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Germline DDX41 variants are the most common mutations predisposing to acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) in adults, but the causal variant (CV) landscape and clinical spectrum of hematologic malignancies (HMs) remain unexplored. Here, we analyzed the genomic profiles of 176 patients with HM carrying 82 distinct presumably germline DDX41 variants among a group of 9821 unrelated patients. Using our proposed DDX41-specific variant classification, we identified features distinguishing 116 patients with HM with CV from 60 patients with HM with variant of uncertain significance (VUS): an older age (median 69 years), male predominance (74% in CV vs 60% in VUS, P = .03), frequent concurrent somatic DDX41 variants (79% in CV vs 5% in VUS, P < .0001), a lower somatic mutation burden (1.4 ± 0.1 in CV vs 2.9 ± 0.04 in VUS, P = .012), near exclusion of canonical recurrent genetic abnormalities including mutations in NPM1, CEBPA, and FLT3 in AML, and favorable overall survival (OS) in patients with AML/MDS. This superior OS was determined independent of blast count, abnormal karyotypes, and concurrent variants, including TP53 in patients with AML/MDS, regardless of patient’s sex, age, or specific germline CV, suggesting that germline DDX41 variants define a distinct clinical entity. Furthermore, unrelated patients with myeloproliferative neoplasm and B-cell lymphoma were linked by DDX41 CV, thus expanding the known disease spectrum. This study outlines the CV landscape, expands the phenotypic spectrum in unrelated DDX41-mutated patients, and underscores the urgent need for gene-specific diagnostic and clinical management guidelines.

Introduction:Myeloid/lymphoid neoplasmswith fibroblast growth factor receptor 1 rearrangement (MLN FGFR1) are aggressive hematologic neoplasms caused by various reciprocal translocations involving chromosome band 8p11, resulting in tyrosine kinase fusion genes (eg, ZMYM2::FGFR1from t[8;13][p11;q12] or BCR::FGFR1from t[8;22][p11;q11]) and constitutive FGFR1activationand downstream signaling. Pemigatinib is a potent and selective oral inhibitor of FGFR1-3 approved for the treatment of adults with relapsed or refractory MLN FGFR1. Efficacy and safety data for 41 patients with MLN FGFR1in the ongoing FIGHT-203 study of pemigatinib were previously reported. Median daily pemigatinib dose was 9.5 mg; treatment-emergent adverse events led to dose interruptions in 66% of patients. Rates of complete response (CR) among the 38 patients evaluable for clinical response and rates of complete cytogenetic response (CCyR) for the 40 patients evaluable for cytogenetic response were 74% and 70%, respectively. CR and CCyR occurred in >80% of patients with chronic phase disease and in >50% of patients with blast phase disease. Here we present an analysis of centralized fluorescence in situ hybridization (FISH) evaluation of bone marrow (BM) cells and serial characterization of levels of FGFR1fusion transcripts in this same cohort.

Introduction

The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs), and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded blood and bone marrow samples with high accuracy in a clinically relevant time frame, which is performed in our Clinical Laboratory Improvement Amendments–certified College of American Pathologists–accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs, and gene fusions, with similar accuracy to lower-throughput assays that focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis, and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically relevant genomic alterations with therapeutic relevance.

Leukemic transformation (LT) of myeloproliferative neoplasms (MPNs) is associated with a poor prognosis and resistance to therapy. Although previous candidate genetic studies have identified mutations in MPN patients who develop acute leukemia, the complement of genetic abnormalities in MPN patients who undergo LT is not known nor have specific molecular abnormalities been shown to have clinical relevance in this setting. We performed high-throughput resequencing of 22 genes in 53 patients with LT after MPN to characterize the frequency of known myeloid mutations in this entity. In addition to JAK2 and TET2 mutations, which occur commonly in LT after MPN, we identified recurrent mutations in the serine/arginine-rich splicing factor 2 (SRSF2) gene (18.9%) in acute myeloid leukemia (AML) transformed from MPNs. SRSF2 mutations are more common in AML derived from MPNs compared with LT after myelodysplasia (4.8%) or de novo AML (5.6%), respectively (P = .05). Importantly, SRSF2 mutations are associated with worsened overall survival in MPN patients who undergo LT in univariate (P = .03; HR, 2.77; 95% CI, 1.10-7.00) and multivariate analysis (P < .05; HR, 2.11; 95% CI, 1.01-4.42). These data suggest that SRSF2 mutations contribute to the pathogenesis of LT and may guide novel therapeutic approaches for MPN patients who undergo LT.

Leukemic transformation (LT) of myeloproliferative neoplasms (MPNs) is associated with a poor prognosis and resistance to therapy. Although previous candidate genetic studies have identified mutations in MPN patients who develop acute leukemia, the complement of genetic abnormalities in MPN patients who undergo LT is not known nor have specific molecular abnormalities been shown to have clinical relevance in this setting. We performed high-throughput resequencing of 22 genes in 53 patients with LT after MPN to characterize the frequency of known myeloid mutations in this entity. In addition to JAK2and TET2mutations, which occur commonly in LT after MPN, we identified recurrent mutations in the serine/arginine-rich splicing factor 2 (SRSF2) gene (18.9%) in acute myeloid leukemia (AML) transformed from MPNs. SRSF2mutations are more common in AML derived from MPNs compared with LT after myelodysplasia (4.8%) or de novo AML (5.6%), respectively (P= .05). Importantly, SRSF2mutations are associated with worsened overall survival in MPN patients who undergo LT in univariate (P= .03; HR, 2.77; 95% CI, 1.10-7.00) and multivariate analysis (P< .05; HR, 2.11; 95% CI, 1.01-4.42). These data suggest that SRSF2mutations contribute to the pathogenesis of LT and may guide novel therapeutic approaches for MPN patients who undergo LT.

The discovery of JAK2 and MPL mutations in patients with myeloproliferative neoplasms (MPNs) provided important insight into the genetic basis of these disorders and led to the development of JAK2 kinase inhibitors for MPN therapy. Although recent studies have shown that JAK2 kinase inhibitors demonstrate efficacy in a JAK2V617F murine bone marrow transplantation model, the effects of JAK2 inhibitors on MPLW515L-mediated myeloproliferation have not been investigated. In this report, we describe the in vitro and in vivo effects of INCB16562, a small-molecule JAK2 inhibitor. INCB16562 inhibited proliferation and signaling in cell lines transformed by JAK2 and MPL mutations. Compared with vehicle treatment, INCB16562 treatment improved survival, normalized white blood cell counts and platelet counts, and markedly reduced extramedullary hematopoeisis and bone marrow fibrosis. We observed inhibition of STAT3 and STAT5 phosphorylation in vivo consistent with potent inhibition of JAK-STAT signaling. These data suggest JAK2 inhibitor therapy may be of value in the treatment of JAK2V617F-negative MPNs. However, we did not observe a decrease in the size of the malignant clone in the bone marrow of treated mice at the end of therapy, which suggests that JAK2 inhibitor therapy, by itself, was not curative in this MPN model.

Disease alleles that activate signal transduction are common in myeloid malignancies; however, there are additional unidentified mutations that contribute to myeloid transformation. Based on the recent identification of TET2mutations, we evaluated the mutational status of TET1, TET2, and TET3in myeloproliferative neoplasms (MPNs), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). Sequencing of TET2in 408 paired tumor/normal samples distinguished between 68 somatic mutations and 6 novel single nucleotide polymorphisms and identified TET2mutations in MPN (27 of 354, 7.6%), CMML (29 of 69, 42%), AML (11 of 91, 12%), and M7 AML (1 of 28, 3.6%) samples. We did not identify somatic TET1or TET3mutations or TET2promoter hypermethylation in MPNs. TET2mutations did not cluster in genetically defined MPN, CMML, or AML subsets but were associated with decreased overall survival in AML (P= .029). These data indicate that TET2mutations are observed in different myeloid malignancies and may be important in AML prognosis.

Germline DDX41variants are the most common mutations predisposing to acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) in adults, but the causal variant (CV) landscape and clinical spectrum of hematologic malignancies (HM) remain unexplored. Here, we analyzed the genomic profiles of 176 HM patients carrying 82 distinct presumably germline DDX41variants among a group of 9,821 unrelated patients. Using our proposed DDX41specific variant classification, we identified features distinguishing 116 HM patients with CV from 60 HM patients with variant of uncertain significance (VUS): an older age (median 69 years), male predominance (74% in CV versus 60% in VUS, P=0.03), frequent concurrent somatic DDX41variants (79% in CV versus 5% in VUS, p<0.0001), a lower somatic mutation burden (1.4 ± 0.1 in CV versus 2.9 ± 0.04 in VUS, P=0.012), near exclusion of canonical recurrent genetic abnormalities including mutations in NPM1, CEBPAand FLT3in AML, and favorable overall survival (OS) in AML/MDS patients. This superior OS was determined independent of blast count, abnormal karyotypes, and concurrent variants, including TP53in AML/MDS patients, regardless of patient’s sex, age or specific germline CV, suggesting that germline DDX41variants define a distinct clinical entity. Furthermore, unrelated patients with myeloproliferative neoplasm (MPN) and B-cell lymphoma were linked by DDX41CV, thus expanding the known disease spectrum. This study outlines the CV landscape, expands the phenotypic spectrum in unrelated DDX41-mutated patients, and underscores the urgent need for gene-specific diagnostic and clinical management guidelines.

Delic: Diaceutics: Employment, Equity Ownership. Blombery:Janssen: Honoraria; Novartis: Consultancy; Invivoscribe: Honoraria. Calasanz:Janssen: Honoraria; Diaceutics: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria. Colomer:Novartis: Honoraria; Incyte: Honoraria. Evans:Diaceutics: Honoraria; Novartis: Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Mason:Novartis: Honoraria; Jazz Pharmaceuticals: Honoraria; AbbVie: Honoraria. Thiede:Daiichi Sankyo: Honoraria; Diaceutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; AgenDix GmbH: Employment, Equity Ownership. Clark:Diaceutics: Employment, Equity Ownership.

No relevant conflicts of interest to declare.

Deininger: Blueprint: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Yaish: Octapharma: Other: Study investigator.

Guzman: Cellectis: Research Funding. Roboz:Cellectis: Research Funding; Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy. Melnick:Janssen: Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Perl: Actinium Pharmaceuticals: Consultancy; Asana Biosciences: Consultancy; Arog Pharmaceuticals: Consultancy; Ambit/Daichi Sankyo: Consultancy; Astellas US Pharma Inc.: Consultancy. Becker:Millenium: Research Funding. Lewis:Roche: Honoraria, Other: Travel; Amgen: Other: Travel. Levine:Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.

South: Affymetrix: Consultancy, Honoraria; ARUP Laboratories: Employment; Lineagen Corporation: Consultancy; Illumina: Consultancy, Honoraria.

He: Foundation Medicine, Inc.: Employment, Equity Ownership. Brennan:Foundation Medicine, Inc.: Employment, Equity Ownership. Nahas:Foundation Medicine, Inc.: Employment, Equity Ownership. Balassubramanian:Foundation Medicine, Inc.: Employment, Equity Ownership. Otto:Foundation Medicine, Inc.: Employment, Equity Ownership. Lipson:Foundation Medicine, Inc.: Employment, Equity Ownership. Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc.: Employment, Equity Ownership. van den Brink:Boehringer Ingelheim: Consultancy, Other: Advisory board attendee; Merck: Honoraria; Regeneron: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tobira Therapeutics: Other: Advisory board attendee. Rowe:Amgen: Consultancy; BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx Ltd.: Consultancy. Levine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.

Shp2 is a non-receptor protein-tyrosine phosphatase encoded by PTPN11and implicated in the Ras, JAK-STAT and PI3K pathways. Activating mutations in Shp2 are found in patients with developmental disorders such as Noonan and LEOPARD syndrome, as well as, hematologic malignancies. Although rare in most other solid tumors, Shp2 mutations are common in juvenile myelomonocytic leukemia (JMML) accounting for ~35% of cases. To understand its role as a cooperating mutation in AML we sequenced PTPN11in human samples. Here we report that Shp2 mutations are present in human AML at a rate of 6.6% (6/91) in the ECOG E1900 dataset. To investigate the biological function of Shp2 mutations we asked how this functions in a cooperative model of leukemogenesis with the MLL-AF9 fusion protein. Despite showing increased genetic stability compared to other leukemias, MLL leukemias commonly contain type I mutations that can functionally cooperate resulting in more aggressive leukemias. These mutations often occur in genes encoding components of the Ras pathway including mutually exclusive mutations of NRAS, KRAS, PTPN11and NF1and account for ~37% of MLLrearranged leukemias. However, the mechanisms of cooperation with MLL fusion proteins are poorly understood. We found that the Shp2E76K activating mutation commonly found in humans significantly accelerates MLL-AF9 mediated leukemogenesis. The E76K mutation results in structural changes that confer increased phosphatase activity to the Shp2 protein and increased Ras signaling. We attribute the MLL-AF9/Shp2E76K cooperation to a more rapid leukemic initiation as evidenced in colony formation assays using mouse bone marrow HSPCs. Cells transduced with MLL-AF9/Shp2E76K expanded faster than MLL-AF9 cells at early stages following transduction, indicating more efficient transformation of myeloid progenitors than MLL-AF9 alone. Cytokine independent growth is achieved in MLL-AF9 cells following expression of Shp2E76K through the constitutive activation of the IL3 signaling pathway and ERK phosphorylation. Importantly, addition of Shp2E76K significantly accelerated MLL-AF9 mediated acute myeloid leukemia in mice, indicating activated Shp2 cooperates with MLL-AF9 in vivo. In addition, leukemic stem cell frequency was increased by greater than 4 fold due to Shp2E76K expression. As Shp2 is reported to regulate an anti-apoptotic gene program, we investigated these in the context of MLL-AF9 leukemic cells with and without Shp2E76K. While Bcl2, BclXLand Mcl-1, were upregulated in Shp2E76K cells, Mcl-1 showed the highest upregulation in response to Shp2E76K. Further, expression of Mcl-1 with MLL-AF9 in colony assays phenocopies expression of Shp2E76K suggesting that, mechanistically, Shp2 mutations may cooperate through activation of an anti-apoptotic gene program, primarily through Mcl-1. Finally, we asked how leukemic cells bearing Shp2E76K would respond to small molecule inhibition of Mcl-1. MLL-AF9 leukemic cells expressing Shp2E76K were desensitized to small molecule mediated Mcl-1 inhibition consistent with increased Mcl-1 protein. These data were confirmed in human cells where U937 cells, which contain an activating Shp2 mutation, exhibited resistance to Mcl-1 inhibition compared to ML2 or K562 cell which both bear wild type Shp2. Together, these data suggest patients with hyperactive Shp2 signaling may respond poorly to drugs targeting Mcl-1 due to an overabundance of the protein.

Mutations in TET2and IDH1/2,which result in reduced conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), are observed in a significant proportion of patients with acute myeloid leukemia (AML). Hence these mutations define a novel class of AMLs with alterations in DNA methylation. However, it has been shown that not all AML cases with low levels of 5-hmC harbor somatic mutations in TET2and IDH1/2. We hypothesized there are additional somatic mutations that can lead to alterations in TET enzyme function and resultant changes in the epigenetic state of hematopoietic cells.

Chemotherapy induced neutropenia (CIN) was a challenge to physicians until the discovery of growth factors. Neutropenia predisposes the patient to infection with increased morbidity, mortality, and cost of hospitalization. Not only does CIN leads to a delay in chemotherapy but also a dose reduction, which results in decreased efficacy and impact on survival. Filgrastim is a granulocyte colony stimulating factor that leads to the proliferation, differentiation and function of neutrophils. With an increase in the neutrophil count and function, Filgrastim reduces the incidence of neutropenia and the complications associated with febrile neutropenia (FN), making it possible to deliver chemotherapy on time, without dose adjustment. ASCO guidelines recommend that Filgastrim be given at 5 mcg/kg/day starting from days 1–3 after chemotherapy for patients with the risk of FN greater than 20% until the absolute neutrophil count is near-normal. It was recently reported that two doses of Filgrastim were comparable to higher number doses to prevent the nadir. We hypothesized that only a single dose is sufficient to prevent the nadir and delay in treatment.A retrospective analysis of chart data from all patients treated at The Brooklyn Hospital Center ambulatory clinic was reviewed after the initiation of chemotherapy. Data collected included the white cell count, neutrophil count, dates of chemotherapy and Filgrastim administration. Patients who received one dose of Filgastrim within 2–3 days, but not less than 24 hours prior to receiving subsequent chemotherapy comprised the study population. Data was analyzed for each individual cycle of chemotherapy.The study population consisted of 11 patients with a total of 33 cycles of chemotherapy. Of the 11 patients, 9 had breast cancer, 1 had embryonal cancer, and 1 had colon cancer. Chemotherapy varied from Etoposide and Cisplatin, FOLFOX with Bevacizumab, and Adriamycin with Cyclophosphamid (AC) followed by Paclitaxel where the AC was given as a dose dense regimen. All the cycles had adequate response in neutrophil count to a single dose of Filgrastim when it was delivered within 2–3 days, but not before 24 hours of receiving chemotherapy. The mean absolute neutrophil count prior to Filgrastim was 0.8 compared to 6.0 (p=0.0001) after a single dose. There was no delay in treatment noticed as a result of the single dose of Filgrastim in all the 33 cycles.Filgrastim as a single dose is sufficient to treat CIN and prevent delay in subsequent treatment.No relevant conflicts of interest to declare.

Relapsed ALL carries a very poor prognosis despite intensive therapy, indicating the need for new insights into disease mechanisms. We have previously used gene expression profiling (Hogan et al. ASH 2009) and copy number analysis (Yang et al. Blood 2008) in paired diagnosis and relapsed ALL samples to better understand the biologic mechanisms leading to recurrent disease. To create an integrated genomic profile of ALL, we have now focused on high throughput RNA sequencing to detect changes in the transcriptome from diagnosis to relapse.To date we have sequenced 6 matched diagnosis/relapse pairs (i.e. 12 marrow samples) from B-precursor ALL patients enrolled on Children's Oncology Group (COG) P9906 and AALL0232 trials. RNA libraries were prepared from poly-A selected RNA and sequenced using 54 base pair single end reads using the Illumina Genome Analyzer IIx. Each sample was sequenced in at least 7 lanes, generating an average of 100 million reads per sample. BWA (v0.5.8) was used to align the reads to the human genome, producing an average of 53 million mapped reads. Samtools (v0.1.8) was then used to predict genetic variants across the genome, filtering out variants with a low mapping quality (0), low coverage (<8X), or overlap with known single nucleotide polymorphisms (SNPs) from dbSNP (r131) or the 1000 Genomes Project.We observed a total of 119,000 genetic variants across all samples, with comparable overall mutational burden at relapse and diagnosis. To identify candidate lesions that may indicate a selection for common chemoresistance pathways, we focused our analysis on relapse-enriched, non-synonymous variants. 8,486 non-synonymous variants (insertions/deletions and single nucleotide variants [SNV]) were identified that occurred more often at relapse compared to diagnosis. Our analysis was focused on relapse-enriched SNVs that coded for non-synonymous changes, of which 154 were prioritized for validation. Validation was completed using matched genomic DNA samples and PCR products were directly sequenced. Mutation calls were made by manual review of tracings using the Mutation Surveyor program from Softgenetics. Thirty-three percent of predicted SNV loci were validated, but upon further sequencing of matched germline samples, five relapse specific mutations were confirmed. Mutations in COBRA1, FAM120A, RGS12, SND2, and SMEK2 were found in individual patient relapse samples. Validation is currently ongoing to confirm additional SNVs and an expanded validation of mutations will be completed in an additional 66 matched diagnosis/relapse pairs from COG 9906 and AALL 0232 and 0331 studies. Relapse specific isoforms identifying alternative exon usage was also detected in 15 genes, all of which were shared amongst multiple patients. In addition, a significant increase (p=6.7×10−6) was observed in the number of poly-adenylation sites in the genes of the relapse samples.While, isoform specific expression was shared amongst patients at relapse, all relapse specific mutations were private and our data to date indicate that a diversity of mechanisms contribute to relapsed disease. Further sequencing analysis of our expanded cohort of samples will determine the mutation and isoform expression prevalence, as well as the functional significance and the potential therapeutic relevance.No relevant conflicts of interest to declare.

EZH2 and ASXL1 mutations were recently described in a spectrum of myeloid malignancies; mutational analysis of small patient cohorts has suggested the highest mutational frequency in myelofibrosis (MF) and chronic myelomonocytic leukemia (CMML). The current study seeks to determine i) EZH2 and ASXL1 mutational frequencies in WHO-defined subcategories of MF, CMML and blast-phase myeloproliferative neoplasm (MPN), ii) if these mutations are mutually exclusive of TET2, IDH, JAK2 and MPL mutations and iii) clinical correlates of ASXL1 and EZH2 mutations in primary MF (PMF) and CMML.The study population included 94 patients: 46 PMF, 22 post-polycythemia vera/essential thrombocythemia MF (post-PV/ET MF), 11 blast-phase MPN and 15 CMML (10 CMML-1 and 5 CMML-2). High throughput DNA resequencing was used to screen archived bone marrow for EZH2, ASXL1, TET2, IDH, JAK2 and MPL mutations.ASXL1 mutations were identified in all disease categories, including PMF (13%), post-PV/ET MF (23%), blast phase MPN (18%), and CMML (20%). We identified somatic mutations in TET2 in 15%, 14%, 18%, and 13% of PMF, post-PV/ET MF, blast phase MPN, and CMML, respectively. By contrast, mutations in EZH2 and IDH1/2 were less frequent. EZH2 mutations were seen in 3 out of 46 PMF patients (7%) and were not observed in patients with post-PV/ET MF or blast phase MPN. Mutations in IDH1/2 were restricted to blast-phase MPN (36%) and PMF (7%). No mutations in EZH2 or IDH1/2 were seen in CMML.Although we identified frequent TET2 and ASXL1 mutations, we only identified one patient with concurrent mutations in both genes. Three ASXL1 mutation-positive patients also had mutations in EZH2 or IDH and one patient had concurrent ASXL1, TET2 and IDH mutations. In addition, 7 ASXL1, 7 TET2, and 1 IDH mutated patients were JAK2V617F-positive. MPL mutations were also documented in all three mutation categories.All EZH2- and ASXL1-mutated PMF patients displayed normal karyotype and none underwent leukemic transformation during follow-up. Furthermore, mutated versus unmutated patients, in both instances, were not significantly different in age and sex distribution or clinical characteristics. The 3 EZH2-mutated PMF patients died after 29, 48 and 67 months from the time of mutation analysis. In univariate analysis, the presence of mutant ASXL1 in PMF was associated with worse survival (p=0.06) but the borderline significance was lost during multivariable analysis that included risk stratification according to DIPSS (Passamonti et al. Blood 2010; 115: 1703–1708). The 3 ASXL1 mutated CMML cases were alive after 40, 34 and 12 months from time of mutation analysis and none of them had progressed to acute leukemia; karyotype was normal in two of the patients and showed isolated trisomy 8 in one.ASXL1 mutations are as frequent as TET2 mutations in MF and CMML. In contrast, EZH2 mutations are infrequent and cluster with PMF. ASXL1 and EZH2 mutations are not mutually exclusive events, seem to be associated with normal karyotype and do not appear to be leukemogenic or prognostically detrimental in PMF or CMML.No relevant conflicts of interest to declare.

Abstract 3070

Levine: Novartis: Research Funding; TargeGen: Consultancy. Verstovsek:Incyte: ; Exelixis: ; Cephalon: ; SBIO: ; AstraZeneca: .

Abstract 2894