Pardanani, Animesh

Abstract BACKGROUND: We have previously reported on the therapeutic value of low-dose thalidomide in combination with prednisone (THAL-PRED) for the treatment of both anemia and thrombocytopenia in MMM (Blood2003;101:2534), with response rates of 62% and 75% for each cytopenia respectively. Alkylators have long been shown to have palliative benefit in MMM, however are used judiciously as long-term use could increase likelihood of leukemic transformation. Additionally, oral cyclophosphamide has been shown to synergize with thalidomide and steroids in the treatment of myeloma (Garcia-Sanz et. al. Leukemia2004;18:856–863). In the current study, we investigated the value of adding oral cyclophosphamide to THAL-PRED for MMM (TPC). METHODS: Study eligibility criteria included a histologically confirmed diagnosis of MMM and either severe anemia (hemoglobin < 10 g/dL) or symptomatic splenomegaly. Protocol treatment was initiated with 50 mg of oral thalidomide (THAL) daily, oral cyclophosphamide (CTX) 25 mg daily for three months, and a three month prednisone taper starting at 0.5 mg/kg/day for the first month followed by a 50% reduction for the second months and a further 50% reduction for the third and final month of treatment with prednisone. Patients with a demonstrable clinical response after three months were able to remain on thalidomide alone as maintenance. Responses were assessed by the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) criteria for MMM clinical responses (Tefferi et. al. Blood 2006; epub May 2006). RESULTS: 13 patients were enrolled to the trial (median age 73 years, range, 46–80; 7 males). Six patients (46%) completed the planned three cycles (i.e. 12 weeks) of treatment, the remaining seven patients discontinued treatment early for the following reasons: progression (n=3), patient choice (n=2), other medical problem (n=1) and died on study (n=1). After the initial 3 months of treatment, 3/13 (23%) patients evidenced a response (n = 1) or stable disease (n = 2) and continued on an additional three months of THAL alone. At enrollment, nine patients (69%) were anemic (8 of whom (62%) were red cell transfusion dependent). Ten patients (77%) were at least moderately thrombocytopenic (platelet count < 100 x 109//L). Eight patients (62%) had palpable hepatomegaly/splenomegaly, and five patients (38%) had severe constitutional symptoms (night sweats). According to IWG-MRT criteria for MMM the best responses observed from therapy was a CI (clinical improvement) in 2 individuals (one each for anemia (1/9 patients (11%); and thrombocytopenia (1/10 patients (10%)). No clear responses in organomegaly or constitutional symptoms were observed. The regimen overall was well tolerated with toxicities in excess of grade 2 all being related to myelosuppression (anemia (n=2), neutropenia (n=2), and thrombocytopenia (n=3)). CONCLUSIONS: This phase II study of combination therapy with thalidomide-prednisone-cyclophosphamide in patients with MMM strongly suggests that the addition of the alkylator cyclophosphamide largely negates the clinical benefits of THAL-PRED. This observation supports the potential immunologic mechanism of THAL-PRED benefit in MMM, and cautions against potential combinations of immunological and cytotoxic therapy in this disorder.

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 Background: Mechanisms of hereditary erythrocytosis have been elucidated recently. These include high oxygen affinity (HOA) hemoglobin (Hb) variants, bisphosphoglycerate mutase (BPGM) deficiency, abnormalities in the erythropoietin receptor (EPOR) and oxygen-sensing pathway (OSP) proteins prolyl hydroxylase domain-2 (PHD2), hypoxia-inducible factor-2 alpha subunit (HIF2A), and von Hippel-Lindau (VHL). We present our experience with these disorders. Methods: Evaluation of erythrocytosis patients' blood samples for HOA Hb variants by protein or DNA sequencing methods has been performed in our laboratory for over 30 years. Testing has included alkaline/acid electrophoresis, isoelectric focusing, capillary electrophoresis, high performance liquid chromatography, mass spectrometry and Sanger sequencing. BPGM deficiency was determined by enzyme activity assay and/or genetic testing. Since 2012 we have evaluated the EPOR, EGLN1 (PHD2), EPAS1(HIF2A), and VHL genes. Reflexive evaluations, which include testing for p50 and pertinent Sanger sequencing of HBB, HBA1, HBA2, EPOR (exon 8), EGLN1 (exons 1-5), EPAS1 (exon 12) and VHL, have been performed on a subset of cases. Genetic results were correlated with clinical and phenotypic data. Results: Retrospective database review identified 62 confirmed HOA Hb variants (48β, 14α) with clinical erythrocytosis (Table 1). Multiple Hb variants were silent on at least one protein method but showed decreased p50 values. One homozygous BPGM mutation case was identified. Of the 394 cases evaluated for EPOR and OSP abnormalities, 39 cases (10%) contained genetic alterations. Of these, 11 were known pathogenic mutations and 28 were novel alterations including 4 pathogenic, 12 likely pathogenic and 12 variants of unknown significance (VUS). Eighteen EGLN1, 10 EPAS1, and 7 EPOR alterations were detected, all heterozygous. Four VHL mutation cases were identified: 3 homozygous/compound heterozygous known mutations and one novel heterozygous VUS (p.R200Q). All EPOR mutations resulted in a premature stop codon. Most of the EPAS1 alterations were located near proline 531, although two predicted splice site disturbances were found. The EGLN1 alterations were novel, variable, and affect the enzyme's catalytic domain. All 4 VHL cases involved the R200 amino acid position in at least one allele. Serum Epo levels varied: decreased in EPOR (5/5); normal in PHD2 (11/11) and most HIF2A (6/7) and VHL (3/4) cases; increased in 1 HIF2A and 1 VHL case. p50 was normal in EPOR and OSP cases (31/31 tested). Conclusion: A spectrum of abnormalities was identified. Salient points include: 1) Diagnostically, a high index of suspicion for HOA Hb variants is required as they can give false negative results by routine protein detection methods. p50 and Epo levels were useful for triage. 2) Phenotypically, serum Epo levels were low in EPOR and normal in PHD2 and HIF2A cases. Unexpectedly, the two classic Chuvash polycythemia VHL cases were associated with normal Epo levels. 3) Mechanistically, the alterations were truncating in EPOR, variable and scattered in EGLN1 and clustered near proline 531 in EPAS1. VHL mutations were rare and consistently affected the R200 amino acid position in at least one allele. Novel alterations were frequent, especially in EGLN1. 4) Clinically, many patients were asymptomatic but a subset demonstrated recurrent headaches and one had chest pain. Clotting complications included cerebrovascular accident in one EPAS1 and EGLN1 case each and portal vein thrombosis in one EGLN1 case. Table 1. Clinically significant high oxygen affinity Hb variants at Mayo Clinic n Beta Alpha >30 Malmo Tarrant 10 - 20 Bethesda, San Diego, Andrew-Minneapolis, M-Saskatoon, Olympia, Syracuse, Abruzzo Dallas 5 - 10 Ty Gard, Ypsilanti, Alberta, Coimbra, North Chicago J-Cape Town 2 - 4 Brigham, Kempsey, Little Rock, Puttelange, Wood, Brisbane, Cowtown, Creteil, Linkoping, Osler, Potomac, Providence, Ranier, Sparta, Vanderbilt, Heathrow, Helsinki, Hiroshima, Homozygous HPFH, McKees-Rocks, Pierre Benite, Regina Chesapeake, Ethiopia, Chiapas, Columbia Missouri, Burlington, Legnano 1 Alcorn County*, Bologna-St. Orsola, Bunbury, Cambridge-MA, Cardarelli, Chandigarh, Johnstown, Nantes, Nebraska, Olomouc, Palmerston North, Pitie-Salpetriere, Saint-Jacques, South Milwaukee Linwood, Longview*, Milledgeville, Sarasota Springs, Voorhees * novel Disclosures Pardanani: Stemline: Research Funding.

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.

Abstract Abstract 1742 Background Karyotype is one of the most potent and reproducible risk factors for both overall (OS) and leukemia-free (LFS) survival in primary myelofibrosis (PMF) (Blood 2011;118:4595). It is currently not clear if the number of metaphases studied or the abnormal metaphase percentage alters this prognostic impact. Methods: An updated Mayo Clinic database of karyotypically- and DIPSS-plus-annotated patients with PMF was used to identify a consecutive series of patients and their cytogenetic information obtained at time of referral was centrally re-reviewed. Cytogenetic results were interpreted and reported according to the International System for Human Cytogenetic Nomenclature; abnormal karyotype was defined by the presence of at least 2 metaphases with structural abnormalities or monosomy or 3 metaphases with polysomy, regardless of number of metaphases examined. For this particular study, the presence of less than 20 evaluable metaphases did not disqualify patients. “Very high risk” karyotype included monosomal karyotype, inv(3) or i(17q) abnormalities (Blood 2011;118:4595). “unfavorable” karyotype included complex or any sole or two abnormalities that included +8, −7/7q-, -5/5q-, inv(3), i(17q), 12p-, or 11q23 rearrangement (Blood 2011;118:4595). All other cytogenetic abnormalities were considered “favorable” Results: A total of 590 patients (median age 65 years; range 19–89 years) including 424 (72%) males. The DIPSS-plus (JCO 2011;29:392) risk distribution was 40% high, 39% intermediate-2, 12% intermediate-1 and 9% low. Cytogenetic findings included 17 (3%) very high risk, 69 (12%) unfavorable, 165 (28%) favorable and 339 (57%) normal karyotypes. The number of bone marrow metaphases studied to report these cytogenetic findings were ≥20 in 468 (79%) patients, 11 to 19 in 71 (12%) patients and ≤10 in 51 (9%) patients; the proportion of cases studied with ≥20 metaphases were 53% for very high risk, 74% for unfavorable, 83% for favorable and 80% for normal karyotype (p=0.006). Among patients with abnormal karyotype, the abnormal metaphase percentage was ≥75% in 148 (59%) patients, 50 to 74% in 36 (15%) patients, 26 to 49% in 27 (11%) patients and ≤25% in 38 (15%) patients; the proportion of patients with ≥75% was 59% for very high risk, 67% for unfavorable and 56% for favorable karyotypes (p=0.70). As expected, OS was significantly different among very high risk, unfavorable, favorable and normal karyotype patients with respective median survivals of 8, 23, 41 and 57 months (p<0.0001). The number of metaphases studied (p=0.62) or the abnormal metaphase percentage (p=0.12), by themselves, did not affect survival. Similarly, the survival difference among the aforementioned cytogenetic risk groups was equally apparent when patients with ≥20 metaphases studied (n=468; P<0.0001) and those with <20 metaphases studied (n=122; p<0.0001) were separately analyzed. Analysis of patients with very high risk or unfavorable karyotype (n=86) revealed no significant effect of abnormal metaphase percentage on survival (Figure; p=0.80). A similar scenario was demonstrated for patients with favorable karyotype (Figure; p=0.24). Conclusions: Neither the number of metaphases examined nor the abnormal metaphase percentage appear to influence the currently recognized cytogenetic risk stratification in PMF. The current study has implications for both clinical practice and clinical research involving cytogenetic prognostication in hematological malignancies. Disclosures: No relevant conflicts of interest to declare.

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 Abstract 2849 Background We have previously reported on the natural history of polycythemia vera (PV), focusing primarily on overall and leukemia-free survival (ASH Annual Meeting Abstracts. 2011;118(21):277-). In the current study, we present, on behalf of the International Working Group for Myeloproliferative neoplasms Resarch and Treatment (IWG-MRT), our analysis regarding risk factors for thrombosis. Methods Under the auspices of IWG-MRT, seven international centers of excellence for myeloproliferative neoplasms participated in the current study. The two principle investigators (AT and TB) reviewed all the cases and selected 1,545 patients who met the 2008 WHO criteria for PV, were age 18 years or older, diagnosed after 1970, and whose submitted data included diagnostically essential information. Results I: Presenting Features Median age was 61 years (range, 18–95; 51% females). Arterial and venous thrombosis history before or at diagnosis was documented in 246 (16%) patients and 114 (7.4%) patients, respectively. Major hemorrhage hemorrhage before or at diagnosis was documented in 17 (4.5%) patients. Other features at diagnosis included pruritus (36%), microvascular disturbances (28.5%), palpable splenomegaly (36%), abnormal karyotype (12%), leukoerythroblastosis (6%), increased LDH (50%), thrombocytosis (53%), extreme thrombocytosis (platelets >1 million mm3; 4%) leukocytosis (49%), JAK2 V617F (95%), other JAK2 mutations (3%), subnormal serum erythropoietin (Epo) level (81%), and endogenous erythroid colonies (EEC; 73%). History of hypertension (46%), hyperlipidemia (18.3%), diabetes (8.4%), and tobacco use (16%) was also obtained. Results II: Clinical Course To date, 347 (23%) deaths, 50 (3%) leukemic progressions, and 138 (9%) fibrotic transformations have been recorded. Overall, cytoreductive treatment was not used in 416 (27%) patients and the remaining were exposed to different agents based on physician discretion. Post-diagnosis arterial or venous thrombosis occurred in 184 (12%) and 137 (9%) patients, respectively. Results III: Risk Factors for thrombosis Arterial and venous thrombosis-free survival, from time of diagnosis, were separately analyzed using the occurrence of thrombosis as the endpoint (uncensored variable) and last follow-up or death before thrombosis as the censored variable. In univariate analysis, the following were significantly associated with post-diagnosis arterial thrombosis: advanced age, leukocyte count, presence of a leukoerythroblastic smear (LES), history of hypertension and history of arterial thrombosis before or at diagnosis; multivariable analysis using all these five parameters identified arterial thrombosis history (RR 2.5, 95% CI 1.6–4.0; p<0.0001), LES (RR 2.3, 95% CI 1.3–4.2; p=0.005) and history of hypertension (RR 1.6, 95% CI 1.1–2.4; p=0.02) as independent predictors of post-diagnosis arterial thrombosis. Only two parameters predicted post-diagnosis venous thrombosis, in univariate analysis, and both remained significant during multivariable analysis: abnormal karyotype (RR 3.1, 95% CI 1.7–5.4; p=0.0001) and history of venous thrombosis (RR 2.4, 95% CI 1.2–4.9). Of note, the type of JAK2 mutation or presence of either subnormal Epo or EEC did not influence either arterial or venous thrombosis. Results IV: Risk Stratification for arterial and venous thrombosis The figures below illustrated arterial or venous thrombosis-free survival of patients stratified by the absence of all risk factors or presence of one or ≥2 risk factors. For arterial thrombosis, the presence of ≥2 risk factors clearly delineated a high risk group (RR 3.1, 95% CI 1.9–5.0) whereas the presence of one (RR 2.4, 95% CI 1.4–4.2) or two risk factors (RR 10.1, 95% CI 3.6–28.2) for venous thrombosis delineated an intermediate and high risk group, respectively. Conclusions: History of arterial thrombosis and venous thrombosis are key risk factors, respectively, for recurrent arterial and venous thrombosis in PV. In addition, abnormal karyotype is a strong independent risk factor for venous thrombosis and the presence of leukoerythroblastosis and hypertension, for arterial thrombosis. This information allows for a simple and practical risk stratification and raises interesting pathogenetic implications that require further clarification. Disclosures: Vannucchi: Novartis: Membership on an entity's Board of Directors or advisory committees. Gisslinger:Novartis: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy, Research Funding, Speakers Bureau. Passamonti:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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.