MLL-PTD Mutations Research Articles

High EGFL7 expression may predict poor prognosis in acute myeloid leukemia patients undergoing allogeneic hematopoietic stem cell transplantation

ABSTRACTEpithelial growth factor-like 7 (EGFL7) is a secretory protein with a well-characterized role in angiogenesis and the oncogenesis of certain solid tumors. Overexpression of EGFL7 is associated with adverse prognosis in patients with cytogenetically normal acute myeloid leukemia (CN-AML). However, whether this association persists after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains unclear. To further clarify the prognostic role of EGFL7, seventy-one AML patients with EGFL7 expression data who underwent allo-HSCT from The Cancer Genome Atlas database were included and divided into either EGFL7high or EGFL7low group based on the median EGFL7 expression level. Two groups had similar clinical and molecular characteristics except that the EGFL7high group had less frequent NPM1 mutations (P= .001). Kaplan-Meier survival curves showed that high EGFL7 expressers had shorter OS than the low expressers (P= .040). Univariate analysis showed that high EGFL7 expression, MLL-PTD, RUNX1 and TP53 mutations were associated with short OS (all P< .05). Multivariate analysis indicated that high EGFL7 expression, FLT3-ITD, RUNX1 and TP53 mutations were independent risk factors for OS (all P< .05). Collectively, our study suggested that EGFL7, like the other widely-used risk stratification factors, could serve as a prognostic tool and therapeutic target in AML, even after allo-HCST.

Mutational spectrum and prognostic stratification of intermediate-risk acute myeloid leukemia.

The mutational spectrum and prognostic stratification of intermediate-risk acute myeloid leukemia (IR-AML), which accounts for a substantial number of AML, are unclear. In order to explore the prognostic significance of the mutational spectrum in IR-AML, 106 IR-AML patients were collected from The Cancer Genome Atlas database. Sixty-two patients underwent chemotherapy-only, forty-four proceeded to allogeneic hematopoietic stem cell transplantation (allo-HSCT). Fifty-five patients had more than five recurrent genetic mutations. NPM1 had the highest mutation frequency, followed by DNMT3A, FLT3, RUNX1, IDH2, IDH1, and TET2. In all patients, allo-HSCT was an independent favorable factor for EFS and OS (P = 0.036, P = 0.001, respectively); age ≥60 years, FLT3-ITD and mutations in DNMT3A and RUNX1 were independent risk factors for survival (all P < 0.05). In the chemotherapy-only group, multivariate analysis showed that age ≥60 years was an independent risk factor for EFS and OS (P = 0.008, P = 0.017, respectively). In the allo-HSCT group, multivariate analysis indicated that MLL-PTD was an independent risk fact for EFS (P = 0.037), FLT3-ITD and RUNX1 mutations independently contributed to poor OS (P = 0.035, P = 0.014, respectively). In conclusion, older age was an important risk factor for IR-AML patients undergoing chemotherapy-only; FLT3-ITD, MLL-PTD and RUNX1 mutations were significant risk factors for IR-AML patients who received allo-HSCT.

Prognostic value of genetic mutations in adolescent and young adults with acute myeloid leukemia.

Clinical outcomes and the genetic background of acute myeloid leukemia (AML) in adolescent and young adults (AYAs) are known to differ in younger children and older adults. To clarify the impact of genetic mutations on clinical outcomes of AYAs with AML, we analyzed data from the JPLSG AML-05 and JALSG AML201 studies. AYAs aged 15-39years (n=103) were included. FLT3-ITD, KIT, CEBPA, NRAS, KRAS, WT1, MLL-PTD, and NPM1 mutations were analyzed. Overall survival (OS) of the AYAs was 61% and event-free survival was 38% at 3years. FLT3-ITD (HR 2.10; 95% CI 1.07-4.12; p=0.031) and NPM1 (HR 0.24; 95% CI 0.06-1.00; p=0.050) mutations were associated with risk of overall mortality in multivariate analysis. OS was significantly different according to FLT3-ITD and NPM1 mutation status (p=0.03). Survival was 100% with NPM1 mutations in the absence of FLT3-ITD and 35% (95% CI 14-57%) with FLT3-ITD in the absence of NPM1 mutations. The OS of AYAs, children (n=413) and older adults (n=124) of the AML-05 and AML201 participants were significantly different (p<0.0001). This is the first report to combine clinical and genetic data of AYA AML from the major Japanese pediatric and adult study groups.

Mutational Frequency in Hispanic Vs. Non-Hispanic Patients with Acute Myeloid Leukemia (AML)

Introduction: There is a paucity of data on the distribution of acute leukemia (AL) across ethnic groups. Historically, B-cell acute lymphoblastic leukemia (B-ALL) and acutepromyelocytic leukemia (APL) are over-represented in Hispanic patients, while acute myeloid leukemia (AML) is less frequent. We analyzed the ethnic distribution of AL in Florida based on incidence rates and nativity differences using Florida Cancer Data System (FCDS). In our study, B-ALL and APL were more common in Hispanics vs. non-Hispanics (IRR of 1.627 and 1.302, respectively); however, contrary to prior reports, AML was also more common (IRR 1.533) (Swords et al, BCJ 2016, in press). No nativity differences were observed. Given this novel finding of increased AML incidence in Hispanics in South Florida, we examined AML patients at Sylvester Comprehensive Cancer Center (SCCC) to look for ethnic differences in mutational frequency.Methods: We obtained molecular genetic data on AML patients treated from 2012-2015 across multiple centers.Genomic DNA from bone marrow or peripheral blood was sequenced for ASXL1, DNMT3A, FLT3 TKD, IDH1, IDH2, KIT, NPM1, PHF6, TET2 on the IlluminaMiSeq platform using a lower limit of detection of 5% and minimum coverage of 500X. Alignment and variant calling were performed usingNextGENe® software as previously described byGenoptix, Inc. MLL PTD, FLT3 ITD and CEBPA mutations were detected by PCR amplification followed by fragment analysis.For patients treated at SCCC, we obtained IRB approval to conduct retrospective chart reviews for clinical and pathologic data. Categorical data was analyzed by Fisher exact test or Chi-square test as appropriate. Continuous data was analyzed by independent t-test and Wilcoxon-Mann Whitney test for parametric and non-parametric data, respectively. Statistical analysis was performed using Stata (version 13.0).Circos plots were created usingCircos.Results: We examined the 11-gene mutational profile of 927 unique AML patients. The frequency of these mutations was consistent with previous reports (Park et al, NEJM 2012). DNMT3A, NPM1, and FLT3 were the most common abnormalities (Figure 1A). 75 of these 927 patients were treated at our center. Of these, median age was 60.2 years; 53.3% were female; 80% were white, 13.3% black,6.7% other. 68% (n=51) self-identified as non-Hispanic and 32% (n=24) as Hispanic (Table 1). Median WBC was 3.95 K/µL (1.4-100.7); 37% had antecedent MDS or MDS-related changes; 8% had favorable-, 69% intermediate-, and 23% poor-risk cytogenetics. There were no differences in WBC, MDS, or cytogenetic risk by ethnicity. In the 75 patient SCCC cohort, TET2 (n=14, 18.7%), CEBPA (n=14, 21.9%), and DNMT3A (n=11, 14.7%) were the most common mutations. 16 patients had an IDH mutation (10 IDH1 and 6 IDH2). We found no statistically significant difference in mutation frequency in Hispanic vs. non-Hispanic patients (Figure 1B); however, there was a non-significant increase in MLL-PTD mutations in Hispanic patients (15.8% vs. 9.3%, p=0.665) and IDH1 mutations in non-Hispanics (11.8% vs. 0%, p=0.168).Conclusions: We present one of the largest examinations to date (n=927 patients) of mutational frequency in AML. Our findings corroborate prior literature; however, we did note the rare co-occurrence of TET2 and IDH mutations-showing the 2 are not always mutually exclusive. We examined demographic data on a subset of these patients treated at SCCC, where we have a large Hispanic population, and assessed for ethnic differences in mutational frequency. We found a suggestion of increased MLL PTD mutations, a poor-risk abnormality, in Hispanic patients, and increased IDH1 mutations in non-Hispanics, but found no significant differences. This may be due to our relatively small sample size. We are now examining the full 927 patient cohort for demographic data and updated results will be presented. If certain AML mutations cluster by ethnicity, this might explain differing incidence rates and outcomes in Hispanics, and examination of predisposing heritable or environmental factors should be pursued.Table 1. Distribution of mutations in an 11-gene AML molecular profile by ethnicity in 75 patients with AML.Figure 1.Circosplots characterizing the distribution of concomitant mutations in 927 patients with AML (A); and the incidence of mutations by ethnicity in 75 patients with AML (B). [Display omitted] [Display omitted] [Display omitted] DisclosuresVaupel:Genoptix, a Novartis Company: Employment. Hall:Genoptix, a Novartis Company: Employment.

A Novel Regimen for Acute Myeloid Leukemia with MLL Partial Tandem Duplication: Results of a Phase 1 Study NCI 8485

Background: MLL partial tandem duplication (MLL PTD) occurs in approximately 5-8% of patients with acute myeloid leukemia (AML) and is associated with an adverse prognosis. Our group has published that the MLL wild type (WT) allele is epigenetically silenced in MLL PTD; we showed that re-expression of this gene can be induced with methyltransferase (DNMT) and/or histone deacetylase (HDAC) inhibitors. Further, re-expression of MLL WT following combined decitabine and HDAC inhibitor treatment sensitized MLL PTD myeloid leukemia cells to chemotherapy in vitro. We hypothesized that epigenetic silencing of the MLL WT contributes to MLL PTD-associated leukemogenesis and that its pharmacologic re-expression with DNMT and HDAC inhibitors would activate apoptotic mechanisms important for chemo-response in the clinic. We aimed to develop a regimen to be tested in this unique molecular subset of disease. Because of the relatively low frequency of MLL PTD AML, this dose finding study was conducted in relapsed/refractory (R/R) AML regardless of molecular subtype but was enriched for MLLPTD.Methods:In this phase 1 study, adults aged 18-59 years with R/R AML, ECOG 0-2, and preserved organ function were enrolled. Patients received decitabine 20mg/m2 daily on days 1-10 and vorinostat 400mg daily on days 5-10 for all dose levels. Dose-escalated cytarabine was given on days 12, 14, 16 (six doses) according to the following schedule: dose level (DL) 1, 1.5g/m2/q12hr; DL 2, 2g/m2/q12hr; DL 3, 2.5g/m2/q12hr; and DL 4, 3g/m2/q12hr. Standard method 3+3 phase I design was used with the primary objective to determine the maximum tolerated dose and define specific toxicities with this combination therapy, in order to ultimately develop a regimen for MLLPTD AML.Results:Seventeen adults with R/R AML and median age of 46 years (range, 21-59 years) enrolled. The median number of prior induction therapies was 2 (range, 1-4). European LeukemiaNet (ELN) genetic risk classification frequencies were: favorable (n=2), intermediate-I (n=3), intermediate-II (n=5), and adverse (n=7), respectively. Four patients had MLL PTD. A total of 6 patients were treated at DL 1 with no non-hematologic dose limiting toxicity (DLT). DL 1 was expanded after two patients experienced prolonged but uncomplicated myelosuppression. Since both patients achieved complete remission (CR) shortly after passing the day 42 DLT cut off for hematologic recovery, the protocol was amended to allow further time for count recovery (up to 56 days). Three patients each were treated on DL 2 and 3; 5 patients were treated on DL 4 with no other DLTs observed. Diarrhea, nausea, fatigue, febrile neutropenia, and elevated ALT were the most common toxicities (any grade, regardless of attribution), occurring in 41%, 29%, 29%, 35%, and 35% of patients, respectively, but none were DLTs. In regards to ≥ Grade 3 toxicities, febrile neutropenia and catheter-related infections were most common at 35% and 24%. Significant mucositis was not observed. DL 4 was the recommended phase 2 dose (RP2D). CR or CR with incomplete count recovery (CRi) was observed in 6/17 patients (35%, 5 with CR). Of 6 responders, all (n=4) patients with abnormal karyotype achieved cytogenetic remission. The median number of prior therapies for patients achieving CR/CRi was 2 (range, 1-3). Four patients subsequently received allogeneic transplantation. Of the four patients known to have MLL PTD mutations, two responded (1 with CR and 1 with CRi). It is interesting to note that the two patients with MLL PTD mutations who did not respond to treatment also had FLT3-ITD mutations, while this mutation was absent in the two responding patients who had MLLPTD.Conclusions: We successfully determined the RP2D for this novel treatment regimen. The regimen had modest toxicities beyond uncomplicated (though prolonged) myelosuppression, and we propose that the study provides a framework for larger efficacy studies for AML patients with the uncommon but biologically distinct molecular feature of MLLPTD.Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number U01CA076576. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Also supported by P30 CA016058/CA/NCI. DisclosuresNo relevant conflicts of interest to declare.

Higher expression levels of the HOXA9 gene, closely associated with MLL-PTD and EZH2 mutations, predict inferior outcome in acute myeloid leukemia.

BackgroundAlthough the biological insight of acute myeloid leukemia (AML) has increased in the past few years, the discovery of novel discriminative biomarkers remains of utmost value for improving outcome predictions. Systematical studies concerning the clinical implications and genetic correlations of HOXA9 aberrations in patients with AML are relatively promising.Materials and methodsHere, we investigated mutational status and the mRNA levels of the HOXA9 gene in 258 patients with AML. Furthermore, hematological characteristics, chromosome abnormalities, and genetic mutations associated with AML were analyzed, followed by the assessment of clinical survival. Besides, the expression level and mutational status of MEIS1, a cofactor of HOXA9, were also detected in patients with AML with the aim of a deeper understanding about the homeodomain-containing transcription factors associated with hematological characteristics.ResultsHOXA9 and MEIS1 mutations were detected in 4.26% and 3.49% AML cases, respectively. No correlations were detected between mutation status and clinical characteristics, cytogenetic and genetic aberrations, and clinical survival. Higher HOXA9 expression levels were correlated with white blood cell count and closely associated with unfavorable karyotype as well as MLL-PTD and EZH2 mutations, whereas, there was an inverse correlation with the French–American–British M3 subtype. Compared with patients with lower HOXA9 expression levels, those with higher HOXA9 expression levels had a lower complete remission rate and inferior survivals in both AML and cytogenetically normal AML.ConclusionHOXA9 expression may serve as a promising biomarker to ameliorate a prognostic model for predicting clinical outcome and consummating individualized treatment in patients with AML.

The Outcome of Relapsed Childhood Core Binding Factor Acute Myeloid Leukemia: A Report from the JPLSG AML-05R Study

BACKGROUND: Core binding factor acute myeloid leukemia (CBF-AML), which is characterized by the chromosomal abnormalities of t(8;21) and inv(16), is the most frequent subtype of pediatric AML. Patients with CBF-AML have a better prognosis than patients with a normal karyotype when they receive chemotherapy of appropriate intensity; however, in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 study, the excessive treatment reduction was observed to induce a higher incidence of relapse in children with CBF-AML. We investigated the outcomes of patients with relapsed CBF-AML who were registered in the JPLSG AML-05.PATIENTS & METHODS: We conducted a retrospective analysis of the JPLSG AML-05R study using a questionnaire. Furthermore, a mutation analysis was performed to identify NRAS, KRAS, KIT, WT1, NPM1, NUP98-NSD1, FLT3-ITD and MLL-PTD mutations using the available samples. We investigated the probability of overall survival (pOS) after relapse according to the patient's age, time from diagnosis to relapse, site of relapse, FAB classification, t(8;21) versus inv (16), reinduction chemotherapy regimen, obtaining a second complete remission before stem cell transplantation (SCT), the method of SCT and the presence of additional mutations.RESULTS: Forty-one of the 154 children with CBF-AML who were registered in the JPLSG AML-05 study experienced a relapse. The information from 32 of these 41 patients was collected using a questionnaire. Twenty-seven had t(8;21), and 5 had inv (16). It was possible to perform molecular analyses for 28 of the patients. KIT mutations were identified in 16 of 28 patients (57.1%). The follwing mutations were also detected: NRAS (n=4), KRAS (n=1), and WT1 (n=1). There were no cases with FLT3 -ITD, NUP98-NSD1, MLL-PTD or NPM1 mutations. Twenty-five cases achieved a complete remission after the initial reinduction (78.1%). The pOS was 65.6% (21/32) after a mean observation period of 2.7 years. The significant prognostic factors influencing pOS included a failure to obtain complete remission prior to SCT (p=0.03), reinduction chemotherapy regimen [p=0.02, a FLAG-based or ECM (etoposide, cytarabine and mitoxantrone)-based regimen was better than other modalities], donor source (p<0.01, cord blood was better than bone marrow and peripheral blood) and HLA matching between the donor and recipient (p=0.03, a mismatch was better than a full-match). Additional mutations, including the presence of the KIT gene, did not have any effect on pOS (p=0.64).CONCLUSIONS: Relapsed childhood CBF-AML in the JPLSG AML-05 study was still associated with a better prognosis, because almost two thirds of the patients were successfully treated with SCT. Obtaining complete remission prior to SCT was associated with a good prognosis. A FLAG- or ECM-based regimen is therefore recommended for reinduction chemotherapy. Unexpectedly, cord blood transplantation and HLA-mismatched SCT were associated with a better prognosis. Further investigations are necessary to clarify the genes that are associated with a poor prognosis in patients with CBF-AML. DisclosuresNo relevant conflicts of interest to declare.

Does Age of Patients Influence the Composition of Gene Mutations in Myeloid Neoplasms?

Introduction: Incidences of myeloid neoplasms, i.e. AML, MDS, MDS/MPN overlap, increase with age. Cytogenetic aberrations are still the hallmark for diagnosis and prognostication especially in AML and MDS. Different patterns of relations between chromosomal aberrations and age have been described. However, in recent years, gene mutations have been depicted to further discriminate patients with respect to their diagnosis and are increasingly used for prognostication. In parallel, recent studies (Jaiswal, NEJM 2014) have demonstrated that mutations in genes occurring in hematological neoplasms are also observed in healthy individuals and increase in frequency with age.Aim: To investigate if specific molecular markers, such as DNMT3A, ASXL1, and TET2, increase in frequency with age in myeloid neoplasms as recently shown for healthy individuals.Patients and methods: We investigated 1639 patients (pts) between 20 and 93 years (yrs), 578 with de novo AML (median age: 63 yrs), 846 with MDS (median age: 73 yrs), and 215 with CMML (median age: 75 yrs). In all cases, we followed the diagnostic criteria of the WHO classification based on morphology. All patients have also been investigated by cytogenetics and for disease-oriented molecular mutations (15-36 genes/pt: 15 in AML, 36 in MDS, and 21 in CMML). Analyses were performed by melting curve analysis, gene scan, Sanger sequencing, or next generation sequencing.Results: In total we detected 3089 mutations (range: 0-9/pt) spread over all except for seven analyzed genes. Grouping the entity-specific cohorts by age of the patients into decades revealed a steady increase of the prevalence of mutations with age in MDS (at least one mut/pt, 25% in 20-29 to 93% in >80 yrs; p<0.001), less prominent in AML (77% in 20-29 to 100% in >80 yrs, p=0.007), but not in CMML (96%-100% in all decades). However, the number of mutations per patient increased according to age in all three entities, significantly in MDS (p<0.001) and AML (p<0.001). Considering AML patients separated into three cytogenetic classes (Grimwade, Blood 2010) resulted in the same findings for the intermediate risk (p=0.012) and adverse risk group (p<0.001), while the good risk group showed no change in mutation numbers over decades (median: 1 mut/pt, range 0-3). This indicates that in good risk AML (PML-RARA, CBFB-MYH11, RUNX1 -RUNX1T1) only very few additional mutations are needed for AML initiation. In contrast, an age-dependent increasing incidence of gene mutations is specific in normal karyotype and in adverse cytogenetics.We next focused on specific gene mutations according to age <60 vs ≥60 yrs within all three entities. In addition, AML patients where again subgrouped by cytogenetics. In AML good and adverse risk groups no age-dependent significant increase of specific gene mutations occurred, while in the intermediate risk group mutations in ASXL1 (3/160 vs 32/114, p<0.001), MLL-PTD (3/160 vs 17/214, p=0.01), RUNX1 (32/160 vs 20/214, p=0.001), and TET2 (4/159 vs 26/214, p=0.001) were significantly more frequent at higher age. In contrast, NRAS mutations appeared more often in younger AML patients (32/160 vs 20/214, p=0.004). In MDS, mutations in SF3B1 (27/115 vs 253/731, p=0.019), SRSF2 (10/115 vs 133/730, p=0.011), TET2 (10/115 vs 250/731, p<0.001), and TP53 (2/115 vs 50/731, p=0.035) were more frequently observed in older patients. In CMML only TET2 mutations occurred more often in older patients (5/12 vs 135/190, p=0.026).Focusing on the genes recently described to be mutated in healthy individuals showed that all of the above mentioned mutations found in myeloid neoplasms (except MLL-PTD and RUNX1) are comprised in the 10 most frequently mutated genes in the healthy aging population. However, the fact that the frequencies of these mutations are not age-dependent in some entities, e.g. ASXL1 only age-dependent in AML but not in CMML and MDS, might indicate different roles of these mutations in the pathogenesis, i.e. driver mutations independent of age, as well as their contribution to accumulation of mutations and onset of a myeloid neoplasm.Conclusions: 1) The number of mutations significantly increase with age in AML and MDS and non-significantly in CMML. 2) Several genes show age-dependent frequencies, which differ between AML, MDS, and CMML and are also related to the cytogenetic background. 3) Based on molecular mutations healthy aging and myeloid neoplasms are neighbouring scenarios. DisclosuresMeggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Perglerová:MLL2 s.r.o.: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Clonal Evolution in NPM1 Mutated Acute Myeloid Leukemia (AML)

Background: Mutations in the nucleophosmin (NPM1) gene represent one of the most common gene mutations in AML, and are considered a founder event in the pathogenesis of AML. Recently, we gained novel insights into clonal architecture and clonal evolution in 53 NPM1 mutated (NPM1mut) AML patients (pts). Here, we showed the highest stability for DNMT3A mutations (DNMT3Amut) suggesting that these mutations represent an early event in leukemogenesis as well as an increase in genomic complexity.Aims: To validate our previous results on clonal evolution in a larger cohort of NPM1mut AML pts and to define relapse specific mutation patterns.Methods: Paired samples at diagnosis and relapse from 129 NPM1mut AML pts were assessed for additional mutations by comprehensive mutation analysis (FLT3-ITD, FLT3-TKD, DNMT3A, IDH1/2, NRAS, ASXL1, TP53, MLL-PTD). In addition, 11 AML cases with loss of NPM1mut at the time of relapse and 12 cases with persisting NPM1mut were subjected to whole exome sequencing (WES).Results: At diagnosis, the incidence of concurrent gene mutations was as follows: FLT3-ITDmut 32% (40/126), FLT3-TKDmut 19% (22/118), DNMT3Amut 69% (78/113), NRASmut 19% (22/117), IDH1mut 21% (26/123) and IDH2mut 19% (22/118). None of the pts analyzed exhibited a TP53 (n=53), MLL-PTD (n=63) or ASXL1 (n=59) mutation. At relapse, a significant shift in the genetic pattern was found in 74 pts (57%). FLT3-ITDmut was lost in 10 pts and newly acquired in 24 pts,16 pts lost FLT3-TKDmut. DNMT3Amut was lost in 2 pts and gained in 1 pt. NRASmut was lost in 12 pts and gained in 4 pts. IDH1mut was lost in 3 pts and acquired in 4 pts, while IDH2mut was lost in 2 pts and gained in only one pt. ASXL1mut and TP53mut were gained in 2 and 1 pts, respectively; gain of MLL -PTDmut (n=4) was restricted to pts with loss of NPM1mut at the time of relapse. Based on these findings we calculated the following stabilities: FLT3-ITDmut 75%, FLT3-TKDmut 27%, DNMT3Amut 97%, NRASmut 45%, IDH1mut 88% and IDH2mut 91%, respectively. In total, 11 pts (9%) lost NPM1mut at relapse while DNMT3Amut was present in 9/11 pts at diagnosis and remained stable at relapse; 4/11 pts gained MLL-PTDmut, one pt ASXL1mut and 3 pts acquired RUNX1mut.By WES we observed a persistence of mutations known to be involved in clonal hematopoiesis, such as DNMT3A and TET2 mutations. These were usually seen during all analyzed time points (diagnosis, remission and relapse). In addition, we found distinct mutational patterns at the time of relapse compared to the time of diagnosis. For example, relapse- and diagnosis-specific mutations in NPM1mut loss cases were significantly enriched for different signaling pathways.Conclusions: 74 (57%) of 129 NPM1mut AML pts showed clonal evolution at the time of relapse. DNMT3Amut demonstrated the highest stability (97%) confirming our previous findings that DNMT3Amut constitutes an early event which persists in preleukemic hematopoietic stem cells. High-resolution sequencing of selected cases is ongoing and will further unravel the clonal hierarchy in NPM1mut AML.Those authors equally contributed to work: SKS, SC, LB and KD. DisclosuresHeuser:Karyopharm: Research Funding.

BAALC Expression Is a Feasible Marker for Risk Stratification and Detection of Minimal Residual Disease in Cytogenetically Normal Acute Myeloid Leukemia

IntroductionDuring the last years it has been shown that PCR-based assessment of minimal residual disease (MRD) is of great importance for risk stratification and early detection of relapse in acute myeloid leukemia (AML). Most frequently, monitoring of MRD was restricted to patients carrying specific genetic markers such as fusion genes and gene mutations. However, many patients lack such markers amenable to sensitive detection by PCR. For this reason, it is still crucial to identify molecular targets that are applicable for MRD assessment in patients with AML. High brain and acute leukemia, cytoplasmic (BAALC) expression defines an important risk factor in cytogenetically normal AML (CN-AML) and has been proposed to represent a clinically useful biomarker to monitor response to therapy. A significant correlation of BAALC expression levels with the mutation ratio of internal tandem duplications in FLT3 (FLT3-ITD), partial tandem duplications in MLL (MLL-PTD) and NPM1 mutations has been shown previously (Weber et al., BCJ 2014). The objective of this study was to further validate the applicability of BAALC expression as a marker for detection of MRD.Patients and MethodsWe investigated BAALC mRNA expression levels in 550 diagnostic and follow-up samples from 116 de novo CN-AML patients showing high BAALC expression at initial diagnosis. BAALC mRNA expression was assessed by quantitative real-time PCR and normalized against the expression of the control gene ABL1. To identify high BAALC expressers the previously defined cutoff ratio of 33.1% BAALC/ABL1 (Weber et al., BCJ 2014) was used. The cohort was composed of 55 females (47%) and 61 males (53%). Age ranged from 18 to 85 years (median: 55). The median number of follow-up samples per patient was 3 (range: 1-21) with a median follow-up time of 32 months (range: 16-48 months). All patients included in this study were treated according to standard AML protocols in curative intent. In addition to BAALC expression, a full molecular characterization for the presence of FLT3 -ITD (42/116, 36%), MLL -PTD (10/116, 9%) and mutations in NPM1 (38/116, 33%) was performed.ResultsAt diagnosis, BAALC expression ranged from 33.2 to 5655.7% BAALC/ABL1 with a median of 163.9%. Of the 116 patients, 62 patients (53%) harbored at least one of the previously established MRD markers (FLT3-ITD, MLL-PTD and NPM1), while 54 patients (47%) lacked these MRD targets. To validate stability of BAALC expression we analyzed 25 patients with paired diagnostic and relapsed samples available. The respective BAALC expression levels at both time points showed good correlation (Mean % BAALC/ABL1 of diagnosis vs. relapse: 343.1 vs. 297.4, r=0.642, p=0.001). Three of the 25 patients relapsed after allogeneic stem cell transplantation. Also in these patients BAALC expression levels were similar between diagnosis and relapse (Mean %BAALC/ABL1: 686 vs. 684), supporting the use of BAALC expression for follow-up evaluation. Moreover, in 5 cases with cytomorphologic complete remission during follow-up a molecular relapse based on elevated BAALC expression levels (32.4-315.2% BAALC/ABL1) could be detected 21-42 days before cytomorphologic relapse. To investigate the predictive value of BAALC expression levels during follow-up, BAALC expression was analyzed in 58 patients after the second course of induction chemotherapy, but before start of consolidation chemotherapy. To separate low from high BAALC expression in this follow-up analysis, the cutoff established for diagnostic samples (33.1% BAALC/ABL1) was used. According to this cutoff, 29% (17/58) of the patients had high BAALC expression levels, while 71% (41/58) exhibited low BAALC expression levels. High BAALC expression was associated with a shorter EFS (median: 5 month vs. not reached, 1-year-EFS: 61% vs. 21%, p<0.001)ConclusionThis data validates the applicability of BAALC expression as a target for MRD monitoring in CN-AML. A significant number of CN-AML would benefit from molecular monitoring using a quantitative BAALC expression assay aimed at following disease course and early detection of relapse. DisclosuresWeber:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Targeted Resequencing of MLL-PTD Positive AML Patients Reveals a High Prevalence of Co-Ocurring Mutations in Epigenetic Regulator Genes

BackgroundPartial tandem duplication mutations of the Mixed Lineage Leukemia gene (MLL-PTD) can be found in about 10% of patients with AML, especially in patients with normal karyotype AML. The mutation generates a self-fusion within the N-terminal part of MLL and has been shown to be leukemogenic in mouse models. In patients, the presence of the mutation is associated with poor prognosis. Little is known on the molecular profile of patients with MLL-PTD and on the cooperating mutations. In order to identify accompanying molecular alterations, we performed whole exome sequencing (WES) of eight AML patients harbouring MLL-PTD mutations. Based on the observed alterations we then designed a custom amplicon panel and performed targeted resequencing in a cohort of 90 MLL-PTD mutated AML patients.Materials and MethodsAll patients included in this analysis were treated in prospective treatment protocols of the Study Alliance Leukemia (SAL). To enrich for malignant cells and to obtain germline reference material (T-cells), FACS sorting was performed on viable cells banked at diagnosis. After whole genome amplification of the primary DNA, whole exomes were enriched (TruSeq chemistry; Illumina), and paired-end sequenced using Illumina HiSeq2000 2x100 bp runs. Resulting data were mapped against human genome (Hg19). Only somatic single nucleotide variants (SNVs) were included in the final analysis. Based on the SNVs identified by whole exome sequencing (WES), a custom amplicon panel (TruSeq Custom Amplicon, TSCA, Illumina) for targeted resequencing was designed. The assay included either the entire coding region or mutational hot spots of 56 genes (Fig.1). In total, 700 targets were amplified in a single reaction for each patient and paired end sequenced on a MiSeq NGS system (Illumina). Paired end reads were BWA mapped against targeted regions and data analysis was done using the Sequence Pilot software package (JSI Medical Systems) with a 20% variant allele frequency (VAF) mutation calling cutoff. Only non-synonymous variants not specified as SNP in the db137 database and predicted as deleterious (Provean) were included in the final analysis. All variations were confirmed by Sanger sequencing.ResultsWES of eight MLL-PTD (7/8 FLT3-ITD negativ) patients revealed a total 490 SNVs (range 13-254 per patient). Most frequently mutated genes were DNMT3A, IDH1/2 and TET2. Somatic mutations were also found in genes rarely mutated in AML, such as ATM, GNAS, TET1 and EP300. Based on the WES-data, 90 MLL-PTD patients were screend for a panel of 56 genes using the TSCA assay, which revealed in total of 169 mutations. 18 genes were not found to be mutated and in 8 patients, no co-occurring mutations were identified. Due bad assay performance EP300, EZH1, JAK3, MLL2, MLL3 and NOTCH1 were excluded from the data analysis. Here again, the most frequently mutated genes were DNMT3A (34.4%), IDH1 (20.0%), IDH2R140 (18.9%), IDH2R172 (7.9%), TET2 (16.7%) and FLT3 (11.3%). Mutations were less frequently found in RUNX1 (8.9%) and ASXL1, SMC1A, U2AF1 (5.6% each) (Fig. 1). In addition to these known genes, most prevalent mutations were found in ATM (8.9%) as well as DNMT3B and TET1 (4.4% each). Overall, we oberserved a low frequency of mutations in typical class 1 genes such as NRAS, KRAS and FLT3, which was lower than reported in the TCGA data set.ConclusionsThis analysis in a large set of patients with MLL-PTD mutations did not reveal any new and specific individual mutation present in patients with this alteration. Instead, our finding of a very high prevalence of alterations in epigenetic regulator genes, found in more than 85% of patients with MLL-PTD, strongly argues for a particular disease biology in these patients. These findings might also implicate that treatment based on demethylating agents or histone-deacetylase inhibitors might be especially attractive in patients with MLL-PTD. [Display omitted] The assay included either the entire coding region or mutational hot spots of the following 56 genes; ASXL1, ATM, BCOR, BRAF, CBL, DDR1, DNMT1, DNMT3A, DNMT3B, EIF4A2, EP300, ETV6, EZH1, EZH2, FLT3, GATA1, GATA2, GNAS, HRAS, IDH1, IDH2, JAK1, JAK2, JAK3, KDM4A, KDM5A, KDM5C, KDM6A, KIT, KRAS, MET, MLL, MLL2, MLL3, NOTCH1, NOTCH4, NPM1, NRAS, PDGFRA, PDGFRB, PHF6, PTEN, PTPN11, RAD21, RUNX1, SF3A1, SF3B4, SMC1A, SMC3, SMC4, TET1, TET2, TP53, U2AF1 and WT1. DisclosuresThiede:AgenDix GmbH: Equity Ownership, Research Funding; Illumina: Research Support, Research Support Other.

Acute Myeloid Leukemia Alters Natural Killer Cell Maturation and Functional Activation

Acute myeloid leukemia (AML) is a devastating disease primarily affecting adults. Immune evasion is a major mechanism of AML persistence, and represents a barrier for long-term clinical success. Natural killer (NK) cells are a key component of the innate immune system and hold promise as a tool for effective anti-leukemia therapy. However, to date, the clinical success of NK therapy has been disappointing, indicating additional immune evasion strategies may affect the ability of NK cells to function in patients. We hypothesized that AML may evade the innate immune system by inhibiting NK cell maturation.To evaluate NK function and maturation during AML progression, our lab utilized a novel knock-in model of AML that expresses both Flt3-ITD and Mll-PTD mutations (PTD/ITD), and develops AML with 100% penetrance and recapitulates human disease. For these studies, both primary PTD/ITD mice and transplanted AML blasts were used. For transplant studies, AML or wild-type (WT) control cells (CD45.2+) were transplanted into irradiated C57BL/6 (CD45.1+) naïve recipients. Normal (CD45.1+) NK cells were defined as NK1.1+/CD3- by flow cytometry, and expression of receptors was determined within this population. NK cells in leukemic mice exhibited a reduction in the activating receptors Ly49D and Ly49H in the spleen, blood, LN, and bone marrow. There was also an increase in the inhibitory receptor Ly49C/I and NKG2C/A/E (both p<0.05). However, these alterations were not consistent with all activating and inhibitory receptors, as CD69 was elevated, while NKp46 expression was decreased (both p<0.05). NK function was determined by ELISA measurement of soluble interferon-gamma (IFN-g) production. While there was no change in IFN-g production at baseline, there was surprisingly a significant elevation in IFN-g detected upon stimulation with interleukin-12 and interleukin-18 in the NK cells isolated from leukemic mice as compared to WT control mice (p=0.0004).NK maturation was then evaluated by examining CD11b and CD27 protein expression on NK1.1+/CD3- cells in the spleen, bone marrow, blood, and LN. CD11b+/CD27+ (DP) NK cells were reduced in the spleen as well as bone marrow, blood and LN (p<0.02 for all organs). AML splenocytes had a corresponding increase in NK cells negative for both proteins (DN) when compared to WT control splenocytes (p=0.0004). Previous studies indicate the DP population of NK cells have the highest cytotoxic and cytokine producing functions, while DN NK cells are functionally immature. These alterations were more distinct as AML burden increased in the mice (p=0.0004). Interestingly, there was no significant difference in the most mature CD11b+/CD27- NK population in the spleen, but it there was a significant increase in the CD11b+/CD27- population in the blood (p=0.002). Additional analysis of two transcription factors known to regulate NK development, T-bet and Eomes, was also performed. A reduction in expression of both transcription factors was seen in total NK cells of leukemic mice, as well as NK subsets in the spleen (p<0.02). These data suggest that NK maturation is altered in the presence of AML, and that this alteration becomes more pronounced as leukemic burden increases. Studies evaluating the mechanistic link between AML and NK maturation are ongoing. Future studies will evaluate the efficacy of combinatorial therapies addressing both this imbalance in maturation as well as enhancing recognition to improve NK killing of AML cells. DisclosuresNo relevant conflicts of interest to declare.

Minimal Residual Disease by Multiparametric Flow Cytometry Predicts Relapse Free Survival better than Over-Expression of WT1 and BAALC in Acute Myeloid Leukemia

Background: Improved molecular and immunophenotypic characterization of AML offers the opportunity to define markers in individual patients which can be used to monitor minimal residual disease (MRD). Immunophenotypic analyses are based on the multiparametric flow cytometry (MPFC) detection of leukemia-associated immunophenotypes. The most useful molecular prognostic markers implicated in AML characterization are NPM1 mutations, FLT3 ITD, CEBPA mutations, MLL -PTD, RUNX1 and ASXL1 mutations. Deregulated expression of genes have also been identified as prognostic markers eg BAALC, WT1 and ERG. In this study, gene mutations (NPM1, FLT3, CEBPA, MLL-PTD), gene expression (WT1 and BAALC)and immunophenotyping were prospectively assessed to detect MRD.Methods: Diagnostic Marrow/peripheral blood samples from 98 AML patients (excluding AML-M3) were included between March 2012 and July 2014. Patients received standard induction chemotherapy with daunorubicin 60 mg/m2 for 3 days and cytosine arabinoside 100 mg/m2 iv for 7 days. Marrow was done 21-28 days after start of induction chemotherapy for assessment of morphology, MPFC and molecular markers. If marrow was in remission (CR), then patients received 1st consolidation therapy with 18 gm/m2 of cytosine arabinoside (HiDAC). Marrow was repeated 21-28 days after 1st consolidation chemotherapy for assessment of same parameters as above. Patients then received 2 more HiDAC or underwent allogeneic transplant according to cytogenetic risk. Cytogenetic analyses were performed using standard techniques of chromosome banding and FISH. All cases were divided into three cytogenetic risk groups (i.e. good, intermediate and poor) based on NCCN guidelines. Immunophenotyping was done using 8 color MPFC. The same panel of antibodies was used to characterize the leukemic cells at diagnosis, post induction and post consolidation. MRD was calculated as a percentage of abnormal leukemic cells per total nucleated cells. cDNA synthesis and quantitative real time PCR (RQ-PCR) assays for WT1 and BAALCwere carried out according to Europe Against Cancer guidelines. Mutation profiling was carried out by capillary electrophoresis and direct DNA sequencing methods as described previously. Chi-square method was used to detect any association between risk groups and gene mutations. Probabilities of relapse-free survival (RFS) were estimated using Kaplan-Meier method. Univariate comparisons of RFS for potential prognostic markers (molecular and MPFC) were made using log-rank test.Results: Of 98 patients enrolled in this prospective study, 86 patients completed induction and 59 patients 3 courses of HiDAC. The median age was 27 years (range 15-58). Based on cytogenetic risk groups 24.4%, 47% and 28.6% were good, intermediate and poor risk cases respectively. Twelve patients died during induction due to sepsis and 16 patients were refractory to induction chemotherapy. Baseline frequencies of FLT3-ITD, MLL-PTD, NPM1-type A and CEBPA gene mutations were 20.4%, 10.2%, 20.4% and 25.5% respectively. The biallelic (TAD1 and ZIP mutations) and monoalleleic CEBPA mutations were found in 4.1% and 21.4% patients respectively. No significant association was found between different risk groups and gene mutations except NPM1-type A mutations were associated with good risk group (P=0.015). MRD by RQ-PCR for BAALC/WT1 and MPFC was assessed after post induction and post first consolidation therapy. There was one log reduction in mean copy number of WT1 (5312 vs 430) and BAALC (19648 vs 3298) between diagnosis and post consolidation samples. No significant differences were found between high expressors of WT1/BAALC and RFS. MPFC analyses revealed that 48% of post induction samples were MRD positive (range: 0.05-38%, mean 6.6%) Similarly, 53% of post consolidation samples were MRD positive (range: 0.01-85%, mean 10.9%). Univariate analysis showed that MRD positivity by MPFC at post induction (RFS at 1.5 years- 85% vs 30%, P=0.012) and post consolidation (RFS at 1.5 years- 90% vs 42%, P=0.015) was associated with poor RFS. Patients without MLL-PTD mutation showed better RFS (P=0.003) as compared to mutated cases and TAD2positivity at post induction showed a trend to better RFS (P=0.097).Conclusion: Our results suggest that MRD by MPFC predicts RFS more accurately than over-expression of WI1 and BAALC. DisclosuresNo relevant conflicts of interest to declare.

Cytogenetic and Molecular Genetic Characterization Of MLL-PTD Positive AML In Comparison To MLL-Translocated AML

Background Partial tandem duplications within the MLL gene (MLL-PTD) are a recurrent molecular alteration in acute myeloid leukemia (AML). MLL-PTD occurs with a frequency of 6-8% in de novo AML. Data on additional cytogenetic and molecular alterations in MLL-PTD+ AML is scarce. Beside partial tandem duplications within the MLL gene, the MLL gene is also a target of balanced translocations leading to the fusion of MLL with a large variety of partner genes. Aims 1. Evaluate the spectrum of additional cytogenetic and molecular genetic alterations. 2. Analyze whether additional aberrations impact prognosis. 3. Compare the spectrum of additional abnormalities between MLL-PTD+ AML and AML with MLL-translocations. Patients and Methods We selected a cohort of 225 de novo AML patients harboring a MLL-PTD. These were compared to a cohort of 130 de novo AML with MLL-translocation (MLL-t). Mutation screening for the following genes was performed in pts with MLL-PTD and MLL-t, respectively: ASXL1 (132; 85), CEBPA (184; 67), FLT3-ITD (225; 125), FLT3-TKD (208; 112), IDH1R132 (145; 88), IDH2R140 (141; 63), IDH2R172 (137; 73), KRAS (59; 82), NRAS (98; 82), RUNX1 (213; 97), NPM1 (221; 123), TP53 (104; 89), and WT1 (159; 86). EVI1 expression was assessed in 55 MLL-PTD+ pts and in 77 pts with MLL-t. Results The frequency of MLL-PTD and MLL-translocations differed significantly with respect to age. While MLL-PTD were more frequent in elderly pts, MLL-t were more frequent in younger pts (&lt;10 yrs: 0%/2.3%; 10-19 yrs: 0%/3.8%, 20-29 yrs: 0.9%/13.1%, 30-39 yrs: 3.1%/9.2%, 40-49 yrs: 8.9%/23.1%; 50-59 yrs: 11.1%/13.1%; 60-69 yrs: 35.1%/13.8%; 70-79 yrs: 29.3%/13.8%; ≥80 yrs: 11.6%/7.7%; p&lt;0.001). FAB subtype distribution differed significantly between of MLL-PTD+ and MLL-t AML. While in MLL-PTD+ AML most frequently subtypes M1 (33.8%) and M2 (43.6%) were observed, AML with MLL-t most frequently showed M4 (30%) and M5a (23.1%). The most frequent cytogenetic abnormalities in MLL-PTD+ cases were gains of 11q (n=37), followed by 8q (n=14), and 13q (n=7) and losses of 5q (n=14), 7q (n=14) and 17p (n=5). In contrast, in MLL-t patients the most frequent gains were trisomies 6 (n=7) and 8 (n=33), as well as gains of 1q (n=10), 19p (n=10), 19q (n=8) and 21q (n=21). There were many significant differences in co-occurring mutations between MLL-PTD+ and MLL-t: DNMT3Amut: 44.7% vs 0% (p&lt;0.001), FLT3-ITD: 33.3% vs 3.2% (p&lt;0.001), IDH1R132 14.5% vs 0% (p&lt;0.001), IDH2R140: 19.9% vs 0% (p&lt;0.001), IDH2R172 9.5% vs 0% (p=0.005), and RUNX1 25.8% vs 2.1% (p&lt;0.001). On the other hand KRASmut (3.4% vs 23.2%, p=0.001) and NRASmut (8.2% vs 25.6%, p=0.002) were less frequent in MLL-PTD+ as compared to MLL-t AML. TP53 mutations were observed in comparable frequencies (3.8% vs 5.6%). NPM1 mutations were not detected in either entity. The mean EVI1 expression was significantly higher in MLL-t pts compared to MLL-PTD+ pts (167.1+/-259.1vs 0.4 +/- 0.47, p&lt;0.001). Overall, chromosome abnormalities in addition to the MLL alteration were more frequent in MLL-t AML as compared to MLL-PTD+ AML (mean number of alterations: 1.2 vs 0.7, p=0.004). This goes along with more additional molecular mutations in MLL-PTD+ AML as compared to MLL-t AML (mean number of molecular mutations: 1.5 vs 0.6, p=0.004). Overall survival at 5 yrs was comparable in both subgroups (MLL-PTD+: 27.9% vs MLL-t: 39.8%). In both subgroups age was significantly associated with OS (&lt;60 yrs vs ≥60 yrs: MLL-PTD+: 56.9 vs 16.3 months, MLL-t: 47.8 vs 9.7 months, for both p&lt;0.001). Neither in MLL-PTD+ AML nor in MLL-t AML the presence of additional chromosome aberration had an impact on outcome. With respect to molecular mutations only IDH2R140 was significantly associated with shorter OS (HR: 2.2, p=0.007) and IDH2R172 with longer OS (HR: 0.2, p=0.04) in MLL-PTD+ AML. Conclusions Although both MLL-PTD+ and MLL-translocations disrupt the same gene AML harboring one or the other MLL abnormality differ significantly with respect to age distribution, the pattern of additional cytogenetic abnormalities and the frequency of accompanying molecular mutations. MLL-PTD is more frequent in older patients, presents most frequently as FAB M1 and M2, and harbors more additional molecular genetic events and less additional cytogenetic events. However, both AML subtypes are associated with adverse outcome, particularly in elderly patients. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

High-Throughput Mutational Profiling Of Post-Myeloproliferative Neoplasm Acute Myeloid Leukemia Reveals Frequent Mutations In NRAS In JAK2V617F-Negative Post-MPN AML

BackgroundA subset of patients with the Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) transform to AML, and the prognosis of post-MPN AML patients is very poor. As such, new genomic insights are needed to define the mechanisms that govern transformation from MPN to AML, and to identify new therapeutic targets for clinical intervention in this poor-risk myeloid neoplasm. Importantly, patients with JAK2V617F mutant chronic-phase disease transform to JAK2 wildtype AMLs in approximately half of cases, indicating that diverse genomic paths lead to transformation. AimsTo characterize the genomic alterations, including point mutations, short indels, translocations, and copy number alterations, in 33 post-MPN AML samples. MethodsGenomic DNA and total RNA was isolated from formalin fixed paraffin embedded (FFPE) tissue, blood and bone marrow aspirates. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 272 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >590X for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. We achieved a mean coverage depth of 511x (range 405-645). ResultsThe most common genomic alterations identified in post-MPN AML samples were mutations in JAK2 (51.5%), ASXL1 (48.5%), and IDH2 (30.3%). Mutually exclusive mutations in the genes of spliceosome components SRSF2, U2AF1, and SF3B1 were identified in 39% of patients in this cohort, suggesting that somatic mutations in splicing factors are a common genomic event in transformation from MPN to AML. These data were in contrast to de-novo AML, in which mutations in FLT3, NPM1, and DNMT3A are the most common disease alleles (NEJM 2013; 368: 2059), suggesting that the spectrum of genomic alteration in post-MPN AML differs from that in de novo AML.Within our cohort of post–MPN AML samples, 52% of the patients had JAK2V617F positive AML and 48% of our cohort presented with JAK2 wildtype AML. Notably, the two subsets of post-MPN AML had distinct mutational patterns. In the JAK2V617F mutant subgroup, we identified frequent mutations in ASXL1 (41.2%), TP53 (41.2%), and IDH2 (41.2%). In JAK2-wildtype AML subgroup we identified frequent ASXL1 mutations (56.3%) and NRAS point mutations (37.5%). NRAS mutations were exclusive of JAK2 mutations in the entire cohort, consistent with alternate disease alleles which activate signaling in JAK2V617F positive AML and in JAK2 wildtype AML. SETBP1 mutations were found in 19% of patients with JAK2 wildtype post-MPN AML but not in any patients with JAK2V617F-positive post-MPN AML.Several alterations not previously described in post-MPN AML, to the best of our knowledge, were identified in this cohort including MLL-PTD, which was observed in both JAK2V617F and wildtype JAK2 patients. Copy number analysis of our high-depth sequencing data allowed us to identify homozygous deletions of TET2 and ETV6 and MYC amplifications in post-MPN AML. Univariate analysis demonstrated TP53 mutations were associated with significantly impaired overall survival (p<0.001). IDH2 mutations were associated with higher peripheral blood blast count (p=0.017). ConclusionThe spectrum of genomic alterations in post-MPN AML is strikingly distinct from de novo AML. Furthermore, capture-based sequencing allowed us to show there are important differences in the mutational profile of JAK2V617F and JAK2 wildtype post-MPN AML. NRAS mutations may represent driver mutations in JAK2V617F negative post-MPN AML and further biologic evaluation of these mutations is needed. Most importantly, the identification of NRAS, IDH2, and MLL-PTD mutations in post-MPN AML suggest specific therapies including MAPK inhibition, IDH2 inhibition and dot1L/BRD4 inhibition may be of value in genotypically defined subsets of post-MPN AML. These finding help to further define the biology of leukemic transformation, as well as offer new therapeutic avenues for the treatment of post-MPN AML. [Display omitted] Disclosures:Rampal:Foundation Medicine: Consultancy. Verstovsek:Incyte Corporation: Research Funding.

Possible Biomarkers To Predict Response In Patients With Myelodysplastic Syndromes (MDS) Or Acute Myeloid Leukemia (AML) Treated With 5-Azacitidine

IntroductionAzacitidine (aza) was the first drug to demonstrate a survival benefit for MDS patients (pts) in a randomized trial [Fenaux, Lancet Oncol 2009]. However, only about half of the pts respond and even after achieving complete remission (CR) and with sustained treatment, pts usually relapse. To date, limited data is available on prognostic factors influencing response. Therefore, until now we have analyzed 71 pts with MDS or AML, treated with at least 3 cycles of aza, to identify prognostic indicators that might help to improve treatment decisions. Patients and Methods71 cases were included and characterized by cytogenetics and various molecular assays including sensitive next-generation sequencing for mutations in ASXL1, DNMT3A, EZH2, FLT3-LM, IDH1, IDH2, KRAS, MLL-PTD, NRAS, RUNX1, SF3B1, SRSF2, TET2, and TP53. Median age was 71 (50-84) years. 38 pts had MDS (8 RAEB I, 26 RAEB II, 4 RCMD), 28 had AML (6 dysplastic, 16 sAML/MDS, and 6 tAML/MDS), and 5 pts had MDS/MPD. The IPSS [Greenberg, Blood 1997] was low in one, intermediate (int) 1 in 4, int 2 in 26, and high in 14 pts. Regarding cytogenetic risk groups we found 39 low, 11 int and 20 high risk pts according to IPSS and 49 int vs 21 high risk pts according to AML criteria [Grimwade, Blood 2010].Median time from diagnosis to treatment was 122 days (0-2348). With a median follow up of 1091 days from start of treatment median survival was 530 days (95% CI 412-874). Pts received a median number of 6 cycles (3-43). 12 pts have received allogeneic transplantation after aza treatment and were censored at that date. To assess the impact of biomarkers, Kaplan-Meier curves and Cox models were estimated from the first day of treatment. Variables were considered for multivariate analysis if the univariate p value was <0.1. ResultsRegarding cytogenetics, 47% of pts exhibited a normal karyotype, 17% had chromosome 7 abnormalities, 13% of karyotypes included del5q, 13% +8, 7% del20q, and 13% were complex. All except one case showed molecular aberrations. The most frequently mutated gene was ASXL1 (42%), followed by RUNX1 (38%), SRSF2 (38%), TET2 (30%), TP53 (18%), DNMT3A (16%), IDH2 (13%), NRAS (13%), EZH2 (12%), SF3B1 (11%), IDH1 (9%), FLT3-ITD (8%), MLL-PTD (7%), and KRAS (6%).Responses were observed in 33 pts (46%; 4 CR, 3 marrow CR with hematological improvement, 8 partial remission, and 18 HI). When cytogenetic abnormalities were examined, responses were seen in 45% of pts with normal karyotype, in 50% of pts with chromosome 7 aberrations, in 33% with del5q, in 67% with +8, in 56% with complex karyotype, and in all 5 cases with del20q (p=0.02).Regarding molecular abnormalities response rate was highest in pts with mutations in NRAS (67%) and IDH2 (63%), followed by RUNX1 (56%), ASXL1 (54%), SF3B1 (43%), TP53 (42%), SRSF2 (36%), TET2 (30%), DNMT3A (30%), MLL-PTD (25%), KRAS (25%), FLT3-ITD (20%), IDH1 (17%), and EZH2 (13%). However none of these mutations had a significant impact on response rate, possibly due to limited statistical power. The only p-value <0.1 was seen for EZH2 (p=0.08).Finally we analyzed the influence of prognostic markers on survival in aza treated pts. In univariate analysis potential candidate variables were: TP53 (p=<0.01, HR: 3.63), cytogenetic group according to AML (p=0.02, HR 2.02) or IPSS (p=0.07, HR (high-low) 1.92, (int-low) 0.86), complex karyotype (p<0.01, HR: 3.17), age [yrs] (p=0.01, HR: 1.08), Hb [g/dl] (p=0.078, HR: 0.836), time to start of aza [yrs] (p=0.01, HR: 0.753), MLL-PTD (p=0.07, HR: 2.87), and AML vs MDS (p=0.01, HR: 2.13).In multivariate analysis TP53 retained a significant influence on survival (HR: 4.99 [CI: 2.20-11.31], p<0.01), accompanied by age (HR:1.10 [CI: 1.03-1.16], p<0.01), Hb (HR: 0.69 [CI: 0.52-0.93], p=0.01), AML vs. MDS (HR: 2.42 [CI: 1.21-4.84], p=0.01), and time to start of aza (HR: 0.70 [CI: 0.52-0.94], p=0.02).TP53 mutations negatively influenced survival, although response rates were comparable to other pts. Response duration was often short. However, pts might still benefit from aza treatment, if combination therapy (e.g. lenalidomide in del5q and TP53 positive pts) or allogeneic transplantation consolidate and prolong treatment results ConclusionAfter the analysis of 71 pts, TP53 mutations represent the only somatic mutations with a significant negative impact on survival in aza treated pts. The small group of pts with del20q might have higher response rates. The analysis of further pts is ongoing. Disclosures:Kuendgen:Celgene: Honoraria, Research Funding. Off Label Use: Azacitidine in AML >30% marrow blasts. Lauseker:Celgene: Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Albuquerque:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Gattermann:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Germing:Celgene: Honoraria, Research Funding. Götze:Celgene Corp: Honoraria.

Prognostic Impact Of Molecular Mutations In 182 Patients With Myelodysplastic Syndromes

IntroductionMyelodysplastic syndromes (MDS) are heterogeneous regarding clinical characteristics, as well as prognosis and treatment approaches. Therapeutic decision making relies greatly on prognostic scoring systems. Recently, the gold-standard IPSS (International prognostic scoring system [Greenberg Blood 1997]) has been revised. The new IPSS-R still uses cell counts, marrow blast count, and karyotypic abnormalities to stratify patients (pts) into risk groups [Greenberg Blood 2012]. However, more than 50% of all MDS pts present with a normal karyotype and even in pts with identical chromosomal abnormalities outcome may vary. Somatic mutations are more common than cytogenetic abnormalities and can be identified in over 70% of pts including pts with normal karyotype [Bejar JCO 2012]. Therefore, the addition of such mutations to common prognostic markers might help to refine prognostication in MDS pts and improve therapeutic procedures by individualizing MDS treatment. Patients and MethodsWe analyzed 182 pts with different subtypes of MDS: RCUD, RARS, MDS-U, 5q- (n=28), RCMD +/-RS (n=85), RAEB 1 (n=16), RAEB 2 (n=25), MDS/MPD (n=24), AML <30% marrow blasts (n=4). Median age was 68 (16-87) years. Patients belonged to the following cytogenetic risk groups: 73% good, 13% intermediate, and 14% poor risk according to IPSS. In 29% of pts IPSS risk group was low, 43% intermediate 1, 21% intermediate 2, and 7% belonged to the high risk group. IPSS-R risk groups were very good 10%, good 32%, intermediate 29%, poor 13%, and very poor 16%.Various molecular assays were performed including sensitive next-generation sequencing for mutations in ASXL1, DNMT3A, EZH2, FLT3-ITD, IDH1, KRAS, MLL-PTD, NRAS, RUNX1, SF3B1, SFRS2, TET2, and TP53. Most of the data was collected prospectively within the clinical routine diagnostic procedures. During that time the marker panel was adjusted, when new analyses became available. Thus, not all markers are currently available for all pts (see table).To assess the impact of the biomarkers, Kaplan-Meier curves were estimated starting at the day of diagnosis. ResultsThe most frequent mutation was TET2 (30.2%), followed by ASXL1 (25.4%), SF3B1 (22.9%), SFRS2 (22.2%), RUNX1 (20.4%), DNMT3A (13.7%), TP53 (9.9%), EZH2 (9.0%), NRAS (4.7%), MLL-PTD (3.8), IDH1 (3.5%), FLT3-ITD (3.3%), KRAS (2.8%), IDH2 (2.8%), and CBL (2.2%).Three pts with normal karyotype, who would have been classified as ICUS (idiopathic cytopenia of undetermined significance) due to only mild dysplasia, were reclassified as MDS-U as they exhibited typical somatic mutations (RUNX1 plus TET2; TET2, and DNMT3A).Median follow up was 3.8 years (95% confidence interval [CI] 3.1-4.5). During this time 74 pts died and 108 pts were censored at the last date they were known to be alive. Median survival was 4.9 years (95% CI 2.7-7.1).A significant influence on survival in univariate analysis could be demonstrated for TP53 (p<0.001), EZH2 (p=0.003), SF3B1 (p=0.016), ASXL1 (p=0.016), and RUNX1 (p=0.042) (see table). Other prognostic variables with significant impact regarding survival were age at diagnosis (p<0.001), Hb (p=0.001), platelet count (p=0.002), marrow blast count (p<0.001), FAB subgroup (p<0.001), IPSS (p<0.001), IPSS-R (p<0.001), and cytogenetic risk groups according to IPSS (p<0.001) as well as IPSS-R (p=0.001). ConclusionWe could confirm mutations in TP53, EZH2, RUNX1, and ASXL1 to be predictors of poor overall survival, while SF3B1 mutations conferred a favorable prognosis in pts with MDS. Integrating mutation assessment into the clinical routine might improve diagnostic procedures as well as prognostication, and individualize treatment approaches. Further analyses are ongoing.Mutationpositivenegativepnmedian survival/ years95% CInmedian survival/ years95% CISF3B122not reached743.111.73-4.490.016TET2547.322.99-11.651254.502.18-6.810.241DNMT3A173.752.01-5.501076.462.14-10.790.224NRAS83.501.26-5.741624.881.87-7.900.334KRAS53.500.87-6.141715.813.59-8.030.237RUNX1343.112.12-4.101336.462.87-10.060.042SFRS2202.742.26-3.21706.070.00-12.990.399ASXL1432.682.14-3.211266.071.93-10.210.016FLT3-ITD52.441.46-3.421454.882.72-7.040.235IDH152.390.69-4.091395.813.09-8.520.190MLL-PTD62.060.00-4.461516.072.38-9.760.219EZH2121.781.02-2.541216.073.03-9.110.003TP53151.270.57-1.961366.462.80-10.13<0.001 Disclosures:Kuendgen:Celgene: Honoraria, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Gattermann:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Götze:Celgene Corp: Honoraria. Germing:Celgene: Honoraria, Research Funding.

The Prognostic Impact Of Complex Gene Mutation In The Intermediate Risk Karyotype Acute Myeloid Leukemia

Abstract Background Many gene mutations were detected and overlapped in de novo acute myeloid leukemia (AML), but the prognosis of complex gene mutation remains to be unclear. In this study, we analyzed the prognostic impact of complex gene mutation in de novo AML patients with the intermediate risk karyotype. Methods We analyzed 143 samples from de novo AML patients with the intermediate risk karyotype diagnosed at Nippon Medical School Hospital from 2000 to 2012. Bone marrow or peripheral blood samples containing 20% or more blast cells were used for analyses. Mutation analyses were performed using PCR method for FLT3-ITD, FLT3-TKD and MLL-PTD, and direct sequence for NPM1, C/EBPα, DNMT3a, IDH1/2, TET2 and N/K-RAS. Results The NPM1 (39.9%), DNMT3a (26.6%), FLT3-ITD (24.5%), IDH1/2 (18.9%), TET2 (17.5%), C/EBPα (14.7%), N/K-RAS (14.0%) and MLL-PTD (6.3%) mutations were detected in our cohort, respectively. When we performed prognostic analyses for mutations of these genes, DNMT3 mutation and FLT3-ITD were isolated as a poor prognostic factor in overall survival (OS) , respectively (DNMT3a mutation positive: n=39, 3yOS 17.9%. negative: n=104, 3yOS 33.2%. p=.0056) (FLT3-ITD positive: n=35, 3yOS 12.2%. negative: n=108, 3yOS 35.0%. p=.0077). Moreover, in the FLT3-ITD positive cases, OS of patients with DNMT3a R882 mutation was significantly shorter than those without R882 mutation (R882 positive: n=20, 3yOS: 0%. negative: n=15, 3yOS 25.0%. p&lt;.0256). Interestingly, High rate of patients with FLT3-ITD (91.4%), NPM1 (89.5%), DNMT3a (92.1%), TET2 (84.0%), and IDH1/2 (88.9%) mutations were detected other overlapped mutations, respectively. The frequency of the overlapped mutations in patients with DNMT3a mutation, especially with mutations on R882, was significantly higher than those in patients without them (DNMT3a: p=.0001, R882: p&lt;.0001). For total cohort, the rates of and OS and relapse free survival (RFS) in patients with three or more overlapping mutations (complex gene mutation: CGM) were significantly lower than those in patients without them (CGM+: n=36, 3yOS 5.6%. CGM-: n=107, 3yOS 37.7%. p&lt;.0001) (CGM+: n=12, 3yRFS: 8.3%. CGM-: n=57, 3yRFS: 36.0%. p=.0013). Moreover, among the patients without FLT3-ITD, the rates of RFS and OS at 3 years in patients with complex gene mutation were significantly lower than those in patients without them (CGM+: n=11, 3yOS 5.6%. CGM-: n=96, 3yOS 37.7%. p&lt;.0408) (CGM+: n=4, 3yRFS: 8.3%. CGM-: n=51, 3yRFS: 36.0%. p=.0179). Conclusions Our study revealed that the gene mutations appeared to be overlapped, and the complex gene mutation significantly affected the prognosis of de novo AML with the intermediate risk karyotype. Intriguingly the DNMT3a mutation may contribute to an occurrence of complex gene mutation by giving genetic instability to AML cells. Disclosures: No relevant conflicts of interest to declare.

Modeling and Targeting MLL-PTD/RUNX1 Related MDS/AML In Mouse

Myelodysplastic syndromes (MDS) are heterogeneous disorders in which the hematopoietic stem cells (HSCs) in the bone marrow are defective, resulting in insufficient normal blood cells. MDS progress to secondary acute myeloid leukemia (sAML) in about one third of patients, as additional genetic abnormalities are acquired. Because of the similar molecular mechanisms under these two related disease categories, MDS with increased blasts (>5%) and AML with multilineage dysplasia and/or antecedent MDS, are also defined as MDS/AML. MLL and RUNX1/CBFb regulate normal hematopoiesis, and we have shown that they form a regulatory complex to regulate downstream target genes. Mutations of MLL1 (in-frame partial tandem duplication, MLL-PTD, or MLL translocations) or RUNX1 are found in about 28% of MDS, particularly in high-risk MDS or therapy-related MDS. sAML frequently contains both MLL-PTD and RUNX1 mutations, arguing for cooperative leukemogenic synergy between these two molecular lesions. However, Mll-PTD knock-in mice or Runx1Δ/Δ mice do not develop spontaneous MDS or AML. RUNX1 mutations can cause mouse MDS/AML in murine retroviral transduction mediated overexpression and BMT, however, the latency is long (8-14 months) and retroviral vector insertion mutagenesis at Evi1 or Mn1loci seems critical for MDS/AML development in this model. Indeed RUNX1 mutations cooperate with Evi1 upregulation in both murine MDS/AML model and human AML. Thus, we hypothesize that combining RUNX1 mutations with MLL-PTD may facilitate its transformation toward MDS and/or sAML.To understand the impact of RUNX1 mutation cooperativity with MLL-PTD, we first expressed MDS relevant patient-derived RUNX1 mutants (D171N and 291fsX300) in the context of Mll-PTD knock-in mouse bone marrow cells and performed BMT and in vitro CFU replating assay. RUNX1 mutations (D171N and 291fsX300) could not transform WT BM cells. However, they could transform MLL-PTD BM cells and undergo serial replating. Interestingly, D171N and 291fsX300 transformed MLL-PTD cells form different type of clones: MLL-PTD/D171N clones are bigger and diffuse, while MLL-PTD/291fsX300 clones are smaller but denser. In BMT assay, the MLL-PTD/D171N and MLL-PTD/291fsX300 BMT mice developed MDS and MDS/AML (2-10 months) after BMT. The MLL-PTD/D171N BMT mice developed anemia, neutropenia with leukodysplasia and left-shifted differential counts, and a hypo-cellular marrow with excess blasts, while MLL-PTD/291fsX300 BMT mice developed rather similar trilineage dysplasia features but present hyper-cellular marrow with high percent of blasts, some of the mice were diagnosed as MDS/AML. Interestingly, the MLL-PTD/291fsX300 BMT mice also develop myelo-fibrosis (MF) in the BM. We further generated Mll-PTD/Runx1Δ/Δ mice using Mx1-Cre mediated deletion. These mice showed thrombocytopenia one month after pI-pC injection, and developed pancytopenia 2-4 months later. The CBC exhibited increased MCV, RDW and severe anemia. All these Mll-PTD/Runx1Δ/Δ mice died of MDS induced complications within 8 months, and tri-lineages dysplasias (TLD) were found in bone marrow aspiration. Similar but accelerated lethal MDS were found in recipients transplanted with PTD/Runx1Δ/Δ BM cells compared with controls (median survival: 68 days VS undefined). Low dose decitabine (DAC 0.3 mg/kg, twice a week, subcutaneous injection) were used to treat these recipients, and we found significantly longer median survival in DAC treated recipients than controls (median survival: 94.5±6.4 VS 53.5±3.5 days, p<0.001). Neither Mll-PTD nor Runx1Δ/Δ BM cells could replate more than 4 times with M3434 methaltheloase, however, PTD/Runx1Δ/Δ BM cells could be replated more than 6 months in vitro. We also treated these cells in vitro with DAC (0.5 uM). Fewer colony numbers and increased differentiated cells (Gr1+/Mac1+) were found in DAC treated cells than PBS treated controls (CFU numbers/1x105seeded cells: 34±7.7 vs 619±30.5, p<0.001).In conclusion, our study demonstrates that: 1) RUNX1 mutations and complete deletions cause MDS or MDS/AML phenotypes in Mll-PTD background; 2) Decitabine is a promising drug to treat MLL-PTD/RUNX1 related MDS/AML. These exciting new models allow us to identify and analyze MDS/AML-initiating cells (MIC) and major targets that are critical for clonal evolution and pathogenesis of MDS/AML and therapeutic interventions. Disclosures:No relevant conflicts of interest to declare.

Integrated Genetic Profiling Of JAK2 Wildtype Chronic-Phase Myeloproliferative Neoplasms

BackgroundThe Myeloproliferative Neoplasms (MPNs), including Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF) are clonal hematopoietic disorders. JAK2V617F mutations are observed in approximately 90-95% of PV cases, but only 40-50% of ET and PMF cases. Although JAK2 exon 12 and LNK mutations are observed in the majority of JAK2V617F-negative PV patients, candidate gene and exome sequencing studies to date have not identified activating oncogenes in the majority of JAK2V617F-negative ET/PMF patients. Thus, further genetic investigations are needed to define the mutational architecture of these JAK2 wildtype MPNs in order to gain insight into the biology of these diseases, the clinical implications of genetic events that do occur, and the elucidation of potential therapeutic targets. AimsTo characterize the spectrum of genetic alterations in JAK2 wildtype chronic-phase myeloproliferative neoplasms. MethodsWe identified 32 patients with a confirmed diagnosis of an MPN (per 2008 WHO criteria), including MF, PV and ET, who were negative for JAK2V617F using a CLIA-certified allele specific assay for the JAK2 disease allele. Genomic DNA and total RNA was isolated from formalin fixed paraffin embedded (FFPE) tissue, blood and bone marrow aspirates. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 258 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >590X for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. ResultsHigh coverage sequencing allowed us to identify JAK2V617F mutations in two patients (allele burden 3-5%) that were below the limit of detection of the CLIA assay. The most common mutations observed in JAK2V617F-negative MPN were in ASXL1 (22% of patients) and in TET2 (9%). Taken together, mutations in known epigenetic modifiers (ASXL1, TET2, DNMT3A, EZH2, MLL) were observed in 43% of samples, including a MLL-PTD mutation in one patient with PMF. We identified mutations in spliceosome components (SRSF2, U2AF1), in a subset of patients, consistent with previous reports, and in each case mutations in spliceosome components were mutually exclusive.We identified mutations in the JAK-STAT pathway (MPL, TYK2) and the RAS pathway components (KRAS, NF1) in 9% of this patient cohort, suggesting that there are alternate disease alleles that activate signaling in JAK2V617F-negative MPN. RNA-sequencing identified a ETV6-ABL1 fusion in one patient, and we identified amplification of PIK3CA in one patient in our cohort; these data suggest fusion genes and amplifications activate signaling in a subset of patients with JAK2V617F-negative MPN. We also identified novel mutations in MPN patients which have not been reported to date, including mutations in DNA repair genes (ATM and BRCA) in 25% of cases and mutations in the Notch signaling pathway (NOTCH1-4) in 31% of cases. The functional implications of these novel mutations remain to be elucidated.In univariate analysis, ASXL1 mutations were found to associate with impaired overall survival (Figure 1, p=0.049). These findings are consistent with data demonstrating an impaired survival in patients with MDS and PMF, and suggest that ASXL1 mutations represent an important biomarker for adverse outcome in JAK2V617F-negative MPN. [Display omitted] ConclusionsThese data demonstrate that the mutational spectra of JAK2V617F-negative MPN includes genes implicated in epigenetic regulation, novel mutations which activate gene signaling, and fusion genes/copy number alterations which provide a novel mechanism of oncogenic activation not previously reported in MPN. ASXL1 mutations occur frequently in JAK2 wildtype Philadelphia-Chromosome negative MPNs, and are associated with impaired overall survival. Collectively, these findings support the importance of ASXL1 mutations in predicting outcome in JAK2V617F-negative MPN, demonstrate that mutations in signaling effectors and in epigenetic regulators are common in MPN, and illustrate the genetic heterogeneity of JAK2V617F-negative MPN. Disclosures:Rampal:Foundation Medicine: Consultancy. Levine:Foundation Medicine, Inc: Consultancy.