Lasho, Terra L.

Abstract Abstract 5154 Background Recent clinical trials with JAK or mTOR inhibitors in primary myelofibrosis (PMF) have identified pruritus as one of the most treatment-responsive disease traits. However, little is known about the prevalence of pruritus in PMF or its clinical and laboratory correlates. Methods The study population was selected among a consecutive series of patients with PMF seen at the Mayo Clinic and in whom bone marrow histologic and cytogenetic information was available for re-review. JAK2, MPL and IDH mutation status and plasma cytokine levels (IL-1b, IL-1RA, IL-2R, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, TNF-α, G-CSF, IFN-α, IFN-γ, MIP-1a, MIP-1b, HGF, IP-10, MIG, MCP-1 and VEGF) were recorded where available. Overall and leukemia-free survival analyses were performed from time of referral to the Mayo Clinic. Results I: Prevalence of pruritus and other baseline information Among 566 consecutive patients with PMF (median age 62 years; 368 males), the presence (n=90) or absence (n=146) of pruritus was documented in 236 patients. The prevalence of pruritus was 38% when considering only those patients in whom pruritus history was documented (n=236), and 16% when all study patients (n=566) were considered regardless of whether or not the absence of pruritus was documented in their history. Among the 90 patients with pruritus, it was severe enough to warrant specific therapy in 51 (57%) patients, was aquagenic in 29 (32%), and onset antedated PMF diagnosis in 15 (17%). All 236 patients had information on karyotype: 136 normal, 76 favorable and 24 unfavorable karyotype. Information on JAK2, MPL, and IDH mutations was available in 197 (125 positive), 147 (9 positive), and 131 (2 positive) patients, respectively. In addition, plasma levels of the aforementioned cytokines were available in 63 patients. Results II: Clinical correlates Parameters at diagnosis that were significantly associated with presence of pruritus included leukocytosis (p=0.03), absence of leukopenia (p=0.002), and absence of MPLW515 mutation (p=0.02). Borderline significance was noted for JAK2V617F positive status (p=0.08). The incidence of pruritus was 0% (0/9) vs. 41% (56/138) in MPL-mutated vs MPL-unmutated cases and 42% (52/125) vs. 29% (21/72) JAK2 mutated vs unmutated cases, respectively. No significant correlations were noted for age, DIPSS-plus scores, transfusion need, karyotype, or plasma cytokine levels. The association with leukocytosis/leukopenia was subsequently shown to be significant only in JAK2V617F-negative cases. To date, 124 deaths and 19 leukemic transformations have been documented. In JAK2-mutated cases (n=125), pruritus did not affect overall or leukemia-free survival. In JAK2-unmutated cases (n=72), a significant correlation between presence of pruritus and inferior leukemia-free survival was noted (p=0.04), and was independent of leukocytosis, thrombocytopenia or abnormal karyotype. Conclusions Pruritus accompanies PMF in a substantial proportion of patients and is more or less likely to occur in the presence of JAK2 or MPL mutations, respectively. In JAK2V617F-negative cases, PMF-associated pruritus clustered with leukocytosis and was independently associated with inferior leukemia-free survival. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2807 CCDC26 is a retinoic acid-dependent modulator of myeloid cell differentiation and death (Yin W et al. Oncogene 2006 :25 :3735). CCDC26 maps to chromosome 8q24.21 and germline polymorphisms in the gene have been associated with low-grade gliomas (Jenkins RB et al. Cancer Genetics 2011;204 :13), which in turn have recently been shown to frequently express IDH1 mutations (Parsons D et al, Science 2008;321 :1807). More recently, we have discovered that CCDC26 SNP associations in gliomas are primarily restricted to patients with IDH1 mutations (Jenkins et al. unpublished observations). We therefore hypothesized that germline CCDC26 SNPS might be associated with patients with myeloid malignancies that harbor IDH1 mutations (Mardis et al. NEJM 2009;361 :1058). Seventy-two IDH -mutated patients with chronic or blast phase myeloproliferative neoplasms (MPN), myelodysplastic syndromes or acute myeloid leukemia (most constituting post-MPN events) were identified: 18 IDH1 R132, one IDH2 R172 and 53 IDH2 R140. These 72 patients and a control group of 153 IDH unmutated cases with myeloproliferative or myelodysplastic neoplasms (total n =225) were genotyped for CCDC26 risk alleles associated with IDH -mutated gliomas: rs10464870, rs891835 and rs4295627. The respective minor allele frequencies (MAF) were significantly different between IDH1 (11%, 8% and 6%) and IDH2 (30%, 27% and 24%) mutated cases (p=0.037, 0.040 and 0.021; OR 3.4, 3.7 and 6.1 with 95% CI of 1.1–10.5, 1.1–13.0 and 1.3–28.9) as well as between IDH- wild type (24%, 24% and 21%) and IDH1 -mutated (11%, 8% and 6%) cases (p=0.10, 0.043 and 0.035; OR 0.42, 0.29 and 0.21 with 95% CI of 0.15–1.19, 0.09–0.96 and 0.05–0.90). The MAF values in our IDH -unmutated cases were similar to that of population-based controls. However, the direction of SNP associations in IDH1 -mutated myeloid malignancies were opposite of those seen in IDH1 -mutated gliomas. Our observations suggest a differential effect of CCDC26 genetic polymorphisms in IDH1 -mutated myeloid malignancies compared to either their IDH2 R140-mutated or IDH1 -mutated gliomas. Such differences in genomic context might contribute to hitherto seen differences in prognostic impact between IDH2 R140 vs. IDH1 R132 vs. IDH2 R172, in AML (Green CL et al. Blood Published online before print May 19, 2011, doi: 10.1182/blood-2010-12-322479). Disclosures: No relevant conflicts of interest to declare.

Abstract Introduction: Several groups are developing orally bioavailable small molecule JAK2 inhibitors as an approach to targeted therapy of myeloproliferative disorders. We previously reported that one such inhibitor (TG101209) potently inhibits JAK2V617F in vitro, and was effective in reducing tumor growth in a mouse xenograft model (Leukemia, 2007 Aug;21(8):1658–68). Here, we expand our analysis using TG101348, a highly selective JAK2 inhibitor currently in preclinical development, using cell lines and primary cells harboring JAK2V617F, MPLW515L/K, or JAK2 exon 12 mutations. Methods: Cell proliferation was assessed using the XTT assay. For primary cell experiments, PBMCs were plated in methylcellulose medium at 0, 300nM, 600nM, and 1200nM TG101348, both with and without erythropoietin (Epo). Hematopoietic colonies were scored after 12–16 days culture, and harvested for genotyping. Results: TG101348 inhibits growth of human erythroleukemia (HEL) and Ba/F3-V617F cells (IC50=300nM and 580nM, respectively), as well as Ba/F3-(MPL)W515L cells (IC50=620nM). JAK2:JAK3 selectivity of TG101348 was demonstrated by its 10-fold more potent inhibition of CHRF (JAK2T875N) as compared to CMK (JAK3A572V) cell proliferation. TG101348’s inhibitory effect on ex vivo hematopoietic colony growth was studied in 12 MPD patients (JAK2V617F=9, MPLW515L/K=1, JAK2 exon 12 mutation [N542-E543del]=1, no mutation=1), and 2 healthy controls (Table). Conclusions: (1) TG101348 inhibits colony growth from MPD patients more potently than healthy controls, reflecting its potential therapeutic window. (2) Endogenous colony growth is inhibited at nanomolar concentrations in all cases, with some alleles showing greater sensitivity than others (MPLW515K = exon 12 mutation > JAK2V617F). The corresponding Epo-supported erythroid colonies are less potently inhibited, reflecting the generally lower prevalence of JAK2 mutations in such colonies. (3) EEC growth in the absence of known JAK2 or MPLW515 mutations is also inhibited, indicating a similar dependence on dysregulated JAK-STAT signaling in such cases. (4) The ability of TG101348 to selectively suppress mutation-positive hematopoietic colonies will be discussed. Inhibitor effect on ex vivo hematopoietic colony growth Paient Mutation IC50 (nM) % Mutation-positive colonies Epo No Epo Epo No Epo (EEC) Normal 1 None >1200 n/a n/a n/a Normal 2 None >1200 n/a n/a n.a PV1 V617F 600 <300 5 40 PV2 V617F 600 300 33 50 PV3 V617F 600–1200 300–600 9 70 PV4 V617F 300–600 600 0 15 PV5 V617F 600–1200 300 80 100 PV6 V617F 600 300–600 7 19 PV7 V617F 600–1200 600 32 36 PV8 V617F 600–1200 <300 45 37 PV9 V617F 600 600–1200 20 12 PV10 Exon 12 (N542–E543del) 900 <300 9 60 PMF1 W515K 300–600 <300 100 33 PMF2 None >1200 300 n/a n/a

Abstract Abstract 3915 Poster Board III-851 Background Thromboembolic disease (TE) is a major cause of morbidity and mortality in polycythemia vera (PV) and essential thrombocythemia (ET). There is limited information on the prevalence of TE in primary myelofibrosis (PMF) and associated prognostic factors. Methods Study patients were recruited form the Mayo Clinic database for PMF. Diagnosis was according to the 2001 WHO criteria. Clinical and laboratory information was collected at time of diagnosis and correlated with the occurrence of TE in general and arterial (AT) or venous (VT) thrombosis in particular. Leukocytosis was defined as a leukocyte count of > 10 × 109/L. Qualitative JAK2V617F analysis was done according to previously published PCR-based methods. In addition, a subset of the study patients underwent quantitative PCR for JAK2V617F and rs12343867 SNP genotyping (performed by a commercially available Taqman assay). All molecular studies were performed on stored bone marrow. Standard statistical methods were used to test significance of associations between thrombosis and other variables. Results 207 patients with PMF were studied (median age 62, range 28-87; 132 males). At presentation, the median (range) of hemoglobin (Hb), leukocytes (WBC) and platelets (plt) were 11 g/dl (6.4-15.4), 8.6 × 109/L (0.8-156.7) and 273 × 109/L (13-1916), respectively. Dupriez prognostic scores at diagnosis were low, intermediate and high in 57%, 33% and 10% of the patients, respectively. 130 (63%) patients were JAK2V617F positive. Among 73 patients in whom quantitative JAK2V617F analysis was performed, the mutation was not detected in 30 (41%) patients and the median mutant allele burden in the 43 mutation-positive patients was 28% (range 1-85). The same 73 patients had rs12343867 SNP genotyping performed that revealed C/C genotype in 14 (19%) patients, C/T in 34 (47%) and T/T in 25 (34%). At or before diagnosis, a total of 29 patients (14 %) had TE: 23 (11%) AT and 9 (4%) VT. The AT included acute coronary syndromes (ACS; n=19), transient ischemic attack/ cerebrovascular accidents (TIA/CVA; n=6) and peripheral arterial thrombosis (PAT; n=1). The VT included abdominal vein thrombosis (AVT; n=4), deep vein thrombosis/pulmonary embolism (DVT/PE; n=6) and dural sinus thrombosis (n=1). Several patients had more than one event and some both AT and VT. During follow-up, 22 (11%) patients experienced TE: 17 (8%) VT and 8 (4%) AT. The post-diagnosis VT included AVT (n=8), DVT/PE (n=13) and both DVT/PE and AVT (n=4). Post-diagnosis AT included CVA (n=4), TIA (n=1), ACS (n=2) and PAT (n=1). AT at or before diagnosis was associated with advanced age (p=0.003) but not with sex, leukocytosis, hemoglobin level, platelet count, JAK2 mutational status, JAK2 mutant allele burden or rs12343867 SNP genotype. VT at or before diagnosis also did not correlate with all these variables or age. These variables were also evaluated regarding their relationship with post-diagnosis AT, which showed significant associations with hemoglobin level (p=0.006), platelet count (p=0.04) and high JAK2V617F allele burden (0.03). The corresponding significant variables for post-diagnosis VT were leukocytosis (p=0.02) and high JAK2V617F allele burden (p=0.01). Of note, AT or VT at or before diagnosis did not predict post-diagnosis events. During multivariable analysis, only higher hemoglobin level predicted post-diagnosis AT. Conclusion The current study provides an accurate description of both arterial and venous thrombotic events in PMF. The association between higher hemoglobin and leukocyte counts at diagnosis and post-diagnosis occurrence, respectively, of arterial and venous thrombosis is similar to observations in patients with PV or ET and suggests a potential benefit from early institution of cytoreductive therapy for the appropriate patient groups. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: JAK2V617F is a recurring mutation in patients with polycythemia vera (PV) (95%) and essential thrombocythemia (ET) (50%), diseases that are frequently complicated by arterial and/or venous thrombosis. JAK2V617F is also frequently identified in patients with splanchnic vein thrombosis, including in those with occult myeloproliferative disorder (MPD). We previously reported a low prevalence of JAK2V617F in thrombosis patients - only 2% of 210 patients with non-splanchnic vein thrombosis (Leukemia, 2007 Aug;21(8):1828–9), and <1% of 136 patients with arterial stroke (J Thromb Haemost. 2007 Aug;5(8):1784–5). Here, we expanded the analysis to 664 thrombosis patients to describe: JAK2V617F prevalence, and clinical course of patients harboring this mutation. Methods: In Phase I, we screened 439 consecutive patients who underwent thrombophilia testing at our institution in early 2005 - patients with splanchnic vein thrombosis or overt MPD were excluded. In Phase II, we identified 228 additional patients considered to be at high risk for harboring JAK2V617F, who were seen at our institution from 2004 to 2006. JAK2V617F screening (assay sensitivity <1%) was performed as described in the aforementioned references. Results: In the initial cohort of 439 patients (median age=56 years; range 17–93 years; 222 female), 136 (31%) presented with stroke, 114 (26%) with deep venous thrombosis (DVT), 95 (22%) with pulmonary embolism (PE), 50 (11%) with DVT and PE, 10 (2%) with central retinal vein occlusion (CRVO), 7 (2%) with cerebral vein thrombosis, and the remaining 27 (6%) with other thromboses. Five patients (MC1-MC5; median age=78 years) were found to harbor JAK2V617F (prevalence=1%) - none had overt clinical features of MPD and all harbored a low level of JAK2V617F in peripheral blood (median=5–2%; range=2.2–7.5%) (Table). One patient (MC2) carried the Prothrombin G20210A mutation In the subsequent cohort, we studied 197 patients (median age=39 years; range=16–84 years) with recurrent DVT and/or PE, and 28 patients (median age=27 years; range=31–49 years) with acute myocardial infarction at a young age (<50 years). One patient (MC6) who presented with recurrent idiopathic DVT was found to harbor JAK2V617F (prevalence <0.5%) (Table) - this patient had no features of underlying MPD and the mutation burden was low (9%). After a median follow up of 27 months (range=4–66 months), none of the 6 patients harboring JAK2V617F has developed overt MPD. Conclusions: In patients with thrombosis, JAK2V617F is infrequently detected in the absence of overt MPD and/or involvement of splanchnic veins. Thus, JAK2V617F screening cannot be recommended as part of the hypercoagulable workup in routine clinical practice in this setting. The risk of developing overt MPD may be quite low for patients harboring a low JAK2V617F burden at the time of thrombosis although longer follow up is needed in this regard. Furthermore, the thrombosis risk may be well controlled with standard warfarin therapy, and the role of myelosuppressive agents (eg. hydroxyurea) is unclear in these patients. Characteristics of patients harboring JAK2V617F Patient Age Sex Thrombosis JAK2V617F allele burden MPD Follow up (months) Status MC1 84 M PE, DVT 5.3% No 66 Alive MC2 82 F PE, cerebral vein 5% No 38 Alive MC3 74 F PE 2.2% No 4 Dead MC4 78 F Central retinal vein 5.2% No 22 Alive MC5 63 M Stroke 7.5% No 30 Alive MC6 69 M Recurrent DVT No No 24 Alive

Abstract Background : Current prognostication in primary myelofibrosis (PMF) utilizes international prognostic scoring systems that rely on clinical parameters that are sensitive to day-to-day variations and subjective interpretation. Recent studies in PMF have disclosed important prognostic information attached to additional cytogenetic details (Blood. 2011;118:4595) and somatic mutations, including CALR and ASXL1 (NEJM. 2013;369:2379; Leukemia. 2013;27:1861). Methods : PMF diagnosis and definition of blast transformation (BT) were 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). Previously published methods were used for analyses of CALR, JAK2, MPL and other prognostically-relevant mutations, including ASXL1, SRSF2, EZH2 and IDH(Leukemia. 2014;28:1472). Results : The training set included 964 Mayo Clinic patients (median age 65 years; 62% males) in whom informative karyotype or mutation information was available; cytogenetic information was available in 903 (94%) cases, JAK2/CALR/MPL mutational status in 532 (55%), ASXL1 in 425 (44%), SRSF2 in 434 (45%), IDH1/2 in 376 (39%) and EZH2 in 268 (28%). DIPSS-plus (JCO. 2011;29:392) risk distribution was high in 37% of patients, intermediate-2 in 37%, intermediate-1 in 15% and low in 11%. We used a revised risk stratification for cytogenetics (see accompanying ASH 2014 abstract) to distinguish four distinct cytogenetic risk categories: very high (monosomal karyotype, inv(3), i(17q), -7/7q-, 11q or 12p abnormalities; n=67), high (complex non-monosomal, 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+ or any other sole translocation, and -Y or other sex chromosome abnormality; n=133) and low (normal or sole abnormalities of 13q- or +9; n=539). Mutational frequencies were 58% for JAK2, 25% CALR, 7% MPL, 36% ASXL1, 11% SRSF2, 5% IDH1/2 and 6% EZH2. The 131 cases with CALR mutations were further subclassified into two prognostically different groups: type 1/type 1-like (n=110) and type 2/type 2-like (n=21) (see accompanying ASH 2014 abstract). At a median follow-up time of 4.2 years for patients who are alive, 664 (69%) deaths and 70 BT (7%) were recorded. Age-adjusted multivariable analysis that included cytogenetic and mutational risk groups disclosed the following as independent predictors of shortened survival: very high risk karyotype (HR 4.2; 3 points), high risk karyotype (HR 1.9; 1 point), triple-negative (HR 2.8; 2 points), JAK2 (HR 3.1; 2 points), MPL (HR 3.1; 2 points), type 2/type 2-like CALR (HR 3.6; 2 points), ASXL1 (HR 1.9; 1 points) and SRSF2 (HR 1.9; 1 point); EZH2 (p=0.24) and IDH1/2 (p=0.68) and intermediate risk karyotype (p=0.87) were not significant. The above-mentioned significant variables and age demarcated at 60 years (2 points), were subsequently used to develop an HR-derived, genetics-based prognostic scoring system (GPSS) for 369 patients who were fully informative for both karyotype and all significant mutations: low risk (0 points; n=31), intermediate-1 (1 or 2 points; n=90), intermediate-2 (3 or 4 points; n=133) and high (5 or more points; n=115); the corresponding median survivals were >17, 9 (HR 4.7, 95% CI 1.7-13.0), 5 (HR 10.7, 95% CI 3.9-29.3) and 2.2 (HR 29.2, 95% CI 10.6-80.0) years (Figure 1). High risk GPSS was also associated with higher BT rate (HR 7.4, 95% CI 2.1-26.3). The prognostic distinction between high/intermediate-2 and low/intermediate-1 risk GPSS, in terms of both overall (median 5 vs 26.4 years; HR 7.1, 95% CI 3.3-14.9) and leukemia-free survival (median 11.6 years vs not reached; HR 9.4, 95% CI 2.2-41.0) was validated in an independent cohort of 183 patients from the University of Florence (Figure 2). Conclusions : The current study demonstrates the feasibility of genetics-based prognostic models in PMF that rely on objective parameters that are amenable to further refinement as new genetic information becomes available. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 3795 Recent studies suggest a powerful prognostic value for plasma cytokine levels in primary myelofibrosis (IL-2R, IL-8, IL-12, IL-15 and CXCL10) and large cell lymphoma (IL-2R, IL-8, IL-10, IL-12, CXCL9 and CXCL10). In order to examine the possibility of a similar phenomenon in myelodysplastic syndromes (MDS), we used multiplex ELISA to measure 30 plasma cytokines in 78 patients with primary MDS. Compared to normal controls (n =35), the levels of 19 cytokines were significantly altered. Multivariable analysis identified increased levels of CXCL10 (p<0.01), IL-7 (p=0.02) and IL-6 (p=0.07) as predictors of shortened survival; the survival association remained significant when the Cox model was adjusted for the international prognostic scoring system (IPSS), age, transfusion-need or thrombocytopenia. MDS patients with normal plasma levels of CXCL10, IL-7 and IL-6 lived significantly longer (median survival 76 months) than those with elevated levels of at least one of the three cytokines (median survival 25 months) (p<0.01). Increased levels of IL-6 were associated with inferior leukemia-free survival, independent of other prognostic factors (p=0.01). Comparison of plasma cytokines between MDS (n =78) and primary myelofibrosis (n =127) revealed a significantly different pattern of abnormalities. These observations reinforce the concept of distinct and prognostically-relevant plasma cytokine signatures in hematologic malignancies.Figure:Survival data for 78 patients with de novo myelodysplastic syndrome (MDS) stratified based on plasma levels of CXCL10, IL-7 and IL-6: (i) normal levels of all 3 cytokines (top curve), or (ii) increased (> 3 standard deviations of the normal mean level) level(s) of one or more of the aforementioned cytokines (bottom curve) (p=0.002).Figure:. Survival data for 78 patients with de novo myelodysplastic syndrome (MDS) stratified based on plasma levels of CXCL10, IL-7 and IL-6: (i) normal levels of all 3 cytokines (top curve), or (ii) increased (> 3 standard deviations of the normal mean level) level(s) of one or more of the aforementioned cytokines (bottom curve) (p=0.002). Disclosures: No relevant conflicts of interest to declare.

Abstract BACKGROUND. In primary myelofibrosis (PMF), survival from time of diagnosis is predicted by the international prognostic scoring system (IPSS), and from time of referral, by the dynamic IPSS (DIPSS) or DIPSS-plus. JAK2/CALR/MPL mutational status and presence and number of other prognostically-relevant mutations (ASXL1, SRSF2, EZH2, IDH1/2) provide IPSS/DIPSS-plus independent prognostic information. The objective in the current study was to revise IPSS, by including mutation-relevant prognostic information. METHODS. The study included 986 PMF patients divided into learning (n=588) and validation (n=398) cohorts. Previously published methods were used to screen for mutations involving JAK2,MPL, CALR, EZH2, ASXL1, IDH1/2 and SRSF2. The clinical variables assessed were those previously identified as being prognostically-relevant by IPSS or DIPSS-plus. The prognostic model (MIPSS) was developed through a stepwise selection process, based on a z-test of the regression coefficients, and its relative quality was measured by means of the Akaike information criterion. RESULTS. In the Italian learning cohort (n=588),median follow-up was 3.6years (95% CI, 0.03-30.8) for alive patients and 196 (33.3%) deaths and 67 leukemic transformations were documented. IPSS risk distribution was low in 26%, intermediate-1 in 30%, intermediate-2 in 24% and high in 20%. Mutational frequencies were approximately 63% for JAK2, 20% CALR, 6% MPL, 7% EZH2, 22% ASXL1, 2.5% IDH1/2 and 9% SRSF2. Constitutional symptoms were recorded in 28.6% of patients, splenomegaly 75% (18% >10cm from LCM) and grade 3 fibrosis 21%. Overall 252 patients were evaluable for karyotype and abnormalities were detected in 35%, including 9.5% unfavorable. Univariate analysis identified the following risk factors for inferior survival: age >60 years, constitutional symptoms, hemoglobin <100g/L, leukocytes >25x109/L, platelets <200x109/L, circulating blasts 1% or greater, splenomegaly >10cm, grade 3 fibrosis, unfavorable karyotype, triple-negativity for JAK2, MPL and CALR (TN), JAK2 or MPL mutation, and mutations in ASXL1, SRSF2, EZH2 or IDH1/2. In multivariable analysis, age >60 years, constitutional symptoms, hemoglobin <100g/L, platelets <200x109/L, TN, JAK2 or MPL mutation, ASXL1 and SRSF2 mutation remained significant; these variables were subsequently assigned the following HR-weighted adverse points: 1.5, 0.5, 0.5, 1.0, 1.5, 0.5, 0.5 and 0.5, respectively. Subsequently, four risk groups with no overlap in the 95% CI of their survival curves were delineated (Figure 1A): low (score 0-0.5); Intermediate-1 (score 1-1.5); Intermediate-2 (score 2-3.5); and high (score 4 or greater). The number of patients in each risk category, median survivals and HR and 95% CI values are included in figure 1A. Akaike information criterion indicated that MIPSS performed better than IPSS in predicting survival (1611.6 vs 1649.0). Leukemia-free survival was also predicted by high (p<0.01) and intermediate 1/2 (p<0.01) risk MIPSS. In the Mayo Clinic validation cohort (n=398), median age 63 years; males 64%,median follow-up for alive patients was 7.5 years and 271 (68%) deaths and 51 (13%) leukemic transformations were documented. DIPSS-plus risk distribution was low in 13%, intermediate-1 18%, intermediate-2 37% and high 32%. Mutational frequencies were approximately 51% for JAK2, 30% CALR, 7.5% MPL, 5% EZH2, 36% ASXL1, 5.6% IDH1/2 and 13% SRSF2. MIPSS performed equally well in this cohort, as illustrated in figure 1B, which includes number of patients in each risk category with their median survival and HR and 95% CI values. Leukemia-free survival in the Mayo cohort was also predicted by high (p<0.01) and intermediate-2 (p=0.04) risk MIPSS. Patients in the Mayo cohort were fully annotated for karyotype, which was MIPSS-independent in its prognostic value for both overall (p<0.01) and leukemia-free (p<0.01) survival. CONCLUSIONS. MIPSS addresses the mutation-relevant prognostic information gap in IPSS and performs equally well in the setting of patients seen at time of diagnosis or referral. Karyotype carries MIPSS-independent prognostic value that is recognized in an alternative GIPSS prognostic model (see accompanying ASH 2014 abstract), which complements MIPSS. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Abstract Background JAK inhibitors, including ruxolitinib, are to date incapable of inducing complete (CR) or partial (PR) remissions, reversal of bone marrow (BM) fibrosis or molecular responses in myelofibrosis (MF). This is consistent with the fact that JAK2 mutations are neither specific nor pathogenetically essential for the disease. Other currently available drugs in MF are equally ineffective in terms of disease-modifying activity. Methods In an investigator-sponsored single-center study (ClinicalTrials.gov Identifier: NCT01731951), imetelstat, a lipid-conjugated oligonucleotide inhibitor of human telomerase, was administered to patients with high or intermediate-2 risk MF (JCO 2011). Adverse events were monitored by common terminology criteria (Version 4.03) and responses by the International Working Group criteria (Blood 2013). Eligibility criteria included platelets ≥50 x 10(9)/L. Study drug and funding were provided by Geron Corporation (Menlo Park, CA, USA). Imetelstat was administered by a 2-hour intravenous infusion (9.4 mg/kg) every three weeks (cohort A) or weekly x 3 followed by every three weeks (cohort B). Mutations with prognostic (ASXL1 and SRSF2) or phenotypic (SF3B1 and U2AF1) relevance were screened by DNA sequencing. Quantitative PCR was used to measure JAK2V617F burden (assay sensitivity 0.01%). Laboratory correlative studies included analyses of granulocyte telomere length, mononuclear cell telomerase activity and human telomerase reverse transcriptase (hTERT) isoforms. Results Thirty-three patients were accrued; the first 18 patients enrolled and followed for a minimum of 3 months or discontinued are presented in this abstract: 11 cohort A and 7 cohort B; 44% PMF, 33% post-PV MF and 22% post-ET MF. Median age was 68 years and baseline risk was high in 56% and intermediate-2 in 44%. Seven patients were transfusion-dependent. Median spleen size was 13 cm and 11 patients had constitutional symptoms. Karyotype was abnormal in 7 patients and 89% were JAK2-mutated. Fifteen (83%) patients were previously treated including 7 with a JAK inhibitor and 3 with pomalidomide. i) Toxicity At a median f/u of 3.2 months, 16 (89%) patients remain on treatment; the two discontinuations were from unrelated death and disease progression. In cohort A, there were no grade-4 treatment-related adverse events; grade-3 events were limited to thrombocytopenia in 27% and anemia in 9%. In cohort B, two (29%) patients experienced grade-4 thrombocytopenia; grade-3 events were limited to thrombocytopenia, neutropenia and anemia in one patient each. Dose reduction was necessary in only two (11%) patients because of grade 3 or 4 myelosuppression. ii) Efficacy Overall response rate was 44%. This included five (28%) patients who met the BM and peripheral blood morphologic criteria for CR (n=4) or PR (n=1) and 3 patients with clinical improvement, pending validation of response duration and resolution of drug-induced grade-1 thrombocytopenia. The four (22%) CR patients experienced reversal of BM fibrosis and recovery of normal megakaryocyte morphology. Two CR patients were transfusion-dependent at baseline and became transfusion-independent. Complete molecular responses were documented in 2 CR patients: one had U2AF1Q157P and 10% JAK2V617F and the other SF3B1K666E and 50% JAK2V617F. A third CR patient had a >50% reduction in U2AF1 469_insAGTATG mutation. Among 13 patients with leukocytosis, 10 (77%) normalized their count or had >50% reduction. Eleven (61%) patients had complete or partial resolution of leukoerythroblastosis. iii) Laboratory correlative studies Three (50%) of 6 spliceosome-mutated vs. 1 (8%) of 12 unmutated (p=0.045) achieved CR. Spliceosome-mutated patients were also more likely to experience grade-3/4 myelosuppression (67% vs. 25% ; p=0.09). Treatment was associated with suppression of telomerase activity, shortening of telomere length and alteration of hTERT isoform pattern. Conclusions The current study signifies the potential value of telomerase-based treatment strategies in MF and identifies imetelstat as an active drug in that regard. The observed morphologic and molecular remissions confirm selective anti-clonal activity, which has thus far eluded other drugs in MF, including JAK inhibitors. The association between response and spliceosome mutations suggests a broader application for the drug in myeloid malignancies.</abstract> Disclosures: Stuart: Geron: Consultancy.

Abstract Abstract 3803 Background: Ring sideroblasts (RS) represent abnormal mitochondrial iron deposits that are commonly present in myelodysplastic syndromes (MDS). The presence of ≥15% RS, by definition (World Health Organization-WHO) is necessary for a diagnosis of a MDS-RS; with refractory anemia with ring sideroblasts (RARS) being a specific morphologic category. RS can also be seen with other morphologic categories such as; refractory cytopenias with multilineage dysplasia (RCMD), MDS-unclassifiable (MDS-U) and refractory anemia with excess blasts (RAEB-1/2). Amongst these categories, RARS is generally believed to have the best survival rate with the lowest risk of leukemic transformation and this has been attributed to the absence of associated dysplasia in more than one lineage. However, it has not been systematically shown whether or not the exact percentage of RS provides additional prognostic information in the context of an accurate morphologic categorization and risk stratification by karyotype or the revised IPSS (IPSS-R). Methods: 200 patients with primary MDS without excess blasts and ≥1% RS were seen at the Mayo Clinic from 1997 through 2007. All patients underwent bone marrow (BM) examination and cytogenetic evaluation at diagnosis and the pathology slides, including iron stains, were centrally re-reviewed to accurately quantify bone marrow (BM) RS percentage and to confirm WHO morphologic categories. Molecular profiling included analysis for IDH1, IDH2, JAK2 and MPL mutations. For the purposes of this study patients were divided into 4 categories based on the RS percentage; <5%, 5–14%, 15–50% and >50%. In addition, each patient was assigned IPSS and IPSS-R prognostic scores at diagnosis and risk stratified by karyotype according to the IPSS-R cytogenetic risk categories. Cox proportional regression method was used for multivariable analysis. Results: Of the overall 200 study patients, 140 (70%) were male and median age was 71 years (range, 17–90 years). At presentation, 34 (17%) patients were red cell transfusion-dependent. There were 56 (28%) patients with <5% RS (RCMD-52, MDS-U-14), 32 (16%) with 5–14% RS (RCMD-29, MDS-U-3), 79 (39%) with 15–50% RS (RARS-43, RCMD-29, MDS-U-2) and 33 (16%) with >50% RS (RARS-13, RCMD-17, MDS-U-3). Three patients (2%) were IDH1 mutant (RARS-1, RCMD-2), 17 (8%) IDH2 mutant (RARS-1, RCMD-14, MDS-U-2), 5 JAK2 V617F (3%) mutant (RARS-2, RCMD-3) and one patient with RCMD had the MPL W515L mutation. At a median follow-up of 33 months, 156 (73%) deaths and 24 (12%) leukemic transformations were documented. Median survivals were 63 months for MDS with >50% RS, 43 months for MDS with 15–50% RS, 35 months for MDS with 5–14% RS, and 14 months for MDS with <5% RS (p=0.005). In univariate analysis, additional risk factors included decreased hemoglobin, decreased platelet count, increased circulating blasts, WHO morphologic categories, red cell transfusion need, IPSS, IPSS-R and cytogenetic risk categories per IPSS-R. On a multivariable analysis only the IPSS-R prognostic score (p<0.0001) and the WHO morphologic categories (p=0.02 for RARS) retained significance. Univariate analysis disclosed significantly inferior leukemia-free survival (LFS) in patients with RCMD, thrombocytopenia, increased circulating blasts, and poor IPSS and IPSS-R prognostic scores. On multivariable analysis once again only the IPSS-R prognostic scores (p=0.02) and WHO morphologic categories (p=0.02) retained their significance. In other words, RS percentage, independent of WHO classification, as a continuous or categorical variable (i.e.: <5%, 5–14%, 15–50% & >50%) did not affect either overall survival or LFS. Conclusions: In MDS without excess blasts, once an accurate WHO morphologic categorization is made based on the presence or absence of multi-lineage dysplasia, there is no additional prognostic value for quantifying bone marrow ring sideroblasts. WHO classification and IPSS-R prognostic scores remain the most important factors in assessing patients with MDS. Disclosures: No relevant conflicts of interest to declare.