molecularly Annotated Elderly Patients with Myelofibrosis Treated with Ruxolitinib: a Multicenter Study on 277 Patients.

Introduction. Ruxolitinib (RUX) is a JAK1/2 inhibitor that may control myelofibrosis (MF)-related splenomegaly and symptoms and can be prescribed regardless of age. Aging correlates with clinically impaired prognosis probably due to biologically increased molecular complexity. However, assessment of efficacy/safety of RUX and correlation with clonal architecture in elderly MF patients (pts) is relatively unknown.Aims. To report the clinical outcome of 277 pts with WHO-defined MF treated with RUX when aged ≥65 yrs. To correlate molecular status assessed by targeted sequencing of 30 genes known to be involved in myeloid disorders with response to therapy and survival.Methods. A clinical database was created in 20 European Hematology Centers. Retrospective data on 487 MF pts treated with RUX according to prescribing information from Jan 2011 to Jun 2016 were collected. Spleen (SR) and Symptoms (SyR) Response to RUX were evaluated according to the 2013 IWG-MRT criteria. Survival was calculated from the start of RUX. Event-free survival (EFS) included progression to acute leukemia (AL), death and RUX discontinuation. Peripheral blood (PB) granulocytes-derived DNA was extracted and processed according to the “Myeloid Solution” (SOPHIA GENETICS) assay on the MiSeq Illumina platform. Variants were pre-classified as potentially pathogenic by SOPHiATM Artificial Intelligence. Output data was analyzed by Sophia DDM v3 software.Results. A total of 277 out of 487 pts (46.6%) started RUX when aged ≥65 yrs. Compared to elderly (age 65-74) pts, very elderly (≥ 75 yr) pts had a higher Charlson Comorbidity Index and a lower platelet count, thus started RUX at lower doses (Table 1). At 6 mos, SR and SyR were achieved by 35.9% and 83.7% of 209 and 221 evaluable pts, respectively. In the first 6 mos, anemia/thrombocytopenia/infections ≥G2 were observed in 33.6%, 21.7% and 29.2% of the 277 pts, respectively. The only feature independently associated with increased infectious risk was a history of previous infection. Evolution into AL occurred in 22 pts. Overall, 110 pts discontinued RUX after a median time of 12.5 mos. Importantly, the rate of toxicities, responses, and disease evolution was not influenced by age. After a median follow-up of 19.5 mos, 65 pts died because of progression of myelofibrosis (40%), heart disease (10.8%), infections (13.8%), AL (15.4%), hemorrhage/thrombosis (7.7%) or transplant-associated toxicity (4.6%). Older age and Body Mass Index <21 (first quartile) was significantly associated with worse survival (p=0.02).NGS will be performed on 100/277 pts with available PB samples at RUX start. Preliminary data on 48 elderly pts (median age, 72.5 yrs) showed that all pts had a mutation in ≥1 gene (Fig. 1). A total of 523 variants have been identified so far. Only 297 (57%) variants were considered potentially pathogenetic. Four genes (BRAF, FLT3, HRAS, WT1) investigated in the NGS panel were never mutated. Recurrent mutations with potential pathogenic impact included TET2 (20,5%), ASXL1 (14%), SETBP1 (7,7%), EZH2 (6,7%), DNMT3A (4,7%), KIT (3,7%). The percentages of pts with pathogenetic variants for the most recurrently mutated genes were: ASXL1 (50%), TET2 (50%), DNMT3A (22,9%), EZH2 (25%), SETBP1 (21%), KIT (10,4%). At least one high molecular risk (HMR) pathogenetic mutation was present in 61% of pts; 37% had ≥2 HMR mutations. The most frequently mutated HMR genes were ASXL1 and EZH2, that co-occurred in 22% of the cases. HMR status was significantly associated with PMF and larger (>10 cm) spleen length (p=0.04). Additionally, patients with EZH2 mutation and ≥3 variants showed a lower baseline hemoglobin and platelet count (respectively p=0.04 and p=0.03). Molecular status (HMR, ≥2 HMR mutations, ≥3 variants and ASXL1- or EZH2-mutations) was investigated for association with clinical responses/outcome parameters. Preliminary data showed a trend for association of HMR+ and ≥3 variants with worse EFS and leukemia-free survival.Discussion. Since response/toxicity rates were not influenced by age, older age should not be a limitation for RUX therapy. The assessment of comorbidities, nutrition status and infectious history may identify specific pts fragilities. The cohort of molecularly annotated pts will be further enriched. However, we have so far observed a high molecular complexity that did not seem to affect responses and survival in the elderly.LC and FHH equally contributed. [Display omitted] DisclosuresBonifacio:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Abruzzese:Novartis: Consultancy; Pfizer: Consultancy; BMS: Consultancy; Incyte: Consultancy. Tiribelli:Incyte: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Breccia:Bristol Myers Squibb: Consultancy; Novartis: Consultancy; Incyte: Consultancy; Pfizer: Consultancy. Cavazzini:Pfizer: Consultancy; Incyte: Consultancy; Bristol-Meyers Squibb: Consultancy; Novartis: Consultancy. Crugnola:Celgene: Honoraria; BMS: Honoraria; Novartis: Honoraria. Martinelli:Amgen: Consultancy; Celgene: Consultancy; Pfizer: Consultancy; Ariad/Incyte: Consultancy; Johnson&Johnson: Consultancy; Roche: Consultancy.

Targeting the Epigenetic and Metabolic Vulnerabilities of Ezh2-Deficiency-Induced Myeloid Neoplasms

Myeloproliferative neoplasms (MPNs) are clonal, progressive hematopoietic malignancies characterized by alterations of multiple signaling and epigenetic pathways in hematopoietic stem cells (HSCs). The identification of driver oncogenic mutations in JAK2, CALR or MPL has transformed our knowledge of MPNs; however, patients with non-mutated JAK2, CALR and MPL (so-called ‘triple-negative‘) have the highest incidence of leukemic transformation and the lowest overall survival (Rumi et al., 2014 Blood 124:1062-1069), indicating that other factors may contribute to MPNs. The molecular processes controlling MPN progression from chronic to acute leukemic transformation remain unknown. This is a major impediment for developing target-based therapeutics to selectively eliminate the mutant stem cells to prevent disease progression and/or relapse. Therefore, the challenge is twofold: 1) to identify and characterize the genetic complexity of MPN pathophysiology, and 2) to understand and control the progression of MPNs from chronic to life-threatening stages.We have developed a new mouse model of MPNs by hematopoietic-selective (Mx1-cre) activation of oncogenic Ras (NRasG12D+/-) and inactivation of Ezh2, the enzymatic subunit of the Polycomb Repressive Complex 2 (PRC2), which mimics concurrent mutations in human MPNs. While NRasG12D+/- alone led to an indolent myeloproliferative disorder, Ezh2 loss markedly accelerated disease progression to life-threatening stages, resulting in thrombocytosis, neutrophilia, splenomegaly, primary myelofibrosis, leukemic transformation and mortality. Strikingly, while loss of Ezh1, a homolog of Ezh2, had no effect on normal hematopoiesis, concurrent inactivation of Ezh1 and Ezh2 with NRasG12D+/- completely abolished MPN development and myelofibrosis in vivo . These results establish distinct functions of Ezh1 and Ezh2 in myeloid neoplasms, and identify a selective epigenetic vulnerability for MPNs induced by Ezh2-deficiency. Follow-up studies of Ezh1/2-mediated chromatin (by ChIP-seq) and transcriptomic (by RNA-seq) changes in normal HSCs and leukemia-initiating cells (LICs) revealed non-overlapping gene targets and cellular pathways required for the distinct cellular phenotypes.Reprogrammed cellular metabolism underlies the progression of various human cancers including leukemia. We noted by RNA-seq transcriptomic profiling that Bcat1, the first and rate-limiting enzyme catalyzing the transamination of branched-chain amino acids (BCAAs), was aberrantly activated in Ezh2-deficient leukemia-initiating cells (LICs). By ChIP-seq analysis, we observed that the Bcat1 promoter is highly enriched with tri-methylated H3-Lys27 (H3K27me3), suggesting that it is epigenetically silenced by Ezh2-PRC2 in normal HSCs. Furthermore, genetic or pharmacologic inhibition of BCAT1 activity selectively abrogated the clonogenic activity of Ezh2-deficient LICs in vitro and the leukemogenic potential in transplanted recipients in vivo, highlighting a metabolic dependency for Ezh2-mutant myeloid neoplasms. Follow-up targeted metabolomic analysis of Ezh2-deficiency-induced metabolic changes established the critical role of Bcat1 and the associated BCAA metabolism in the progression of myeloid neoplasms and leukemia.Collectively, our results not only elucidate the mechanisms by which Ezh1 is specifically required for Ezh2-deficient LICs, but also validate an important epigenetic vulnerability for MPN patients carrying loss-of-function Ezh2 mutations. Moreover, in-depth analysis of BCAA metabolism in myeloid neoplasms identifies a new metabolic dependency for Ezh2-deficient LICs. Because normal hematopoietic stem cells are unaffected by loss of Ezh1 or Bcat1, the selective vulnerabilities raise the possibility of leveraging Ezh1 or BCAA metabolism as targeted therapies to specifically eradicate Ezh2-mutant LICs. Hence, our study promises to provide critical insights into developing new and generalizable therapeutic strategies to selectively eliminate leukemia-initiating cells harboring alterations of epigenetic pathways. DisclosuresDeberardinis:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

FLT3 Mutation in Acute Myeloid Leukemia: Primary or Secondary Mutation?

The receptor tyrosine kinase FLT3 gene is commonly mutated in acute myeloid leukemia (AML) and its mutation is associated with significantly more aggressive disease and poor outcome. However, in the co-presence of NPM1 mutation, the disease is less aggressive and is associated with better outcome. FLT3 inhibitor midostaurin (Rydapt; Novartis Pharmaceuticals, Inc.) was recently approved for patients with FLT3 mutations. While the presence ofa FLT3 mutation is commonly associated with AML,it can develop in less acute myeloid neoplasms, such as MDS and CMML, and implies transformation into more acute phase. In this context, the FLT3 mutation is typically detected in a subclone of the original chronic myeloid clone. The prevalence of subclonal FLT3 mutations is unknown, as is the clinical relevance of subclonal FLT3, especially when treated with FLT3 inhibitors. We used variant allele frequency (VAF) and next generation sequencing (NGS) to determine if FLT3 mutations are founding or secondary mutations and to characterize the co-mutational background in large number of consecutive patients who presented with a clinical impression of myeloid neoplasm.Methods: A total of 6390 consecutive bone marrow aspirate samples or peripheral blood samples were submitted with clinical impression of AML, MDS, or MPN between January 2014 and mid 2017 were tested for mutations in myeloid genes using NGS. We used the TruSight Myeloid panel (Illumina, San Diego, CA) for detecting missense mutations and fragment length analysis (FLA) for detecting internal tandem repeat (ITD) in FLT3 . DNA was extracted from samples using the QIAamp DNA Mini Kit. This NGS testing covers mutations in 54 myeloid-related genes. The average depth of sequencing was 10,000x.Results: A total of 2670 (42%) of the samples showed a mutation in one or more of the tested genes. Of the samples with mutations, FLT3 ITD and kinase domain (KD) mutations were detected in 313 (12%) patients. Of these patients, 208 (66.5%) had ITD and 117 (37.4%) had KD mutations. Twelve patients (3.8%) had both ITD and KD mutations. NPM1 mutation was detected in 138 (44%) of the patients. Most of FLT3 -KD mutations were in codon 835, but mutations in codons 836, 839, 842 and 845 were also found. Additionally, missense mutations in codons 576, 592, and 593 at the site of ITD were detected. To distinguish a FLT3 mutation as a founding mutation rather than a secondary mutation, we used a lower VAF of 10% as a cut-off. Using this approach, we found that 27% (115/313) of patients had secondary FLT3 mutations. Of these subclonal FLT3 mutants, NPM1 co-mutation was detected in 61 (53%) patients, but was not statistically significant (P=0.06). Patients with FLT3 mutation as a subclone had a significantly higher number of co-mutated genes (P-value <0.0001). This group of patients had higher (P≤0.01) mutations in TET2, DNMT3A, NRAS, IDH2, EZH2, and BCOR, consistent with an MDS/CMML background. On the other hand, patients with both FLT3 and NPM1 mutations had a significantly lower number of co-mutant genes (P<0.0001) and particularly lower (P≤0.01) mutation rate in U2AF1, SF3B1, SRSF2, RUNX1, IDH1, BCOR, and AXL1 .Conclusion: FLT3 mutations are frequently (27%) a secondary mutation in a subclone of the neoplastic founding mutations. This appears to be more common when FLT3 mutation is developing in a background of MDS/CMML. The co-presence of an NPM1 mutation is more commonly associated with fewer co-mutations in MDS-related genes. This data suggests that in addition to detecting ITD by FLA, using NGS for profiling mutations in FLT3 -KD and other myeloid genes should be considered whenever FLT3 mutations are evaluated. Further, the clinical decision for combination therapy should consider the entire mutation profile and not only mutations in FLT3 . [Display omitted] DisclosuresMa:NeoGenomics: Employment. De Dios:NeoGenomics: Employment. Funari:NeoGenomics: Employment. Sudarsanam:NeoGenomics: Employment. Jiang:NeoGenomics: Employment. Agersborg:NeoGenomics: Employment. Hummel:NeoGenomics: Employment. Blocker:NeoGenomics: Employment. Albitar:NeoGenomics: Employment.

Reproducibility of m7-FLIPI Risk Scores in Follicular Lymphoma Using Tumor Biopsies and Blood Specimens

Background: Follicular lymphoma (FL) is clinically and genetically heterogeneous. While most patients experience an indolent disease course, with median survival of over 15 years, a portion of patients experience early relapse and poor outcomes. The m7-FLIPI risk score has been described as a clinicogenetic method to accurately determine which patients are at highest risk for early relapse and poor outcomes (Pastore et al., Lancet Oncology 2015; Vindi et al., Blood 2015). However, molecular characterization of the m7 genes currently relies on tumor biopsies, which require an invasive procedure and may be unavailable. Furthermore, it is unclear whether the m7-FLIPI score is consistent over the course of disease. Circulating tumor DNA (ctDNA) is an emerging noninvasive biomarker for tumor detection in diverse lymphomas including FL. In this study, we evaluated the stability of m7-FLIPI status over multiple FL relapses as well as the utility of ctDNA for classification of patients with FL into high- and low-risk m7-FLIPI categories.Methods: We applied Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq), a capture-based method for ultra-deep sequencing (Newman et al., Nat Med 2014; Scherer et al., Sci Transl Med 2016 ) to identify risk via m7-FLIPI in pretreatment tumor and plasma samples of 29 FL patients. In addition, we retrospectively analyzed whole exome sequencing (WES) data of 37 patients from two FL cohorts (Green et al., PNAS 2015; Bouska et al., Leukemia 2017). We first validated in silico our capture-based method for identification of m7-FLIPI-relevant somatic alterations in 258 patients from the original publication (Pastore et al., Lancet Oncology 2015). We then identified non-synonymous mutations in CREBBP, EZH2, ARID1A, MEF2B, FOXO1, EP300, and CARD11 presentin each tumor and plasma sample. Using both CAPP-Seq and WES data (n = 66), we compared m7-FLIPI risk group concordance between paired diagnostic tumor biopsies, paired diagnostic and relapse tumor biopsies, paired FL and transformed FL (tFL) tumor biopsies, and paired diagnostic tumor and plasma samples. FLIPI was calculated at first FL time-point available.Results: We observed a 95% agreement in m7-FLIPI risk category between our capture-based method and methods utilizing full exon coverage. A 100% concordance of m7 gene profile and 100% concordance of m7-FLIPI risk category were seen when comparing paired FL tumor biopsy samples from two separate lymph nodes obtained at diagnosis (n = 5). In addition, a 75% agreement in m7 gene profile and 100% agreement of m7-FLIPI risk category were observed when comparing tumor biopsy samples obtained at FL diagnosis and first relapse (n = 36; median time to relapse of 547 days, range 55 - 4200 days). There was a low concordance in m7 genetic profile between tumor biopsy samples from time of initial diagnosis and transformation (48.4%, n = 31). Importantly, concordance of m7 gene profile and m7-FLIPI risk category was 64.3% and 78.6%, respectively, when assessed by paired tumor and plasma samples obtained at FL diagnosis (n = 14; Figure 1). Furthermore, non-concordant mutations could be present in either the tumor or the plasma sample. Across all FL samples, there was no significant difference in concordance based on individual m7 genes. When analyzing pairs in which mutations were observed, concordance between paired samples was lowest for EZH2 (p = 0.028).Conclusions: m7-FLIPI scores appear consistent between multiple FL tumor samples obtained at the time of diagnosis and across FL relapses. However, concordance in m7 gene profile between paired plasma and tumor samples is lower than between paired tumor biopsies, even at ultra-deep sequencing depths. Optimization of blood samples for assessing m7 gene profile should be performed to improve the reliability of ctDNA for determining m7-FLIPI risk status.Figure 1. Concordance of m7-FLIPI risk category. (A) Re-classification of m7-FLIPI risk category when using paired tumor biopsy and plasma samples from 14 patients. (B) Percentage of discordant samples when comparing diagnostic tumor pairs (n = 5), diagnostic versus relapse tumor pairs (n = 37), and diagnostic tumor versus plasma pairs (n = 14). Concordance was lowest for diagnostic tumor and diagnostic plasma pairs (p = 0.013). [Display omitted] DisclosuresDiehn:Varian Medical Systems: Research Funding; Quanticel Pharmaceuticals: Consultancy; Roche: Consultancy; Novartis: Consultancy. Alizadeh:Gilead: Consultancy; Roche: Consultancy; Genentech: Consultancy; Celgene: Consultancy; CiberMed: Consultancy.

Relative Lymphocytosis Is Associated with Improved Outcome in Myelofibrosis and May Account for the Favorable Prognosis of the Calr mutation

Introduction: Neutrophil-Lymphocyte ratio is prognostic in solid tumors and lymphomas which may serve as an index of anti-tumor immune response. The role of immune surveillance in myeloproliferative neoplasms (MPNs) is being increasingly explored. In this study, we aimed to investigate whether such surrogate markers of immune response offers prognostic significance in MPNs. Myelofibrosis (MF) is characterized by leukoerythoblastosis in the peripheral blood which leads to relative lymphopenia. To account for this, we modified the neutrophil:lymphocyte ratio by instead assessing the prognostic significance of relative lymphocytosis in terms of the total white blood cell (WBC):lymphocyte ratio (WLR). Further, given recent evidence of enhanced immunogenicity of CALR mutant MPN cells, we explored if WLR varies based upon driver mutation.Methods: We retrospectively reviewed MF patients seen at our institution between 1/1/2001 and 5/1/2017. MF was defined by 2016 WHO criteria. Laboratory values were calculated at first presentation and overall survival (OS) was calculated from time of first presentation. WLR defined as WBC/absolute lymphocyte count (ALC). A WLR cutpoint of 7 was based on median WLR value.Results: Of 436 total MF patients, 431 had baseline lab values at time of first presentation, and 319 were informative for somatic gene mutations. Two-hundred forty-nine (78%) were JAK2 mut, 36 (11%) were CALR mut, 17 (5%) were MPL mut and 17 (5%) were triple-negative. On univariate analysis, absolute lymphocyte count (ALC) did not correlate with OS (p = 0.82), and there was no difference in ALC based on driver mutation. WBC:ALC ratio (WLR), however, was associated with inferior OS (p = 0.001) in univariate analysis. High WLR directly correlated with age (p < 0.001), monocytosis (p < 0.001), increased peripheral blasts (p < 0.001), immature myeloid cells (p < 0.001), bone marrow cellularity (p < 0.001), splenomegaly (p = 0.001), constitutional symptoms (p = 0.02), IPSS (p < 0.001) and DIPSS prognostic scores (p =0.01), mutations in JAK2 (p < 0.001), TET2 (p = 0.04) and EZH2 (p < 0.001) and total number of somatic mutations (p = 0.009). Low WLR correlated with presence of CALR (p = 0.04) and DNMT3A mutations (p = 0.04). On multivariate analysis, controlling for DIPSS, albumin, and bone marrow fibrosis, WLR remained a significant prognostic co-variate (p = 0.04).Comparison of WLR according to specific driver mutation showed a trend toward lower WLR in CALR mutant versus JAK2 mutant MF (p =0.07). Low WLR was also associated with improved OS (HR 0.67 [0.51-0.89]; p = 0.005) in the entire MF cohort as well as in JAK2 mut (p = 0.002) and CALR mut subgroups (p = 0.06).Lastly, we assessed the relationship between WLR and variable allele frequency (VAF) of JAK2 and CALR driver mutations as well as the commonly occurring somatic mutations ASXL1 (n = 53) and SRSF2 (n = 29). High WLR was directly associated with VAF of JAK2 (p = 0.01) and CALR (p = 0.04), though the correlation was stronger with CALR than JAK2 (r2 = 0.30 vs 0.05). A trend towards a positive correlation was found between WLR and ASXL1 VAF (p = 0.07) whereas there was no correlation with SRSF2 VAF (p = 0.70).Conclusion: WLR is an independent prognostic co-variate in patients with MF with higher ratios conferring inferior outcomes. Decreased WLR in CALR mut compared with JAK2mut MF patients coupled with the strong correlation with CALR VAF suggests this may underlie the more favorable prognosis in CALR mut patients. Further investigations are warranted to discern the biological responses driving the changes in WLR index. [Display omitted] DisclosuresEpling-Burnette:Incyte: Research Funding; Forma Therapeutics: Research Funding; Celgene: Research Funding. Padron:Incyte: Honoraria, Research Funding. Sallman:Celgene: Research Funding. Lancet:Pfizer: Other: Institutional research funding; Biopath, Biosight, Boehringer Ingelheim, Celator/Jazz, Celgene, Janssen, Karyopharm Therapeutics, and Novartis: Consultancy. Komrokji:Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria.

Novel Stem Cell-Targeted Therapy for Multiple Myeloma Based on Dual Inhibition of EZH1/2

Multiple myeloma (MM) is largely incurable because relapse eventually occurs, despite the recent development of novel therapies. This is mainly caused by a remaining population of drug-resistant myeloma stem cells. There is a side population (SP) enriched with myeloma stem cells. Therefore, targeting SP cells may be a promising strategy to prevent and treat MM relapse.Polycomb repressive complexes 1 (PRC1) and 2 (PRC2) are important epigenetic regulators that maintain the “stemness” of embryonic and hematopoietic stem cells. Enhancer of zeste homolog 1 and 2 (EZH1/2) are catalytic components of PRC2, which trimethylate histone H3 at lysine 27 (H3K27me3) to repress transcription of target genes. Mutation and overexpression of EZH2 are associated with many cancers, including MM. Here, we found that expression of EZH1/2 was significantly higher in SP cells than in non-SP cells. These results suggest that overexpression of EZH1/2 is important for maintaining the stemness of MM cells and that both EZH1 and EZH2 are potential therapeutic targets.We developed a novel EZH1/2 dual inhibitor, OR-S1, which potently inhibited both EZH1 and EZH2, and used it to investigate the effect of pharmacologic inhibition of EZH1/2 on MM. OR-S1 suppressed the proliferation of all MM cell lines tested, and its GI50 value was significantly lower than that of the EZH2-specific inhibitor GSK126. Furthermore, flow cytometric analysis revealed that treatment with OR-S1 significantly depleted the SP cells. These results suggest that dual inhibition of EZH1/2 using OR-S1 eradicates myeloma stem cells.RNA sequencing analysis revealed that the transcriptional profiles of OR-S1-treated MM cell lines were characterized by up-regulation of genes related to the Wnt pathway. RT-PCR confirmed that OR-S1 treatment markedly increased expression of Wnt and Frizzled family members. Furthermore, western blot analysis revealed that non-phosphorylated (active) β-catenin was strongly expressed in OR-S1-treated cells. To determine whether there is direct crosstalk between PRC2 and Wnt signaling, we performed chromatin immunoprecipitation (ChIP)-sequencing using MM cell lines. This revealed that most Wnt and Frizzled loci were marked by H3K27me3. Furthermore, ChIP-qPCR analysis demonstrated that dual inhibition of EZH1/2 using OR-S1 markedly decreased the level of H3K27me3 at most of these loci. These results demonstrate that PRC2 directly targets Wnt signaling and that dual inhibition of EZH1/2 using OR-S1 activates canonical Wnt signaling.Increased activation of the canonical Wnt signaling pathway reduces self-renewal and differentiation of hematopoietic stem cells. Therefore, we generated β-catenin-overexpressing MM cells to examine the effects of increased Wnt signaling. Surprisingly, proliferation of these cells was significantly lower than that of control cells. Furthermore, flow cytometric analysis revealed that overexpression of β-catenin significantly depleted the SP cells. These results suggest that over-activation of Wnt signaling induced by dual inhibition of EZH1/2 reduces the proliferation of MM cells by inhibiting self-renewal, which is important to maintain the stemness of MM cells.Oral administration of OR-S1 to mice bearing MM xenografts significantly impaired subcutaneous tumors. Long-term administration of OR-S1 at lower doses to mice bearing orthotopic xenografts eradicated minimal residual disease from bone marrow and completely cured MM, without eliciting any serious side effects (Figure). Furthermore, we developed an orthotopic patient-derived xenograft model using specimens from a relapsed and heavily pretreated MM patient. In this model, OR-S1 treatment reduced the serum levels of human immunoglobulins. These results indicate that EZH1/2-targeting therapy with OR-S1 is also effective against a human MM xenograft.Taken together, our results strongly suggest that dual inhibition of EZH1/2 is a promising therapeutic approach to eradicate myeloma stem cells and could lead to important advances in the treatment of MM. [Display omitted] DisclosuresNakagawa:Daiichi Sankyo Co.: Research Funding. Fujita:Daiichi Sankyo Co.: Research Funding. Honma:Daiichi Sankyo Co., Ltd.: Employment. Araki:Daiichi Sankyo: Employment. Kitabayashi:Daiichi Sankyo Co.: Research Funding.

Clonal Evolution of Myelofibrosis Patients in Relapse after Allogeneic Stem Cell Transplantation Revealed By Whole Exome Sequencing

Introduction: Clonal evolution has been described in various hematologic malignancies. Persistence of one founding clone is the basis for relapse of AML, and diversity of accompanying mutations greatly varies in AML [Ding et al. Nature 2012]. On the contrary, a targeted sequencing approach revealed that in myeloproliferative neoplasia, few additional mutations arise during progression [Lundberg et al. Blood 2014]. The dynamics of clonal evolution has been linked to response to Ruxolitinib therapy [Newberry et al. Blood 2017].Patients and methods: Here, we describe the clonal evolution of 30 pts (median age at alloSCT 60, range 36-75 years) with myelofibrosis (16 primary, 6 post-ET, 7 post-PV MF) who either progressed directly (n=14) or after a period of complete donor hematopoiesis for 417 days (median, range 7-2240 days) after allogeneic stem cell transplantation. Amplicon-based next generation sequencing of a custom panel consisting of 17 different genes (178 amplicons in 2 primer pools, targeting regions in TET2, IKAROS, JAK2, MPL, SH2B3, CBL, IDH1, IDH2, ASXL1, EZH2, SRSF2, SF3B1, DNMT3A, RUNX1, FLT3, NRAS, KRAS) was performed for all pts using a Personalized Genome Machine (PGM, ThermoFisher/ LifeTechnologies). Additional whole exome sequencing was performed for 6 out of these 30 pts, both before alloSCT and at relapse of the disease after alloSCT, using a Proton (ThermoFisher/ LifeTechnologies). Mutations in CALR were determined using digital PCR.Results: Pts carried JAK2 V617F (n=23), CALR L367fs* (n=3), CALR K368fs*, CALR K385fs* (each n=1), or were triple-negative (n=2). The mutations in the driver genes JAK2, CALR, and MPL that were present before alloSCT were also present at relapse. One pt carrying a JAK2 V617F before alloSCT had developed an additional MPL L494V missense mutation at relapse. With targeted amplicon sequencing, a median of 3 nonsynonymous mutations (range 0-13) was found in a median of 2 (0-8) genes before alloSCT. Before alloSCT, 19 pts were mutated in TET2 (I1762V/ L1721W/ P363L/ L34F/ V218M/ H1778R in 13/ 7/ 3/ 2/ 2/ 2 pts, 9 further mutations occurring only once), trailed by ASXL1 (7 pts) and SRSF2 (6 pts). DNMT3A and IDH2 were mutated in 3 pts, each, with the latter exclusively altered at R140. KRAS (G12V, G12H), RUNX1 (D198Y, A424S), SF3B1 (K666N, V695L) were mutated in 2 pts each. Mutations in CBL (C381S), IKZF1 (Q7R), EZH2 (Y733C), SH2B3 (S186I), and IDH1 (R132H) occurred in one pt each. At relapse, using the identical amplicon based sequencing strategy, a median of 3 (0-13) nonsynonymous mutations was found in a median of 2.5 (0-6) genes. Counting gains and losses of nonsynonymous mutations at relapse, a median of 1 (0-9) changes was observed. Identities of gains and losses were more variable when compared to the mutations before alloSCT. Most changes occurred in TET2, followed by ASXL1, SRSF2, RUNX1, and DNMT3A . Taking only pts who had achieved full donor chimerism (n=16) into account, short molecular remission and number of mutational changes correlated (R²=0.013, figure). This correlation was stronger than the correlation between number of mutations before alloSCT and time in molecular remission after alloSCT (R²=0.0001). Notably, of the 6 pts in whom additional whole exome sequencing was performed at first presentation and upon progression, none showed clonal evolution beyond genes depicted in the targeted amplicon sequencing panel.Conclusion: Clonal evolution of myelofibrosis pts who relapse after alloSCT is modest. A net loss of mutations can be found at relapse. In comparison to the situation in MDS and AML, the gain of novel mutations at relapse seems to be moderate in myelofibrosis. Future research has to identify mutational spectra with specific prognostic and predictive value. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

FOXP1 Expression Is a Prognostic Biomarker in Follicular Lymphoma Treated with Immunochemotherapy and Is Associated with Distinct Molecular Features

Introduction: Follicular lymphoma (FL) is a clinically and molecularly highly heterogeneous disease, yet prognostication relies predominantly on clinical tools. We have recently demonstrated that integration of the mutation status of seven genes (including EZH2 and MEF2B) into a combined clinico-genetic risk model (m7-FLIPI) improves risk-stratification, and represents a promising approach to identify a subset of patients at highest risk of treatment failure. However, the mechanism underlying the association between mutations of the 7 genes included in the m7-FLIPI and outcome has thus far not been elucidated. We assessed whether downstream phenotypic commonalities of gene mutations predict outcome following immunochemotherapy and whether select genotypic and phenotypic alterations are associated with FOXP1 expression.Methods: We mined gene expression data to uncover genes that are differentially expressed in EZH2 - and MEF2B -mutated FLs. We focused on FOXP1 and assessed its protein expression by immunohistochemistry (IHC) in a total of 763 tissue biopsies. For outcome correlation, a population-based training cohort of 142 FL patients treated with R-CVP, and a clinical trial validation cohort comprising 395 patients treated with CHOP +/- rituximab were used. To differentiate cases with high vs. low FOXP1 expression, we chose the cut-off that maximized the log-rank test statistic for failure-free survival (FFS) in the training cohort. The effect of FOXP1 expression on FFS in the validation cohort was estimated using Cox regression analysis. In addition, for exploratory analyses, we used specimens from 350 BC Cancer Agency patients, 124 of whom overlapped with the above-mentioned training cohort.Results: We found FOXP1 to be significantly down-regulated in both EZH2 - and MEF2B -mutated cases. The cut-off value that best separated favorable from unfavorable FFS in the training cohort was determined to be >10% percent tumor cells expressing FOXP1 based on the log-rank test statistic. 76 specimens in the training cohort (54%) had high FOXP1 expression, which was associated with reduced 5-year failure-free FFS (55% vs. 70%, P=0.035). In the validation cohort, high FOXP1 expression status was observed in 248 patients (63%) and correlated with significantly shorter FFS in patients treated with R-CHOP (HR 1.95, P=0.017), but not in patients treated with CHOP (HR 1.15, P=0.44), demonstrating that FOXP1 is a predictive biomarker. In BCCA patients, high FOXP1 expression remained significantly associated with inferior FFS in a multivariate Cox regression model including rituximab maintenance (by intention to treat) and FLIPI high vs low/intermediate (HR 1.84, 95%-CI [1.10-3.08], P=0.021). Similarly, in the GLSG cohort, high FOXP1 expression was significantly associated with poor FFS in patients treated with R-CHOP, after adjusting for rituximab, FLIPI and the interaction of FOXP1 with rituximab (HR 1.84, 95%-CI [1.05-3.24], P=0.035). No significant association was observed in GLSG patients treated with CHOP (HR 1.06, 95%-CI [0.73-1.54], P=0.76). High FOXP1 expression was associated with distinct molecular features such as TP53 mutations, expression of IRF4, and gene expression signatures reminiscent of dark zone germinal centre or activated B cells. Conversely, low expression of FOXP1 was associated with - in addition to EZH2 and MEF2B mutations - GNA13 and TNFRSF14 mutations, as well as with expression of genes related to normal germinal centre light zone cells and germinal centre B cells. Taken together, these observations suggest that FOXP1 expression delineates FL cases with distinct genomic and phenotypic characteristics.Conclusion: In patients treated with immunochemotherapy, high FOXP1 expression identified patient populations with significantly shortened FFS. On the other hand, FOXP1 did not separate distinct outcome groups in patients treated without rituximab, suggesting that FOXP1 is a predictive biomarker. In multivariate analyses, high FOXP1 expression remained significantly associated with FFS in immunochemotherapy patients, after adjusting for the FLIPI. Moreover, FOXP1 high and low expression phenotypes are reflective of distinct sets of molecular alterations. Our results illustrate the value of understanding the intricate relationships between lymphoma biology and response to treatment. DisclosuresKlapper:Hoffmann La Roche: Other: Grant; Novartis: Other: Grant; Celgene: Other: Grant; Takeda Millenium: Other: Grant. Hiddemann:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Steidl:Affimed Therapeutics: Consultancy; Juno Therapeutics: Consultancy. Connors:Janssen: Research Funding; F Hoffmann-La Roche: Research Funding; Bayer Healthcare: Research Funding; Seattle Genetics: Research Funding; NanoString Technologies: Research Funding; Merck: Research Funding; Lilly: Research Funding; Genentech: Research Funding; Cephalon: Research Funding; Amgen: Research Funding; Bristol-Myers Squibb: Research Funding; NanoString Technologies, Amgen, Bayer, BMS, Cephalon, Roche, Genentech, Janssen, Lilly, Merck, Seattle Genetics, Takeda,: Research Funding; Takeda: Research Funding. Sehn:Roche/Genentech: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria. Hoster:Roche: Other: Travel support, Research Funding.