Lasho, Terra L.

Abstract Abstract 1751 Background: JAK inhibitors have significant palliative benefit in myelofibrosis (MF), mainly in the form of improved constitutional symptoms and reduced splenomegaly. Preliminary data suggests that CYT387, a JAK-1/2 inhibitor, also has the ability to produce anemia responses (ASH Annual Meeting, 2011). In general, the mechanism(s) underlying treatment effects of JAK inhibitors remain unclear but likely represent a drug-specific balance between anti-clonal activity and modulation of immuno cellular-cytokine pathways. We conducted a gene expression profiling (GEP) study using primary cells from MF patients undergoing therapy with CYT387 followed by correlation with clinical data. Methods: Study subjects were enrolled in the Phase-1/2 study of CYT387 treatment in patients with primary (PMF), post-polycythemia vera (PPMF) or post-essential thrombocythemia (PTMF) myelofibrosis. Paired research samples were collected; the time points were pre-study and 12 weeks after commencing study treatment. PBMCs were purified from whole blood by Ficoll separation; RNA was isolated from this cell fraction for GEP analysis. Gene expression profiles were generated using Illumnia Human HT-12 v4 microarray. Pair wise analysis was conducted using the Wilcoxon signed-rank test with a p-value cutoff of 0.05 to generate lists of differentially expressed genes between assigned groups. Pathway analysis was conducted to identify relevant pathways enriched for differentially expressed genes. Comprehensive plasma cytokine profiling was performed using Multiplex Bead-Based Luminex technology (Invitrogen, Carlsbad, CA). Results: Seventeen patients were studied based on sample availability; 11 (65%) mere male with median age of 66 years (range 53–85). Twelve (71%) were JAK2V617F mutation positive and the DIPSS-plus risk categorization was 10 (59%) high and 7 (41%) intermediate-2. All patients were evaluable for anemia response; 14 (82%) were red cell transfusion dependent at study start. Nine (53%) patients achieved anemia response by IWG-MRT criteria; of these, 8 patients achieved transfusion independence (minimum non-transfused hemoglobin level of 8 g/dL maintained for at least 12 weeks) and 1 had a sustained >2 g/dL increase in hemoglobin level above baseline. The initial pair wise analysis to identify differential patterns of gene expression compared pre- and post-treatment groups (Figure 1A). This revealed a cluster of significantly (p <0.05) down-regulated genes (minimum 2-fold; median 17-fold) following treatment (displayed in green; upper left quadrant). Pathway enrichment analysis revealed significant associations of these genes with cytokine regulation of immune response, cell proliferation, chemotaxis and cytoskeleton remodeling. We then conducted a pair wise analysis of anemia responders versus non-responders; this revealed a predominance of over expressed gene targets (median 35-fold) in the anemia responder group (Figure 1B) (displayed in red; upper right quadrant). Similar pathway analysis identified enrichment for genes involved in immune system function in this cluster. Conclusions: The current preliminary analysis suggests that genes relevant to immune response-cytokine pathways are significantly over expressed in patients who achieve anemia response following CYT387 therapy. This further suggests a dominant immune component that underpins ineffective hematopoiesis in responding patients. On the basis of broad treatment-related changes in gene expression we suggest that an important component of CYT387's treatment effect is down regulation of these dysregulated pathways. Ongoing studies include validation of select gene targets which will be tested prospectively in future treatment protocols, as well as correlation of gene expression with circulating cytokine-chemokine levels. Disclosures: Pardanani: Bristol-Myers Squibb: Clinical trial support, Clinical trial support Other; YM BioSciences: Clinical trial support, Clinical trial support Other; Sanofi-Aventis: Clinical trial support Other. Off Label Use: Data from Phase −1/2 study of CYT387 use in myelofibrosis is mentioned.

Abstract Background: Current drugs in myelofibrosis (MF), including JAK inhibitors, do not induce complete or partial remissions. Imetelstat is a 13-mer lipid-conjugated oligonucleotide that targets the RNA template of human telomerase reverse transcriptase. A phase-2 study in essential thrombocythemia showed platelet-lowering activity of imetelstat accompanied by reduction in JAK2V617F allele burden, thus providing the main rationale for conducting the current study. Methods: The current study (NCT01731951) is investigator-driven and IRB approved. Drug and research funding were provided by Geron Corporation (Menlo Park, CA, USA). The objective was to obtain preliminary information on therapeutic activity and safety of imetelstat in DIPSS-plus high or intermediate-2 risk MF. Adverse events (AEs) were monitored by Common Terminology Criteria for Adverse Events (Version 4.03) and responses by the International Working Group criteria (Blood2013). Laboratory correlative studies included measurement of plasma cytokines and analysis of mutations. Results : 33 patients were accrued and met eligibility criteria (median age 67 years; 67% males); type of MF was primary 55% and post-PV/ET 45%; risk distribution was high 48% and intermediate-2 52%. Karyotype was abnormal in 16 (48.5%) patients. 79% of patients had received prior therapy including JAK inhibitors in 33%. Protocol treatment : Imetelstat was administered by a 2-hour intravenous infusion (9.4 mg/kg) every three weeks (cohort A; n=19) or weekly x 3 followed by every three weeks (cohort B; n=16). As of 7/24/2014, 11 (33.3%) patients remain on active treatment (median number of cycles completed for all patients = 7; range 1-21). Three patients have died post-registration: intracranial hemorrhage (related), upper gastrointestinal hemorrhage (unrelated) and disease progression. Median follow-up for living patients was 11.0 months (range, 3.6 – 18.4). 22 (66%) patients have discontinued treatment, mainly because of suboptimal response (n=15) or disease progression (n=3). Toxicity : Treatment-related grade 4 neutropenia was seen in 6 (18%) patients and grade 4 thrombocytopenia in 7 (21%). Grade 3 anemia was seen in 9 (27%) patients. Two patients experienced grade 4+ non-hematologic AE: grade 5 intracranial hemorrhage (related), grade 5 upper gastrointestinal hemorrhage (not related); other grade 3+ non-hematologic AEs were seen in only one patient. Regardless of attribution, treatment-emergent grade 1 or 2 liver function test abnormalities affected bilirubin 46%, ALP 52%, AST 55% and ALT 24%. There were 3 instances of grade 3 ALP and one of bilirubin elevations. Efficacy : There were 7 (21.2%) complete (CR; n=4) or partial (PR; n=3) remissions, occurring at a median of 5 cycles of treatment (range 1-9). All 4 CR patients experienced reversal of BM fibrosis and 3 of them a complete molecular response. Six of the 7 CR/PR patients remain in remission (median 9.9 months, range 4.8-14.7). Other responses included anemia response in 4 (31%) of 13 transfusion-dependent patients, >50% reduction in palpable spleen size in 9 (39%) of 23 evaluable patients, ≥50% reduction in leukocyte count in 8 (80%) of 10 patients with marked leukocytosis (WBC >25 x 10(9)/L), resolution of leukoerythroblastosis in the majority of patients, and normalization of platelet count in 9 (75%) of 12 patients with thrombocytosis. Laboratory correlative studies : Mutational frequencies were 79% for JAK2, 18% for CALR, 3% for MPL, 33% for ASXL1, 9% for IDH1/2 and 33% for spliceosome component mutations. CR/PR rates were 27% in JAK2-mutated vs 0% unmutated (p=0.3) and 32% in ASXL1-unmutated vs 0% mutated (p=0.07). CR rate was 38% in SF3B1/U2AF1-mutated vs 4% unmutated (p=0.036). Grade 3+ neutropenia or thrombocytopenia was more likely to occur in JAK2-unmutated (p=0.02) and ASXL1-mutated cases (p=0.049). Multi-cytokine panel screening showed significant differences between baseline and post-treatment samples involving several cytokines, including IL-1b, IL-5, IL-7, IL-17, VEGF, IL-8 and TNF-α (p<0.001 for all). CR/PR did not correlate with baseline cytokine levels. Conclusions : The current study identifies imetelstat as an active drug in patients with MF, but also reveals its potential to cause significant myelosuppression. The association between CR/PR and specific mutations suggests potential targeted activity that might be exploited for patient and disease selection. Disclosures No relevant conflicts of interest to declare.

Abstract Background : The 2016 revision to the World Health Organization (WHO) classification of myeloid neoplasms has recommended distinction between "proliferative" (WBC ≥ 13 x 10(9)/L) and "dysplastic" (WBC < 13 X 10(9)/L) subtypes of chronic myelomonocytic leukemia (CMML). In the current study of 261 molecularly-annotated cases, we sought to clarify the prognostic relevance of distinguishing proliferative from dysplastic CMML and also describe differences in the distribution of disease-associated mutations. Methods : 261 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, KRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF, and SETBP. The 2016 WHO criteria were used to sub-classify CMML into proliferative and dysplastic subtypes. Results :Among the 261 study patients, 65% were males and median age was 70 years. 154 (59%), 64 (25%) and 43 (16%) patients were classified as CMML-0, 1 and 2, respectively. At a median follow-up of 23 months, 174 (67%) deaths and 37 (14%) leukemic transformations were documented. Mutational frequencies were; TET2 45%, ASXL1 45%, SRSF2 40%, NRAS 14%, SETBP1 13%, CBL 10%, JAK2 7%, RUNX1 6%, U2AF1 6%, DNMT3A 6%, SF3B1 5%, ZRSR2 4%, Tp53 4%, IDH2 4%, KRAS 3%, PTPN11 2%, SH2B3 1%, CSF3R 1%, IDH1 1%, EZH2 1%, SUZ12 1%, KIT 1%, FLT3 1%, and CALR 1%. Risk stratification was based on the Mayo Molecular Model: 31% high, 30% intermediate-1, 28% intermediate-2 and 11 % low risk. i) Dysplastic versus proliferative CMML: phenotypic and molecular differences 139 (53%) patients had proliferative and 122 (47%) dysplastic subtypes. There was no difference between the CMML subtypes in terms of age and gender distribution, hemoglobin level, platelet count or BM blast content. Patients with proliferative CMML had higher absolute monocyte counts (AMC) (p<0.0001), circulating immature myeloid cells (IMC, p<0.001), circulating blasts (p<0.001) and serum LDH levels (p=0.01). The following gene mutations were more common in proliferative vs dysplastic CMML: ASXL1 (54% vs 37%, p=0.009), JAK2 (11% vs 3%, p=0.01) and CBL (11% vs 8%, p=0.047); SF3B1 mutations were more common in dysplastic CMML (8% vs 1%, p=0.02). There was no difference in the incidence of TET2, DNMT3A and SRSF2 mutations whereas there was a trend towards a higher prevalence of NRAS (p=0.06) and CSF3R (p=0.06) mutations in proliferative CMML. Cytogenetic abnormalities (p=0.03), including higher risk categories by the Spanish (p=0.03) and the Mayo-French (p=0.01) systems were more common in proliferative CMML. ii) Impact on overall and leukemia-free survival: Median survival for the entire cohort (n=261) was 24 months. In univariate analysis, survival was shorter in patients with proliferative (median 20 months) versus dysplastic (median 29 months) CMML (p=0.008; HR1.5, 95% CI 1.1-2.1; Figure 1A). Other variables of significance, in univariate analysis, included hemoglobin (p=0.001), leukocyte count (p=0.001), AMC (p=0.003), PB blast % (p=0.003), IMC (p=0.01), BM blast % (p=0.045), abnormal karyotype (p=0.02), ASXL1 (p=0.01) and DNMT3A (p=0.0003) mutations. In multivariable analysis, the difference in survival between proliferative and dysplastic subtypes remained significant with the addition of hemoglobin level (p=0.01), PB blast % (p=0.02), IMC (p=0.04), BM blast % (p=0.01) or DNMT3A mutations (p=0.01). This was, however, not the case with addition of leukocyte count (p=0.32), AMC (p=0.18) or ASXL1 mutational status (p=0.14); whereas the adverse impact on survival from the latter three parameters remained significant. The prognostic impact of ASXL1 mutations was most apparent in dysplastic CMML (Figure 1B). There was no difference in leukemic transformation rates (p=0.4). Conclusions: In the context of current prognostic models, sub-classification of CMML into proliferative and dysplastic subtypes might not provide additional prognostic value. The apparent difference in survival between the two subtypes of CMML is probably accounted for by the higher prevalence of leukocytosis/monocytosis and of ASXL1 mutations in proliferative CMML. Disclosures No relevant conflicts of interest to declare.

Abstract Background In a seminal report (NEJM 2011;30:2496), ASXL1, TP53, EZH2, ETV6 and RUNX1 mutations in myelodysplastic syndromes (MDS) were shown to carry an adverse prognostic impact that was independent of the international prognostic scoring system (IPSS) (Blood 1997;89:2079). Two recent studies (Leukemia20 May 2016; doi: 10.1038/leu.2016.138; Leukemia. 2014;28:241) have proposed mutation-enhanced prognostic models that include the revised IPSS (IPSS-R) (Blood 2012;120:2454 ). In the current study, we applied targeted next generation sequencing (NGS) in order to examine the prognostic interaction between adverse mutations and IPSS-R. Methods The study population was recruited from our institutional database of patients with primary MDS, based on availability of archived DNA from the time of diagnosis. The diagnosis of MDS and leukemic transformation (LT) was made according to WHO criteria (Blood. 2009;114:937). Targeted capture assays were carried out, on bone marrow DNA specimens, 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.Specific variants were deemed as mutations if they are associated with a hematologic malignancy (as identified by COSMIC database), or if they have not been associated with a dbSNP. Results Patient characteristics and type and number of mutations: 179 patients were evaluated (median age 73 years; 68% males). IPSS-R risk distribution was 11% very high, 18% high, 16% intermediate, 39% low and 16% very low. At least one mutation was seen in 139 (78%) patients including 17% with one mutation, 29% with 2, 20% with 3, 10% with four, 2% with 5 and one patient with 6 mutations. The most frequent mutations were ASXL1 (30%), SF3B1 (20%), TET2 (17%), U2AF1 (16%) and SRSF2 (16%). Mutation clusters and clinical correlates: ASXL1 mutations were associated with the absence of SF3B1 (p=0.007), U2AF1 (p=0.01) and SRSF2 (p=0.003) mutations; SF3B1 with absence of U2AF1 (p=0.004) and SRSF2 (p=0.004) mutations; and U2AF1 with absence of SRSF2 mutations (p=0.01). Clinical correlates of ASXL1 mutations included older age, lower hemoglobin, and lower risk karyotype; SF3B1 with higher leukocyte count, higher platelet count, lower bone marrow blast percentage and lower risk karyotype; TET2 with older age and higher hemoglobin. Univariate overall and leukemia-free survival analysis before and after adjusting for IPSS-R: In univariate analysis, ASXL1, RUNX1 and TP53 mutations adversely and SF3B1 favorably affected survival; only ASXL1 mutations retained significance when analysis was adjusted for IPSS-R (HR 1.5, 95% CI 1.0-2.1; p=0.03). For leukemia-free survival, univariate analysis identified SRSF2, which was near-significant for overall survival (p=0.06), and RUNX1 mutations as adverse and SF3B1 mutations as favorable risk factors; SRSF2 (HR 4.1, 95% CI 1.6-10.2) and SF3B1-unmutated (HR 5.9, 95% CI 1.1-31.5) retained their significance when adjusted for IPSS-R. A borderline significance (p=0.07) indicating inferior survival for patients with three or more mutations was fully accounted for by the significant (P<0.0001) association between the presence of ASXL1 mutations and higher number of mutations. Multivariable analysis with individual IPSS-R variables: When the five IPSS-R variables (hemoglobin, platelets, absolute neutrophil count, bone marrow blast percentage and karyotype) were analyzed (both as continuous and IPSS-R-defined categorical variables) with each one of the aforementioned mutations with significant effect on overall or leukemia-free survival, only ASXL1 retained its significance for overall survival (HR 1.6, 95% CI 1.1-2.3) and SRSF2 (HR 5.2, 95% CI 2.1-13.3) for leukemia-free survival. Other IPSS-R variables that retained their significance for survival, in the presence of ASXL1 mutation status as a covariate, included hemoglobin level, platelet count and karyotype. Conclusions: In our cohort of 179 molecularly-annotated patients with newly-diagnosed primary MDS, ASXL1 and SRSF2 mutations were identified as IPSS-R-independent risk factors for overall and leukemia-free survival, respectively. The current study also suggests the need to re-evaluate currently established risk factors, in the context of prognostically-relevant molecular information. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 2836 Background: The clinical phenotype of systemic mastocytosis (SM) is highly variable; establishing prognosis in terms of overall survival or risk of transformation to aggressive disease for those with non-indolent and indolent disease variants, respectively, is not trivial. Similar to other clonal hemopathies, mast cell (MC) activation and/or stromal response to clonal MC expansion likely results in a dysregulated immuno cellular/cytokine profile; analysis of this aspect of SM may provide additional prognostic information within the context of well established parameters such as the World Health Organization (WHO) SM classification system. Here, we conducted a comprehensive analysis of circulating cytokines/chemokines with clinicopathologic and clinical outcome correlations in a cohort of SM patients seen at our institution. Methods: The diagnosis of SM and its subclassification were established 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. Follow up information including data on survival and disease progression were updated in July 2012. Concentrations of plasma cytokines were analyzed in duplicate by using Multiplex Bead-Based Luminex technology (Invitrogen, Carlsbad, CA). Results: Forty six SM patients met the above stipulated criteria; 25 (54%) were male and the median age at referral was 61 years (range 21–85). Subclassification of patients per WHO criteria was: indolent SM (ISM) 23 (50%), aggressive SM (ASM) 8 (17%) and SM with associated clonal hematological non-MC lineage disease (SM-AHNMD) 15 (33%). When the distribution of 30 cytokines was considered across the 3 SM sub groups, only interleukin (IL)-8 was significantly different (SM-AHNMD > ISM/ASM; p=0.0002). For ISM patients, increased levels of sIL-2R were associated with presence of B-findings (p=0.0046) including splenomegaly (p=0.001) and serum tryptase levels >200 ng/mL (p=0.0046), and decreased levels of IL-8 and hepatocyte growth factor (HGF) with MC mediator-release symptoms (p <0.05). Increased levels of sIL-2R (r2=0.6; p<0.0001) and RANTES (r2=0.37; p=0.0013) were correlated with bone marrow MC burden, and sIL-2R (r2=0.34; p=0.004) and MIG (r2=0.42; p=0.0012) with serum tryptase levels in ISM patients; similar findings were noted for the overall cohort. At a median follow up of 28 months (range 0–116), 20 (43%) deaths, and 3 (13%) and 1 (2%) transformations to ASM and mast cell leukemia, respectively, were recorded for the overall cohort. In univariate analysis, increased sIL-2R levels were predictive for inferior overall survival (p=0.005); this prognostic significance was maintained in multivariate analysis after adjusting for other known prognostic variables individually (i.e. WHO SM subtypes, age >65 years, hemoglobin <10 g/dL, thrombocytopenia, weight loss or hypoalbuminemia) (all p <0.05). Increased sIL-2R (>75th percentile) effectively stratified patients in the overall cohort into 2 well-delineated risk groups for overall survival (median survival 109 vs. 26 months; p=0.0004) (Figure). This sIL-2R threshold was also able to risk stratify patients within ISM (median survival not reached vs. 38 months) and non-ISM (median survival 31 vs. 5 months) categories (p <0.0001). Conclusions: The current study demonstrates s-IL2R to be a key inflammatory cytokine in SM; it is significantly correlated with a phenotype of high systemic MC burden and in this regard, is an attractive surrogate for treatment response in clinical practice, if validated. The predictive value of sIL-2R for overall survival is akin to similar observations in primary myelofibrosis and diffuse large B-cell lymphoma; in this study, it was noted to be independent of conventional measures of organopathy from MC infiltration, and thus may reflect a novel pathogenetic process in SM, mediated by dysregulated inflammatory and/or immuno cellular pathways. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: CD123 (alpha chain of the interleukin-3 receptor [IL-3RA]) heterodimerizes with CD131 (βc, common beta chain) to constitute the high-affinity receptor for IL-3. CD123 is not expressed on hematopoietic stem/progenitor cells from normal bone marrow (BM). In contrast, CD123 is highly expressed in CD34+/CD38- cells from acute myeloid leukemia (AML) patients, which recapitulate the leukemic phenotype in NOD/SCID mice; CD123, therefore, is a marker for leukemic stem cells in AML (Jordan CT et al., Leukemia 2000). CD123 is also highly expressed on CD4+/56+ leukemic cells in patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN), an aggressive hematodermic neoplasm. The validity of CD123 as a rational therapeutic target was illustrated by clinical efficacy data for SL-401, a biologic target therapy directed to IL-3R, in patients with BPDCN and AML, respectively (Frankel AE et al., Blood 2014 andFrankel AE et al., J Clin Oncol 31, 2013 (suppl; abstr 7029)). Objectives: To study CD123 expression as a potential therapeutic target in myeloproliferative neoplasms (MPN). Methods: The study was approved by our institutional review board and patients provided written consent for sample collection. MPN diagnosis was based on WHO 2008 criteria. Surface antigen expression on hematopoietic cells of various lineages was interrogated using the 4-color multiparametric flow cytometer, FACSCaliber TM (BD Biosciences, San Jose, CA). Data analysis was performed using CellQuest Pro Software (BD Biosciences). Results: We studied a total of 21 MPN patients and 3 normal controls. Of the former, 14 patients had systemic mastocytosis (SM) (indolent SM=9, SM with associated myeloid neoplasm=4 and aggressive SM=1), 6 had primary myelofibrosis (PMF) and 1 had eosinophilic leukemia transformed to AML. Normal controls: Three normal BM samples were studied; the number of CD123 positive cells in the total population was <1%. Rare CD34+/CD38- cells were uniformly CD123-. Of the CD123+ cells, only a minor subset (<10%) coexpressed myeloid/granulocyte lineage (CD13+, CD15+ or CD16+) or monocyte/macrophage lineage (CD14+ or CD11b+) markers. Systemic mastocytosis: The data were informative for 6 patients (3 with peripheral blood [PB], 1 with BM, and 2 with paired PB and BM samples) for whom a sufficient number of mast cells (MC) were identified for immunophenotyping. Neoplastic MC were defined as CD117 hi/SSC hi/CD45 hi/CD34-/CD25+/FcεRI+. CD123 expression was seen in the majority of MC for 4 patients (#1-4, CD123 percentage positive, PB/BM): 91%/n.a. (#1), 75%/81% (#2), 84%/n.a. (#3), and 88%/n.a. (#4). In contrast, one patient (#5) had a minority of CD123+ MC (33%/28%). Clonal eosinophilia: PB was studied; the WBC differential count showed 41% leukemic blasts and 57% eosinophils (both confirmed to be clonally related based on FISH studies). Both cell populations were predominantly CD123+ (≥90%). Primary myelofibrosis: We studied PB samples from 6 PMF patients. Approximately 1-2% of circulating cells marked as CD123+; of these, expression was notable on CD34-/CD38+ cells (median 54%; range 41-74% positive). We found a larger proportion (30-50%) of CD123+ cells that coexpressed CD13+, CD16+ or CD11b+ representing monocytes, immature myeloid cells and granulocytes, as compared to normal BM controls. Studies on BM samples from PMF patients are ongoing. Conclusions: CD123 (IL-3RA) is expressed on relevant primary cells of interest in select MPNs, namely neoplastic mast cells in SM patients and eosinophils in clonal eosinophilia patients. In PMF, a minor population of circulating cells was CD123+ with a biased distribution on myeloid lineage cells as compared to normal BM samples. We are currently studying CD123 expression in additional MPN patients, and also analyzing CD123 expression on BM trephine biopsies by immunohistochemistry. In addition, the cytotoxicity assessment of SL-401 against MPN cell lines and primary cells is ongoing. Overall, the aforementioned data support the clinical development of SL-401 in patients with MPNs and clinical trials are currently being planned. Disclosures Brooks: Stemline Therapeutics: Employment, Equity Ownership. Pardanani:Stemline Therapeutics Inc.: Research Funding.

Abstract Abstract 2803 Background: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disorder with overlapping features between myelodysplastic syndromes (MDS) and myeloproliferative neoplasms. Ring sideroblasts (RS) represent abnormal mitochondrial iron accumulation in MDS; with ≥15% RS necessary for the conventional diagnosis of MDS-RS. Somatic spliceosome mutations are recurrent in MDS, with SF3B1 mutations being the most frequent in MDS-RS (∼75%) and SRSF2 in CMML (∼28%). The distribution of these mutations in the presence of both RS and monocytosis is unknown and their prognostic relevance, in the particular setting, undetermined. Methods: Using the Mayo Clinic database for myeloid malignancies (1997–2007), we identified patients who met the 2008 WHO criteria for CMML, and who also displayed at least 1% RS in their bone marrow (BM). All patients underwent BM examination and cytogenetic evaluation at diagnosis and the pathology slides, including iron stains, were centrally re-reviewed to accurately quantify BM RS. DNA was interrogated in the three most frequent spliceosome genes with somatic mutations; SF3B1, SRSF2 and U2AF35. Results: Sixty four patients met the above stipulated criteria for CMML with ≥1% RS; 46 (72%) were males and median age was 71 years (range, 17–90 years). Fifty three (83%) had CMML-1 and the remainder CMML-2. The percentage of patients with ≥15% RS was 41%: 30% had 15–49% RS and 11% had >50% RS. Thirty patients (47%) displayed SRSF2 mutations (mutational frequencies were 58% in the presence of <15% RS, 42% with 15–49% RS and 0% with >50% RS), 9 (14%) SF3B1 mutations (3% with <15% RS, 26% with 15–49% RS and 43% with >50% RS), and 5 (8%) U2AF35 mutations (8% with <15% RS, 11% with 15–49% RS and 0% with >50% RS). Mutational hotspots were P95 for SRSF2 (93%), K700 for SF3B1 (67%) and Q157 for U2AF35 (60%). The three spliceosome mutations were mutually exclusive. At a median follow-up of 26 months, 49 (77%) deaths and 11 (17%) leukemic transformations were documented. In univariate analysis, significant risk factors for survival included increased levels of white blood cell (WBC), absolute neutrophil (ANC), absolute monocyte (AMC), absolute lymphocyte (ALC) counts, the Spanish cytogenetics risk stratification system (Haematologica 2011;96:375), and the presence of circulating blasts. Neither the presence of spliceosome mutations (SF3B1/SRSF2/U2AF35) nor the percentage of RS (considered both as a continuous and a categorical variable), had an impact on either overall or leukemia-free survival. Conclusions: Among spliceosome mutations in CMML, those involving SRSF2 are by far the most frequent, even in the presence of ring sideroblasts. However, in patients with >50% RS, only SF3B1 mutations were seen whereas in those with 15–49% RS, SRSF2 mutations were more common. These observations suggest that SF3B1 mutations play a dominant but not exclusive role in the pathogenesis of RS. Regardless, the current study did not suggest prognostic impact from either the presence of the spliceosome mutations studied or the percentage of RS. Disclosures: No relevant conflicts of interest to declare.

Abstract Background : Cytogenetic abnormalities in PMF have been broadly classified into “favorable” and “unfavorable” risk categories. The latter include +8, -7/7q-, i(17q), inv(3), -5/5q-, 12p-, 11q rearrangements and complex karyotype (Leukemia. 2011;25:82). In a more recent study, we had demonstrated an even worse prognosis for patients with monosomal karyotype (MK), inv(3) and i(17q) abnormalities (Blood. 2011;118:4595). In the current study, we revisited the topic using a larger database. Methods: PMF diagnosis was according to World Health Organization criteria (Blood. 2009;114:937). Cytogenetic analysis and reporting was done according to the International System for Human Cytogenetic Nomenclature (Cytogenetic and genome research. 2013. Prepublished on 2013/07/03 as DOI 10.1159/000353118). Assignment to “normal” karyotype required a minimum of 10 metaphases analyzed. A complex karyotype was defined as the presence of 3 or more distinct structural or numeric abnormalities. MK was defined as 2 or more distinct autosomal monosomies or single autosomal monosomy associated with at least one structural abnormality (JCO. 2008;26:4791). Results : A total of 903 patients (median age 65 years; range 19-92; 63% males) met the above stipulations for study eligibility. Dynamic International Prognostic Scoring System (DIPSS)-plus risk distribution (JCO. 2011;29:392) was high in 344 (38%) patients, intermediate-2 in 334 (37%), intermediate-1 in 128 (14%) and low in 97 (11%). 472 patients were annotated for JAK2/CALR/MPL mutations; 276 (58%) harbored JAK2, 117 (25%) CALR and 33 (7%) MPL mutations. Abnormal karyotype was documented in 437 (48%) patients and included sole abnormalities of 20q- (n=71; 16%), 13q- (n=57; 13%), +8 (n=25; 5%), -7/7q- (n=17; 4%) and +9 (n=16; 3%). 22 (5%) patients displayed MK and 31 (7%) complex karyotype without MK. At a median follow-up time of 3 years, 634 (70%) deaths and 65 blast transformations (BT; 7%) were recorded. A step-wise survival analysis, using “normal karyotype” and “MK/inv(3)/i(17q)” as comparators for “low” and “very high risk” categories, respectively, resulted in four distinct cytogenetic risk designations: very high (MK, inv(3), i(17q), -7/7q-, 11q or 12p abnormalities; n=67), high (complex without MK, two abnormalities not included in very high risk category, 5q-, +8, other autosomal trisomies except +9, and other sole abnormalities not included in other risk categories; n=164), intermediate (sole abnormalities of 20q-, 1q duplication or any other translocation, and -Y or other sex chromosome abnormality; n=133) and low (normal or sole abnormalities of 13q- or +9; n=539); the corresponding median survivals were 0.9, 2.6, 3.8 and 4.6 years, with respective HR (95% CI) of 4.4 (3.3-5.8), 2.0 (1.6-2.4) and 1.3 (1.03-1.6). Very high (HR 6.1, 95% CI 2.9-13.2) and high (HR 2.2, 95% CI 1.1-4.1) risk categories were also associated with higher risk of BT. There was no significant correlation between the revised cytogenetic risk stratification and JAK2/CALR/MPL mutational categories (p=0.22), but none of the triple-negative or MPL-mutated patients displayed “very high risk” karyotype. The revised cytogenetic model was also effective in risk-stratification of JAK2 or CALR mutated patients, when analyzed separately, and its prognostic relevance for survival was generally independent of JAK2/CALR/MPL mutational status (p<0.01), DIPSS (p<0.01) or DIPSS-plus (p<0.01). Comparison of the currently used “favorable” vs “unfavorable” with the revised cytogenetic risk model showed a significant number of patients with “favorable” karyotype (n=213 of total 753) being re-assigned to high (n=82) or intermediate (n=131) risk categories. Conclusions : The currently used cytogenetic risk stratification in PMF is too broad and underestimates prognosis in a significant number of patients with “favorable” karyotype. We have developed a revised four-tier risk model that is independent of conventional risk models and JAK2/CALR/MPL mutational status and is expected to facilitate the development of genetics-based prognostic scoring systems in PMF. Disclosures No relevant conflicts of interest to declare.

Abstract Background A recent study from the International Working Group on Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) identified prior arterial events and hypertension as risk factors for subsequent arterial thrombosis and prior venous events and age 65 years or older as risk factors for venous thrombosis in polycythemia vera (PV) (Blood 2014;124:3021). In the current single center study, we sought to validate the aforementioned findings from the IWG-MRT and look into further details regarding arterial versus venous thrombosis in PV. Methods Study patients were selected from our institutional database of myeloproliferative neoplasms (MPN) and fulfilled the 2008 World Health Organization (WHO) criteria for the diagnosis of PV (Blood. 2009;114:937). Screening for the two most frequent mutations other than JAK2 (TET2 and ASXL1) were performed according to conventional methods (Leukemia. 2014;28:2206). Statistical analyses considered clinical and laboratory parameters obtained at time of diagnosis. Results Patient characteristics: Analysis was conducted on 587 patients (median age 60 years; 48% males). ASXL1 and TET2 mutational status was available in 133 patients. Among informative cases, 31% had palpable splenomegaly, 34% microcirculatory symptoms, 30% pruritus, 8% erythromelalgia, 42% hypertension, 9% diabetes and 11% were active tobacco users. TET2 and ASXL1 mutations were documented in 18% and 11%, respectively. During follow-up, 224 (38%) patients died and median follow-up for living patients was 109 months. Median survival was 16 years and leukemic or fibrotic transformations were documented in 4% and 14%, respectively. Thrombotic events: A total of 235 (40%) patients experienced at least one thrombotic event before or after diagnosis and included 153 (26%) arterial and 104 (18%) venous events. Thrombotic events were recorded before or at time of diagnosis in 146 (25%) patients and included 97 (17%) arterial and 55 (9%) venous events. Thrombotic events after diagnosis were recorded in 128 (22%) patients and included 82 (14%) arterial and 57 (10%) venous events. Associations for arterial, venous or all thrombotic events occurring at any time before or after time of diagnosis: The occurrence of any thrombotic event before or after diagnosis correlated with active tobacco use (p=0.04), diabetes history (p=0.03), hyperlipidemia (p=0.04) and major hemorrhage (p=0.03). The occurrence of any arterial event before or after diagnosis correlated with advanced age (p=0.006), diabetes history (p=0.005), hypertension history (p=0.004) and hyperlipidemia (p<0.0001). The occurrence of any venous event before or after diagnosis correlated with female sex (p=0.008), younger age (p<0.0001), lower platelet count (p=0.0014) and palpable splenomegaly (p=0.006). TET2 or ASXL1 mutations did not correlate with overall, arterial or venous thrombosis. Risk factors for arterial versus venous events after time of diagnosis: In univariate/multivariable analysis, thrombosis-free survival (TFS) was adversely affected by prior vascular events (p=0.002/0.03 multivariable) and increased leukocyte count (p=0.03/0.03); arterial thrombosis-free survival was adversely affected by prior arterial events (p<0.0001/P<0.0001), hyperlipidemia (p=0.001/0.03), hypertension (p=0.02/NS) and advanced age (p=0.02/NS); venous thrombosis-free survival was adversely affected by prior venous events (p=0.04/0.05), increased leukocyte count (p=0.0009/0.002), and younger age (p=0.02/NS). In addition, major hemorrhage at diagnosis was associated subsequent venous thrombosis in both univariate and multivariable analysis. TET2 or ASXL1 mutations did not affect the risk of overall, arterial or venous thrombosis. Conclusions In contemporarily managed patients with PV, prior vascular events predict future events for both arterial and venous thrombosis; additional risk factors included hyperlipidemia for arterial and increased leukocyte count for venous thrombosis. The current study also identifies association of arterial thrombosis in general with older age and history of hypertension, diabetes or hyperlipidemia whereas venous thrombosis was associated with younger age, female sex and palpable splenomegaly. Additional findings included association of major hemorrhage with thrombosis and higher platelet count with lower risk of venous thrombosis. Disclosures No relevant conflicts of interest to declare.

Abstract Background The International Working Group for Myeloproliferative Neoplasms (MPN) Research and Treatment (IWG-MRT) has previously identified older age, leukocytosis, venous thrombosis and abnormal karyotype as risk factors for overall survival (OS) in polycythemia vera (PV), and older age, abnormal karyotype and leukocytes ≥15 × 109/l as risk factors for leukemia-free survival (LFS) (Leukemia2013;27:1874). Serum lactate dehydrogenase (LDH) is a surrogate quantitative measure of cell turnover and tumor burden. We hypothesized that, compared to leukocyte count, serum LDH might be a biologically more accurate indicator of tumor aggression and sought to examine its potential utility as a prognostic biomarker in PV. Methods Study patients were selected from our institutional database of MPN based on their meeting the 2008 or 2016 World Health Organization criteria for diagnosis of PV (Blood. 2009;114:937; Blood 2016 127:2391) and availability of serum LDH level, obtained within six months of diagnosis. Information on karyotype and targeted next-generation sequencing was available in a subset of the patients (Blood 2015 126:354). Statistical analyses considered clinical and laboratory parameters obtained at time of diagnosis. Age and leukocyte count prognostic categories were based on previously published IWG-MRT study (Leukemia2013;27:1874). Results Patient characteristics: A total of 216 patients (50% females) met the above-outlined criteria: median (10th-90th percentile) levels were for age 63 years (38-81), hemoglobin 18 g/dL (16.6-20.7), platelets 467 x 10(9)/L (237-833), leukocyte count 11.7 x 10(9)/L (7.2-20.3) and spleen size 0 cm (0-2). Pruritus was documented in 28%, microcirculatory symptoms in 31%, erythromelalgia in 6%, hypertension in 45%, diabetes in 7%, active tobacco use in 13%, hyperlipidemia in 25%, palpable spleen in 26% and abnormal karyotype in 15% of informative cases. Grading for bone marrow reticulin fibrosis was documented for 144 patients: 50% grade "0", 42% grade "1", 7% grade "2" and 1 grade "3". Thrombosis history at diagnosis was documented in 26% of patients and occurred after diagnosis in 19%. Mutation analysis was available in 72 patients and revealed TET2 mutations in 18 (25%), ASXL1 in 5 (7%), SRSF2 in 2 (3%) and IDH2 in one (1.4%). Serum LDH levels and correlates: Median serum LDH level was 226 U/L (range 71-1008); the upper normal limit (UNL) for Mayo Clinic was 222 U/L. Increased serum LDH was recorded in 110 (51%) patients that included 26 (12%) patients with LDH ≥1.5 x UNL. A significant correlation was demonstrated between increased serum LDH and older age (p=0.007), female sex (p=0.04), leukocytosis (p=0.01), venous thrombosis history (p=0.005) and, interestingly, absence of active tobacco use (p=0.003); no correlation was noted for bone marrow reticulin fibrosis, presence of palpable splenomegaly, abnormal karyotype or ASXL1 mutation. Survival analysis: After a median follow-up of 77 months, 82 (38%) deaths, 8 (3.7%) leukemic transformations, 13 (6%) fibrotic progressions and 41 (19%) thrombotic events were documented. In univariate analysis, the following adversely affected OS: advanced age (P<0.0001), increased LDH as a continuous variable (p=0.0006), LDH ≥1.5 x UNL (p<0.0001), leukocyte count ≥15 x 10(9)/L (p=0.02) and arterial thrombosis history (p=0.001). In multivariable analysis, only age and LDH remained significant; multivariable HR (95% CI) were 5.4 (2.8-10.4) for age ≥67 years, 3.2 (1.8-6.0) for LDH ≥1.5 x UNL and 2.4 (1.2-4.7) for age 57 to 66 years. In the subset of patients with available information on karyotype and non-JAK2 mutations (n=72), advanced age, LDH ≥1.5 x UNL and ASXL1 mutations remained significantly associated with inferior survival. Serum LDH also predicted leukemic transformation (HR 17.8, 95% CI 4.1-78) and fibrotic progression (HR 11.6, 95% CI 25-53.7); in multivariable analysis, only serum LDH ≥1.5 x UNL (HR 22.3, 95% CI 2.1-234.6) and abnormal karyotype (HR 9.5, 95% CI 1.2-74.7) predicted leukemic transformation and only the former predicted fibrotic progression. Conclusions Serum LDH is a simple and inexpensive biomarker that might supersede leukocyte count as an independent predictor of overall, leukemia-free and myelofibrosis-free survival in PV; advanced age and ASXL1 mutations also retained significance for OS and abnormal karyotype for LFS. Disclosures No relevant conflicts of interest to declare.