Finke, Christy M.

Abstract Background : Gene mutations are common (~90%) in patients with chronic myelomonocytic leukemia (CMML) and involve epigenetic regulators (TET2 ~ 60%, ASXL1 ~40%), spliceosome components (SRSF2 ~40%) and signal pathways (RAS ~30%). Of these, thus far, only ASXL1 mutations have been shown to adversely impact overall survival (OS). In the current study, we used a 27-gene panel assay to identify additional prognostically-relevant mutations in CMML and to also determine if number of mutations carries prognostic relevance. Methods : 175 patients with WHO-defined CMML were included in the study. All patients had bone marrow (BM) biopsies and cytogenetics performed at diagnosis. Targeted capture assays were carried out on BM DNA specimens obtained at diagnosis for the following genes; TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, SUZ12, SRSF2, SF3B1, ZRSR2, U2AF1, PTPN11, Tp53, SH2B3, RUNX1, CBL, NRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF, and SETBP1. Paired-end indexed libraries were prepared from individual patient DNA using the NEBNext Ultra Library prep protocol on the Agilent Bravo liquid handler. Capture libraries were assembled according to Nimblegen standard library protocol. Base-calling was performed using Illumina's RTA version 1.17.21.3. Genesifter software was utilized to analyze targeted sequence data. Specific variants were deemed as mutations if they were associated with a hematological malignancy (as identified by COSMIC database), or if they were not associated with a dbSNP Results: Among the 175 study patients, 66% were males and median age was 70 years. 146 (83%) patients were subclassified as CMML-1. At a median follow-up of 23 months, 146 (83%) deaths and 25 (14%) leukemic transformations were documented. Median survivals were 24 months for CMML-1 and 16 months for CMML-2 (p=0.38). Mutational frequencies were; TET2 46%, ASXL1 45%, SRSF2 45%, SETBP1 19%, CBL 14%, RUNX1 14%, NRAS 12%, U2AF1 8%, SF3B1 6%, ZRSR2 6%, Tp53 5%, DNMT3A 5%, IDH2 5%, PTPN11 5%, SH2B3 5%, JAK2 4%, NPM1 3%, CSF3R 2%, IDH1 2%, EZH2 1%, SUZ12 1%, KIT 1%, FLT3 1%, CALR 1%. 172 patients (98%) had at least one mutation, 21 (12%) had 2, 24 (14%) had 3, 20 (11%) had 4, 9 (5%) had 5, while one (1%) patient had 6 concurrent mutations. Risk stratification was based on Mayo prognostic model: 25% high, 32% intermediate and 43 % low risk. In univariate analysis, presence of ASXL1 mutations (p=0.01), absence of TET2 mutations (p=0.005) and presence of DNMT3A mutations (p=0.02) were associated with inferior survival; in multivariable analysis, ASXL1 (p=0.01) and TET2 (p=0.03) mutations remained significant. In order to determine prognostic interaction between these two mutations, patients were stratified into four mutational categories: ASXL1wt/TET2wt (n =56), ASXL1mut/TET2wt (n =31), ASXL1mut/TET2mut (n =50) and ASXL1wt/TET2mut (n =38). Survival data in these four groups showed significant difference in favor of ASXL1wt/TET2mut (median survival 38 months; p=0.016), compared to those with ASXL1wt/TET2wt (19 months), ASXL1mut/TET2wt (31 months)and ASXL1mut/TET2mut (16 months); there was no significant difference in survival among the latter three groups (p=0.3) (Figure). The number of mutations per patient did not affect outcome (p=0.3). In multivariable analysis, presence of ASXL1 mutations (P=0.01) and absence of TET2 mutations (p=0.003) remained significant when risk factors used in the Mayo prognostic model (HB <10 gm/dl, AMC >10 x 10(9)/L, platelets <100 x 10(9)/L, circulating IMC) were added to the model; the same was true for ASXL1wt/TET2mut (p=0.036). In a separate multivariable analysis that included the Mayo prognostic model as a single variable along with presence of ASXL1 and absence of TET2 mutations; or absence of ASXL1wt/TET2mut mutational status, the respective hazard ratios were 1.4 (95% CI 1.07-2.1; p=0.012), 1.5 (95% CI 1.07-2.1; p=0.03) and 1.8 (95% CI 1.2-2.7; p=0.001). Leukemia-free survival was worse in ZRSR2 -mutated cases (p=0.03). Conclusions: Almost 100% of patients with CMML express one or more myeloid neoplasm-relevant mutations. The current study suggests a favorable prognostic impact from TET2 mutations, unless accompanied by ASXL1 mutations. Disclosures No relevant conflicts of interest to declare.

Abstract Background The Dynamic International Prognostic Scoring System-plus (DIPSS-plus) uses eight adverse factors to predict survival in primary myelofibrosis (PMF): age >65 years, hemoglobin <10 g/dL, leukocytes >25 x 109/L, circulating blasts 31%, constitutional symptoms, red cell transfusion need, platelets <100 x 109/L and unfavorable karyotype (J Clin Oncol. 2011;29:392). The latter two also predicted leukemic transformation. Most recently, an international study of 879 PMF patients identified ASXL1, SRSF2, EZH2 and IDH1/2 mutations as inter-independent predictors of shortened overall or leukemia-free survival (Leukemia 2013Apr 26 doi: 101038/leu2013119. 2013). Molecular prognostication is now well established for acute myeloid leukemia with normal karyotype (NK). There is currently little information on clinical or molecular prognostication in NK-PMF. Methods Diagnosis of PMF was according to WHO criteria. The study population included all referrals to the Mayo Clinic, Rochester, MN, USA or the University of Florence, Florence, Italy. Information on clinical and laboratory parameters and karyotype was available in all study patients, at time of referral, which coincided with time of sample collection for mutation screening. The current study considered only those patients with NK-PMF. ASXL1, EZH2, SRSF2, IDH1/2 and JAK2 mutations were performed on variable number of patients depending on bone marrow or granulocyte DNA availability. Clinical parameters examined for prognostic relevance included those listed for DIPSS-plus, less unfavorable karyotype. Results A total of 690 patients with NK-PMF patients were studied. Median age was 65 years (14-89). The percentage of patients with age >65 years was 48%, red cell transfusion dependent 35%, hemoglobin <10 g/dL 49%, platelets <100 x 10(9)/L 19%, leukocyte count >25 x 10(9)/L 15%, circulating blasts ≥1% 49% and constitutional symptoms 33%. At a median follow-up of 29 months, 351 (51%) deaths and 39 (6%) leukemic transformations were recorded. Mutational frequencies and clinical correlates The respective frequencies (mutated/number of patients studied) of JAK2V617F, ASXL1, EZH2, SRSF2 and IDH1/2 mutations were 60% (284/473), 34% (72/214), 8% (14/179), 15% (37/249) and 5% (14/262). There was no significant association between JAK2V617F and any one of the aforementioned mutations. Inter-mutation association was evident only between SRSF2 and IDH1/2 (p=0.0005). Each one of the DIPSS-plus risk parameters was examined for correlation with a specific mutation: ASXL1 was associated with leukocyte count >25 x 10(9)/L (p<0.0001) and circulating blasts ≥1% (p=0.0005); EZH2 with leukocyte count >25 x 10(9)/L (p=0.008); and SRSF2 with age >65 years (p=0.0007), transfusion need (p=0.04) and hemoglobin <10 g/dL (p=0.03). JAK2V617F was associated with age>65 years (p=0.003) and leukocyte count >25 x 10(9)/L (p=0.02). Predictors of overall and leukemia-free survival In univariate analysis, all 7 DIPSS-plus parameters, as well as ASXL1, EZH2, SRSF2 and IDH1/2 mutations showed significant association with shortened survival: When each of these analyses was adjusted for age, all except constitutional symptoms and SRSF2 mutations retained their significance. Multivariable analysis of the six age-independent DIPSS-plus variables identified all but transfusion-need as independent predictors of inferior survival. A similar analysis restricted to mutations identified ASXL1 and EZH2 as being independently adverse. When both mutations and DIPSS-plus variables were included in the multivariable model, only four parameters remained significant: age >65 years (HR 4.2; p<0.0001), platelets <100 x 10(9)/L (HR 3.4; p<0.0001), ASXL1 mutations (HR 2.2; p=0.0001) and EZH2 mutations (HR 2.7; p=0.001). A similar analysis identified SRSF2 mutations (HR 5.9; p=0.0003) and platelets <100 x 10(9)/L (HR 4.3; p=0.005) as the only predictors of leukemia-free survival. Conclusions In NK-PMF, molecular markers might be prognostically more useful than conventional models that rely on clinical features. In the current study, thrombocytopenia was the only clinical variable, other than age, with additional value to molecular prediction of survival and leukemic transformation. Disclosures: No relevant conflicts of interest to declare.

Abstract Background Chronic myelomonocytic leukemia (CMML) is a clonal stem cell disorder with overlapping features between myelodysplastic syndromes and myeloproliferative neoplasms. Numerous models exist for CMML prognostication, with more recent studies suggesting (JCO 201; 31:2428) or refuting (Leukemia 2013; 27:1504) the prognostic contribution of ASXL1 mutations. Furthermore, SETBP1 mutations were recently shown to be associated with shortened overall survival (OS) in CMML (Leukemia 2013; 10:1038). In the current international study, we examine these issues in a larger cohort of 431 patients. Methods 431 patients with WHO-defined CMML were included in the study. 235 (55%) were seen at the Mayo Clinic from 1997 through 2012. The remainders were from the French CMML registry (JCO 201; 31:2428). All patients underwent bone marrow (BM) examination and cytogenetic evaluation at diagnosis. DNA analysis for spliceosome component mutations (SRSF2, SF3B1 and U2AF1), ASXL1 and SETBP1 mutations were carried out on BM specimens obtained at diagnosis. In order to address the aforementioned discrepancy regarding the prognostic impact of ASXL1 mutations, relevant analyses in the Mayo cohort were first performed with and without inclusion of missense ASXL1 mutations. ASXL1 mutations from the French cohort did not include missense mutations. We evaluated the prognostic relevance of ASXL1 and SETBPI mutations, as well as several other clinical and laboratory parameters including those previously identified by the MDAPS (Blood 2002;99:840) the Spanish cytogenetic risk stratification (Haematologica 2011;96:375), and the Mayo prognostic model (Leukemia 2013;27;1504). Results Among the 431 study patients, 286 (66%) were males and median age was 73 years (range, 17-93 years). There were 368 (85%) patients with CMML-1 and the remainder had CMML-2. At a median follow-up of 23 months, 260 (60%) deaths and 70 (16%) leukemic transformations were documented. Median survivals were 38 months for CMML-1 and 24 months for CMML-2 (p=0.11). Mutational frequencies were 44% (173/390) for SRSF2, 6% (23/379) for SF3B1, 7% (27/387) for U2AF1, 38% (164/411) for ASXL1 (excluding missense mutations), and 5% (21/431) for SETBP1. Risk stratification was, based on i) Mayo prognostic model: 172 (40%) high, 151 (35%) intermediate and 94 (25 %) low risk, ii) MDAPS: 15 (3%) high, 73 (17%) intermediate-2, 125 (29%) intermediate-1 and 218 (50%) low risk and iii) Spanish cytogenetic stratification system: 316 (73%) low, 43 (10%) intermediate and 50 (12%) high risk. In the Mayo cohort, univariate analysis revealed that the exclusion of missense mutations changed the prognostic impact of ASXL1 mutations from non-significant (p=0.08) to significant (p=0.001). Accordingly, all subsequent analyses excluded missense ASXL1 mutations. In univariate analysis, lower hemoglobin (p<0.0001), lower platelet count (p=0.0027), higher absolute monocyte count (AMC) (p<0.0001), higher absolute lymphocyte count (ALC) (p=0.0002), circulating immature myeloid cells (IMC) (P<0.0001), cytogenetic risk stratification (p<0.0001) and ASXL1 mutations (p<0.0001) were significant for OS. In multivariable analysis, lower hemoglobin (p=0.0001; RR 2, 99% CI 1.6-2.6), lower platelet count (p=0.002; RR 1.5, 99% CI 1.2-1.9), higher AMC (p=0.0002; RR 2.2, 99% CI 1.6-3.1) and ASXL1mutations (p=0.0009; RR 1.9, 99% CI 1.5-2.4) retained their independent negative prognostic impact. Similarly, in univariate analysis, leukemia-free survival (LFS) was negatively affected by age (p=0.0015), lower hemoglobin (p=0.0002), lower platelet count (p=0.0002), higher AMC (p<0.0001), higher ALC (p=0.0001), circulating IMC (p<0.0001), BM blasts (p<0.0001), and cytogenetic risk stratification (p=.0002). ASXL1 (p=0.17) and SETBP1(p=0.87) mutations were not found to be significant. In multivariable analysis, lower platelet count (p=0.0005), higher AMC (P=0.0042), circulating IMC (P=0.008) and cytogenetic risk stratification (p=0.009) retained their independent negative prognostic impact. Conclusions In the current international study of a large cohort of patients with CMML, we confirm and clarify the independent prognostic relevance of ASXL1 mutations. The relatively high frequency of ASXL1 mutations in CMML warrants its inclusion in contemporary prognostic models. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2895 Poster Board II-871 Background: Recent studies have reported a significant association between JAK2V617F acquisition and a specific JAK2 germline haplotype; rs12343867 (located at intron 14 of the JAK2 gene) is one of several SNPs that tag this haplotype. We performed rs12343867 SNP analysis in a consecutive cohort of patients with primary myelofibrosis (PMF) in order to study the clinical and laboratory correlates of the particular JAK2 haplotype and its effect on survival. Methods: Study patients were recruited form the Mayo Clinic database for PMF. Molecular studies were performed on DNA extracted from stored bone marrow. rs12343867 SNP genotyping was performed by a commercially available Taqman assay. Quantitative JAK2V617F analysis was done according to previously published methods. Standard statistical methods were used to test significance of associations between SNP genotype and other variables. Survival analysis was performed by the Kaplan-Meier method and comparisons made by the log-rank test. Cox regression model was used for multivariable analysis. Results: 132 patients with PMF were studied (median age 62, range 28-82; 82 males). Risk distribution according to the International Prognostic Scoring System (IPSS) for PMF were low in 39 (29%) patients, intermediate-1 in 41 (31%), intermediate-2 in 25 (20%) and high in 27 (20%). 77 (58%) patients were JAK2V617F positive; median mutant allele burden was 26% (range 1-85) and 21 patients displayed > 50% mutant allele burden. The rs12343867 genotype distributions were C/C 22%, C/T 44% and T/T 34% . The corresponding figures in JAK2V617F positive/negative cases were 31%/9%, 38%/53%, 31%/38% (p=0.01). Among the 21 patients with > 50% JAK2V617F allele burden, 14 (67%) displayed the C/C allele, 2 C/T and 5 T/T. The specific SNP genotype was not significantly affected by age (p=0.33), sex (p=0.46), leukocyte count (p=0.39), platelet count (p=0.09), or IPSS risk category (p=0.63). Patients were followed for a median of 54 months and during this period 73 (55%) deaths were documented. The T/T SNP genotype was significantly associated with shortened survival, compared to either C/C or C/T (p=0.001; Figure). Multivariable analysis showed this association to be independent of IPSS, JAK2V617F mutational status, age or sex. The adverse prognostic effect of the T/T genotype was also apparent when JAK2V617F negative (p=0.01) or positive (p=0.06) cases were analyzed separately. Conversely, JAK2 mutational status or allele burden did not affect survival in patients with T/T or C/C allele. Conclusion: The current study illustrates the non-random haplotype distribution of JAK2V617F in patients with PMF. More importantly, a non-JAK2 haplotype, tagged by the rs12343867 T allele, was associated with inferior survival that is not accounted for by IPSS or JAK2V617F mutational status. These findings, together with previous observations regarding shortened survival associated with low JAK2V617F allele burden, suggest the presence of molecular events in PMF that are more aggressive than JAK2V617F and not necessarily linked to the JAK2 haplotype. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2906 Poster Board II-882 Background: The World Health Organization (WHO) classification system recognizes indolent SM (ISM) as an entity characterized by low systemic mast cell (MC) burden but frequent skin involvement. The WHO system also recognizes 2 provisional ISM sub-variants: smoldering SM (SSM) and bone marrow (BM) mastocytosis (BMM). The two are respectively characterized by increased systemic MC burden (presence of ≥ 2 ‘B-findings') and BM MC infiltration in the absence of skin or multiorgan visceral lesions. The prognostic relevance of this sub-classification remains unclear. Methods: The study population was drawn from a larger cohort of 342 adult SM patients (Blood. 2009 Jun 4;113(23):5727). Clinical data and BM histology were reviewed, and the diagnosis of ISM and its subclassification confirmed per the 2008 WHO proposal. The primary objectives of the study were to: (i) describe the clinical characteristics of a large cohort of ISM patients, within the context of the WHO classification; (ii) evaluate the prevalence of molecular and cytogenetic abnormalities; (iii) evaluate whether ISM subclassification is prognostically relevant on the basis of survival and risk of transformation to acute leukemia or aggressive SM (ASM). Results: (i) Clinical characteristics: 159 ISM patients were evaluated (69 males; median age 49 years, range 19-84); 22 (14%) had SSM, 36 (23%) BMM, and 101 (63%) ‘ISM-other' (ISMo). By definition, ‘B-findings' were absent in BMM patients. ‘B-findings' in SSM (and ISMo) patients was as follows: hepatosplenomegaly and/or lymphadenopathy 91% (21%), BM MC >30% or serum tryptase level >200ng/mL 68% (8%), and hypercellular BM or dysmyelopoiesis without cytopenias 50% (2%). SSM patients were older (p<0.01) and more frequently displayed constitutional symptoms (p<0.01), and anemia (p<0.01). MC mediator-release symptoms (p=0.01), including anaphylaxis (p<0.01), were more frequent in BMM. The following MC-mediators were studied: serum tryptase (57%), beta prostaglandin F2alpha (45%), urine methylhistamine (32%), and urine histamine (21%) – significant differences were noted amongst the ISM subgroups with the following relationship: SSM>ISMo>BMM (p<0.01). (ii) Molecular and cytogenetic abnormalities: Complete karyotype information was available for 55 patients; only 1 patient (with ISMo) had an abnormal karyotype. Fifty nine patients were screened for KITD816V and JAK2V617F; JAK2V617F was universally absent, and distribution of KITD816V was not significantly different amongst the ISM subgroups: SSM (100%), BMM (92%), and ISMo (69%). (iii) Survival and risk of disease transformation: At a median follow of 27 months (range <1-417), 26 deaths were recorded (ISMo 14, SSM 10, and BMM 2), and the combined median survival was 198 months: ISMo 301 months, SSM 120 months, and BMM (not reached) (p<0.01; Figure). Transformation to acute leukemia and ASM was seen in 1 patient (SSM) and 3 patients (2 SSM and 1 ISMo), respectively. Conclusions: Among WHO-defined ISM patients, SSM is associated with significantly shorter survival whereas BMM and ISMo are prognostically similar. If these observations are confirmed by others, consideration may be given to classifying SSM as a distinct subcategory of SM, instead of a sub-variant of ISM as it currently stands. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2893 Poster Board II-869 Background: A specific JAK2 germline haplotype has been associated with the occurrence of JAK2V617F in myeloproliferative neoplasms (MPN). Several SNPs, including rs12343867, tag this haplotype. We performed rs12343867 SNP analysis in a consecutive cohort of patients with essential thrombocythemia (ET) in order to study the clinical and JAK2V617F allele burden correlates of the particular SNP genotype. Methods: Study patients were recruited form the Mayo Clinic database for ET. Molecular studies were performed on DNA extracted from stored bone marrow. rs12343867 SNP genotyping was performed by a commercially available Taqman assay. Quantitative JAK2V617F analysis was done according to previously published methods. Standard statistical methods were used to test significance of associations between SNP genotype and other variables. Survival analysis was performed by the Kaplan-Meier method and comparisons made by the log-rank test. Cox regression model was used for multivariable analysis. Results: 226 patients with ET were studied (median age 57, range 14-91; 145 females). Median hemoglobin, leukocyte and platelet counts at initial diagnosis were 13.8 g/dL, 9.7 × 109/L and 1000 × 109/L, respectively. 118 patients (52%) were JAK2V617F positive and among them, 90 (76%) displayed JAK2V617F allele burden of < 10% (low allele burden group) and the median (range) mutant allele burden in the remaining 28 patients (higher allele burden group) was 12% (12-68). As expected, JAK2V617F-positive cases, compared to their mutation-negative counterparts, were older (p=0.006) and displayed higher hemoglobin (p=0.0001) and leukocyte (p<0.0001) counts and lower platelet (0.03) count. The rs12343867 genotype distributions were C/C 15%, C/T 53% and T/T 33% (expected frequency in the HapMap-CEU listed control population is ∼ 6%, 46% and 48%, respectively). The corresponding figures in JAK2V617F positive/negative cases were 17%%/11%, 53%/53%, 30%/36% (p=0.29). Unlike the case with JAK2V617F mutational status, rs12343867 SNP genotype did not correlate with age (p=0.69), hemoglobin level (p=0.47), leukocyte count (p=0.25) or platelet count (p=0.33). Patients were followed for a median of 7.4 years and during this period 56 (25%) deaths were documented. rs12343867 SNP genotype did not correlate with survival (Figure). Similarly, survival did not correlate with the presence or absence of JAK2V617F. However, patients with higher JAK2V617F allele burden displayed a shorter survival with borderline significance, during multivariable analysis that included age as a covariate. Conclusion: The current study shows the JAK2 haplotype enrichment in a large series of ET patients. However, the particular phenomenon was not significantly different between JAK2V617F positive and negative cases, which suggests specific haplotype association with the disease rather than the mutation. Consistent with this contention, rs12343867 genotype did not correlate with clinical or laboratory variables that typically associate with JAK2V617F mutational status. Furthermore, unlike the case with primary myelofibrosis, rs12343867 SNP genotype did not correlate with survival, an observation that is not surprising considering the lower level of complexity in molecular pathogenesis in ET compared to PMF. Disclosures: Pardanani: TargeGen: Research Funding; Cytopia: Research Funding. Off Label Use: Data from ongoing clinical trial will be presented. Tefferi:TargeGen: Research Funding.

Abstract Background: acquisition of somatic mutations including JAK2V617F or MPLW515L/K results in constitutive activation of JAK-STAT signaling that commonly characterizes myeloproliferative disorders (MPD). JAK2 is the central signaling mediator for most hematopoietic cytokines in this signaling pathway. Hypothesis: small molecule JAK2 inhibitors may have a therapeutic role in MPD regardless of JAK2 mutation status. Methods: We studied effects of the JAK2 inhibitor, TG101209, in a cell-free kinase assay, relevant cell lines, an in vivo phosphorylation assay, and primary cells from MPD patients carrying JAK2V617F or MPLW515L/K mutations. Results: in a cell-free kinase assay, TG101209 inhibited JAK2 potently (IC50=6nM) and displayed selectivity relative to JAK3, ABL, and VEGFR2 kinases (IC50=170nM, 820nM, and 150nM, respectively). In cell proliferation assays, TG101209 inhibited JAK2-expressing Ba/F3 cells (IC50=280nM) and JAK2V617F-homozygous human erythroleukemia (HEL) cells (IC50=300nM), but not BCR-ABL-carrying K562 cells (IC50>2μM). At 24 hours, TG101209 (600nM) induced apoptosis of HEL cells but not K562 cells (40% and 5%, respectively). SCID mice injected intravenously with Ba/F3-JAK2V617F cells developed significant disease burden including massive splenomegaly due to tumor infiltration (day 12). We observed marked decrease in STAT5 phosphorylation in splenic tumors 5 hours after administration of 2 oral doses of TG101209 (50 mg/kg) in this in vivo model. Based on the above data, we plated patient-derived CD34+ cells in methylcellulose (0nM, 300nM, and 600nM TG101209) with and without cytokines. We studied 2 controls and 9 MPD patients: PV=5 (all JAK2V617F+) and AMM=4 (3 were MPLW515L/K+) for colony number, colony size, and colony mutation burden, under each assay condition. TG101209 inhibited overall colony growth from MPD patients (IC50=300–600nM), and it preferentially suppressed growth of mutant colonies relative to wild type in both of 2 MPLW515K+ patients (AMM1 and AMM2) and in 3 of 5 JAK2V617+ patients (PV1-PV3) (Table). Conclusions: TG101209 potently inhibits cell growth that is dependent on constitutive JAK-STAT signaling. Furthermore, for MPD patient-derived cells, TG101209 preferentially suppresses growth of progenitors carrying JAK-activating mutations (MPLW515K>JAK2V617F>MPLW515L). Hence, our data supports the strategy of targeting aberrant JAK-pathway signaling in MPD as a viable therapeutic approach. Patient Mutation IC50 (nM) No Drug (% colonies mutation-positive) TG101209 (% colonies mutation-positive) Erythroid Myeloid Erythroid Myeloid Erythroid Myeloid Normal WT 1000 600 n/a PV1 JAK2V617F ~600 300–600 82 90 36 70 PV2 JAK2V617F ~300 70 55 60 20 PV3 JAK2V617F NA 20 30 30 0 PV4 JAK2V617F >600 ~600 7 10 17 10 PV5 JAK2V617F ~300 300–600 64 92 82 100 AMM1 MPLW515K <300 ~300 100 90 0 20 AMM2 MPLW515K 300–600 ~300 100 90 25 36 AMM3 MPLW515L 300–600 300–600 100 100 92 90 AMM4 WT >600 300–600 n/a

Abstract Abstract 2867 Primary myelofibrosis (PMF) is a clonal hematologic malignancy, which results from the transformation of a pluripotent hematopoietic progenitor cell. A major consequence of this transformation is increased hematopoiesis and an overproduction of abnormal blood cells. PMF is associated with bone marrow fibrosis, extramedullary hematopoiesis, increased numbers of circulating CD34+ cells, splenomegaly, and a propensity to evolve to AML. Patients also display anemia and thrombocytopenia and harbor abnormal, immature megakaryocytes (Mks) in their bone marrow and spleen. PMF patients can present well known mutations including JAK2V617F (65%), MPL (10%), TET2 (17%), CBL (6%), IDH (4%,), which are not specific to the disease and are also present in polycythemia vera, essential thrombocythemia and AML. We hypothesize that the genetic events associated with PMF, including MPL and JAK2 mutations, contribute to defects in Mk maturation, but that additional changes are needed to explain the striking abnormalities seen in PMF relative to the other myeloproliferative diseases. Although there have been studies to examine the aberrant gene expression program of CD34+ cells of PMF patients, we chose to examine the changes that occur in gene expression specifically in Mks as a way to better understand their abnormal differentiation and to determine their contribution to the disease. Primary CD34+ cells from PMF patients and healthy donors were cultivated in serum free media supplemented with recombinant TPO, BSA, liposomes, insulin and transferrin to support the growth of Mks. After 10 days of differentiation, we evaluated the cultures for proliferation, apoptosis and differentiation by flow cytometry. We found that PMF specimens gave rise to a lower percentage of mature (CD41+CD42+) cells as compared to healthy donors, but showed, a lower ploidy level, a greater proliferation and increased survival. These observations are consistent with the clinical observations that PMF bone marrow is characterized by an increased number of immature, dysplastic Mks. We used flow cytometry to collect two populations of cells for analysis: immature CD41+CD42− Mks, and CD41+CD42+ mature MKs. After sorting, we extracted RNA and performed whole genome microarray analysis with Illumina Human HT12-v4 arrays on cohorts of PMF and control specimens. Gene expression data were analyzed by GeneSpring and Gene Set Enrichment Analysis (GSEA). We found that the CD41+CD42− MKs derived from PMF progenitors showed reduced expression of GATA1 as compared to control cells, as expected based on previous study by Dr. Alessandro Vannuchi. GeneSpring analysis revealed that myeloid transcription factors, including CEBPa, GFI1, and SPI1 (PU.1), which are not expressed in normal MKs, are strikingly and significantly overexpressed in PMF samples. Moreover, c-myb, which regulates the erythroid/Mk cell fate decision, FOG-1 and AML1, are also overexpressed in PMF Mks. This aberrant myeloid gene expression program in PMF Mks is reminiscent of a similar defect we observed in Mks with reduced expression of GATA-1 and GATA-2. We predict that reduced levels of GATA-1 protein in PMF Mks, as reported by Dr. Alessandro Vannucchi and colleagues, is in part responsible for the aberrant growth and differentiation of the PMF Mks. Our data support the model that PMF Mks are defective in their ability to properly regulate expression of hematopoietic regulators. Further analysis by GSEA revealed that hematopoietic and cytokine pathways are among those that are highly enriched in PMF Mks. We recently reported that the molecules dimethylfasudil (diMF) and MLN9237 are able to selectively increase ploidy, Mk surface marker expression, and apoptosis of malignant Mks. We treated Mks derived from PMF progenitor cells with diMF and observed a high increase in polyploidization accompanied with a reduction of Mks proliferation. Thus, diMF is able to partially restore Mk differentiation of PMF cells, supporting the testing of polyploidy inducers in myelofibrosis patients. Disclosures: Pardanani: Sanofi-Aventis: Clinical trial support Other; YM BioSciences: Clinical trial support, Clinical trial support Other; Bristol-Myers Squibb: Clinical trial support, Clinical trial support Other.

Abstract Abstract 2831 Background and Aims: Primary myelofibrosis (PMF) is characterized by ineffective erythropoiesis, an underlying inflammatory state and potentially, iron overload in the setting of red cell transfusions. The potential interaction between these clinical variables and assessment of their independent prognostic contribution, if any, is of interest within the context of well established prognostic parameters including the DIPSS-plus model and increased circulating levels of inflammatory cytokines (J Clin Oncol. 2011 Apr 1;29(10):1356–63). Consequently, we studied the relationship between circulating levels of hepcidin (a surrogate for inflammation, iron stores and ineffective erythropoiesis), ferritin (iron stores and inflammation), red cell transfusion need (ineffective erythropoiesis) and soluble inflammatory cytokine levels in PMF patients. Methods: The diagnosis of PMF was according to WHO criteria. Inclusion in this study required availability of archived plasma, bone marrow biopsy, and cytogenetic information at the time of first referral. Plasma total hepcidin levels were measured by ELISA (USCN Life Sciences, Wuhan, China). Serum ferritin was measured as part of routine clinical assessment (normal range: males: 24–336 mcg/L; females: 11–307 mcg/L). Concentrations of plasma cytokines were analyzed in duplicate by using Multiplex Bead-Based Luminex technology (Invitrogen, Carlsbad, CA). Results: Two hundred and three PMF patients met the above stipulated criteria; 136 (67%) were males and the median age at referral was 63 years (range 17–83). Their dynamic international prognostic scoring system (DIPSS)-plus risk categorization was: low-risk 22 (11%), intermediate-1 26 (13%), intermediate-2 85 (42%) and high-risk 70 (34%). Hepcidin levels (median 156279 pg/mL; range 8082–2088002) were strongly correlated with serum ferritin (n=146; median 207 mcg/L; range 14–5367) (r2=0.43, p <0.0001). Both were directly correlated with red cell transfusion need and age, and inversely with hemoglobin level (all p <0.05); however, there was no correlation with increased levels of inflammatory cytokines (>3 standard deviation above mean of normal controls for IL-2 and/ or IL-8) (p>0.05). At a median follow up of 35 months, 104 (51%) deaths and 18 (9%) leukemic transformations were recorded. In univariate analysis, increased levels of hepcidin (HR=2.0; p=0.0006) and ferritin (HR=2.7; p<0.0001) were predictive of inferior overall survival and this significance was sustained during multivariable analysis that included either increased inflammatory cytokine levels (HR=3.2 and 2.7, respectively) or transfusion need (HR=1.7 and 2.2, respectively) as covariates. When all 4 variables (i.e. increased levels of hepcidin, ferritin, inflammatory cytokines and transfusion need) were considered together in the Cox model, only increased inflammatory cytokine levels retained their prognostic significance (HR=2.7; p=0.0046). Furthermore, neither increased ferritin nor increased hepcidin levels were prognostically significant for overall survival when tested either individually or together against the DIPSS-plus model (p>0.05). In univariate analysis, increased hepcidin level, but not increased ferritin or transfusion need, was predictive for inferior leukemia-free survival (HR=2.8; p=0.04) and this significance was sustained in multivariable analysis that included either cytogenetic risk categories (very-high risk versus high-risk versus other) (HR=2.7; p=0.046) or thrombocytopenia (platelet count <100 × 109/L) (HR=2.7; p=0.047) as covariates. Conclusions: Circulating levels of hepcidin and ferritin are highly correlated variables in PMF and appear to provide redundant prognostic information for overall survival in this setting. The lack of correlation with inflammatory cytokines suggests that ineffective erythropoiesis may be the common link for their apparent prognostic value noted during univariate analysis. In contrast to inflammatory cytokines, neither hepcidin or ferritin levels were useful as a complement to the DIPSS-plus prognostic model in the current analysis. The relationship between hepcidin and leukemia-free survival is intriguing however the pathogenetic mechanism remains obscure and needs confirmation in additional patients. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1711 Background: Mutations in genes of the splicing machinery, such as SF3B1, SRSF2 and U2AF35 are common in patients with myelodysplastic syndromes [MDS] (Nature 2011;478:64) and chronic myelomonocytic leukemia [CMML] (Haematologica 2012;Epub). In MDS, SRSF2 gene mutations are an independent risk factor for shortened over-all (OS) and leukemia-free survival (LFS) (Blood 2012;119:3578). In MDS with ring sideroblasts (RS), SF3B1 mutations have a high prevalence (∼50%), but do not influence either, the OS or the LFS (Blood 2012;119:569). We carried out this study to evaluate the prevalence, clinical correlates and prognosis of the aforementioned spliceosome mutations in CMML. Methods: The study included 227 patients with WHO defined CMML who were seen at the Mayo Clinic from 1997 through 2007. All patients underwent bone marrow (BM) examination and cytogenetic evaluation at diagnosis. DNA was interrogated in the three most frequent spliceosome genes with somatic mutations; SRSF2, SF3B1 and U2AF35. Results I: Prevalence and clinical correlates Among the 227 study patients, 153 (67%) were male, median age was 71 years (range, 17–90 years) and 192 (85%) met the WHO criteria for CMML-1. Ninety (40%) patients had SRSF2 mutations (86% CMML-1), 13 (6%) had SF3B1 mutations (75% CMML-1) and 20 (9%) had U2AF35 mutations (95% CMML-1). One-hundred and twenty three (54%) patients had at least one of three spliceosome mutations (86% CMML-1). Mutational hot spots were P95 for SRSF2 (P95L-n=36/H-n=32/R-n=13/A-n=1), K700E (n=7) and H662Q (n=2) for SF3B1, and Q157 (Q157R-n=5/P-n=5/G-n=1) and S34F (n=7) for U2AF35. Seven patients (54%) with SF3B1 mutations had ≥1% RS, with 5 (38%) showing ≥15% RS. Mutations involving all three spliceosome genes were mutually exclusive. The cytogenetic distribution based on the Spanish risk stratification system (Haematologica 2011;96:375) was; SRSF2 mutations: 69 (77%) low risk, 11 (12%) intermediate risk, and 10 (11%) high risk (+8-n=3, del/monosomy 7-n=2, monosomal karyotype-n=5); SF3B1 mutations: 8 (62%) low risk and 5 (38%) intermediate risk; U2AF35 mutations: 15 (75%) low risk, 3 (15%) intermediate risk and 2 (10%) high risk (p=0.89). The distribution of mutations according to the MD Anderson prognostic scoring system [MDAPS] (Blood 2002;99:840) was; SRSF2 - low-n=41, intermediate-1-n=26, intermediate-2-n=18, high-n=5, SF3B1- low-n=7, intermediate-1-n=3, intermediate-2-n=2, high-n=1, and U2AF35- low-n=11, intermediate-1-n=5, intermediate-2-n=3, high-n=1 (p=0.73). There was no statistically significant difference, among the three mutation groups, in prognostically relevant parameters, including gender distribution, median age, hemoglobin values, platelet counts, peripheral blood (PB) and BM blast counts, absolute neutrophil counts (ANC) and absolute monocyte counts (AMC). The only notable difference was that patients with the SF3B1 mutation had a lower median white blood cell count (p=0.04) and a lower absolute lymphocyte count (p=0.045). Results II: Prognostic impact of spliceosome mutations At a median follow-up of 15 months, 166 (73%) deaths and 33 (14.5%) leukemic transformations were documented. Median survivals for patients with mutations involving SRSF2, SF3B1 and U2AF35 were 24, 17 and 12 months, respectively. In univariate analysis, the presence of SRSF2 (p=0.67), SF3B1 (p=0.96) or U2AF35 (p=0.49) mutations had no prognostic impact on OS. Similarly, none of the three spliceosome mutations affected LFS; corresponding p values were 0.55 for SRSF2, 0.9 for SF3B1 and 0.38 for U2AF35 mutations respectively. We then examined possible prognostic value of having none of these mutations (n=104) vs otherwise (n=123) and the results were once again negative (p=0.87). Conclusions: SRSF2 is the most frequently mutated spliceosome gene in CMML, but neither it nor SF3B1 or U2AF35 mutations affect overall or leukemia-free survival in CMML. Furthermore, the current study suggests limited genotype-phenotype association, save for the already established association between SF3B1 mutations and RS. Disclosures: No relevant conflicts of interest to declare.