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Abstract Background : In primary myelofibrosis (PMF), ̴ 88% of patients harbor one of three "driver" mutations, with mutational frequencies of approximately 60%, 22% and 6% for JAK2, CALR and MPL, respectively. Other "non-driver" mutations have also been described in PMF and some of them and their number have been associated with inferior survival (Leukemia. 2014;28:1804). We applied next generation sequencing (NGS) with a broader panel of MPN-relevant genes, in order to identify additional mutations of prognostic relevance as well as obtain additional information regarding the prognostic value of 'number of mutations'. Methods: Targeted capture assays were carried out on bone marrow or whole blood DNA specimens obtained at time of referral for the following genes: TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, SUZ12, SRSF2, SF3B1, ZRSR2, U2AF1, PTPN11, Tp53, SH2B3, RUNX1, CBL, NRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF, and SETBP1. Paired-end indexed libraries were prepared from individual patient DNA using the NEB Next Ultra Library prep protocol on the Agilent Bravo liquid handler (NEB, Ipswich, MA/Agilent Technologies Inc, Santa Clara, CA). Capture libraries were assembled according to Nimblegen standard library protocol (Roche Nimblegen, Inc, Basel, Switzerland). Base-calling was performed using Illumina's RTA version 1.17.21.3. Genesifter® software was utilized (PerkinElmer, Danvers, Massachusetts) to analyze targeted sequence data. Nucleotide variants were called using the Genome Analysis Toolkit (GATK-Broad Institute, Cambridge, MA). 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: 180 PMF patients were evaluated (median age 63 years; 65% males). DIPSS-plus risk distribution was 32% high, 38% intermediate-2, 17% intermediate-1 and 13% low. Driver mutation distribution was 62% JAK2, 22% CALR, 9% triple-negative and 7% MPL. Karyotype was abnormal in 41% of patients and unfavorable in 12%. Mutations other than JAK2, CALR or MPL (i.e. "non-driver" mutations) were seen in 150 (83%) patients including 88% of "triple-negative" cases. 62 (34%) patients harbored one, 55 (31%) two, 16 (9%) three and 17 (10%) four or more. Mutational frequencies were: ASXL1 36%, TET2 18%, SRSF2 17%, U2AF1 17%, ZRSR2 11%, SF3B1 10%, DNMT3A 9%, CEBPA (9%), Tp53 7%, SETBP1 6%, CBL 5%, IDH1/2 5%, SH2B3 4%, CSF3R 4%, NRAS 4%, RUNX1 3% and ≤2% for SUZ12, KIT, PTPN11, NPM1 and EZH2. DIPSS-plus high/intermediate-2 risk patients displayed higher number of mutations (p=0.0004) and higher mutational frequencies for ASXL1 (p=0.02), SRSF2 (p=0.004) and CBL (p=0.02). Associations noted included JAK2 with U2AF1 (p=0.03), unfavorable karyotype with CBL (p=0.01) and normal karyotype with ZRSR2 mutations (p=0.04). At a median follow-up of 4 years, 111 (62%) deaths were documented. For examination of impact on survival, we considered 'number of mutations' and specific mutations with >2% frequency. Accordingly, in univariate analysis, survival was adversely affected by 'number of mutations' (Figure 1) and presence of ASXL1, SRSF2, IDH1/2, U2AF1, RUNX1 and CBL mutations. For multivariable analysis, we considered three categories (zero, 1-3 and ≥4) for number of mutations based on the results from univariate analysis (Figure 1); the results showed ≥4 mutations, 1-3 mutations, RUNX1, CBL, ASXL1 and SRSF2 mutations were independently associated with shortened survival; the respective HR (95% CI) were 4 (1.4-11.1), 3 (1.3-6.8), 2.9 (1.1-8.1), 2.8 (1.3-6.3), 1.8 (1.2-2.7) AND 1.7 (1.03-2.7). When the multivariable analysis was repeated including only the 150 patients with at least one non-driver mutation, the 'number of mutations' was no longer significant (p=0.35) but ASXL1, CBL, RUNX1 and SRSF2 mutations retained their significance. The prognostic relevance of ASXL1 and CBL continued to be apparent even after the addition of DIPSS-plus and driver mutation profile to the multivariable model. Conclusions: Mutations other than JAK2, CALR or MPL occur in more than 80% of patients with PMF, including those with "triple-negative" driver mutational status. The absence of such mutations is independently favorable for survival while the prognostic effect of their presence is influenced by ASXL1, CBL, RUNX1 and SRSF2 mutations. Figure 1. Figure 1. Disclosures Pardanani: Stemline: Research Funding.

Abstract Background Current risk stratification in Polycythemia Vera (PV), is designed to estimate the likelihood of thrombotic complications. In ECLAP large prospective study of 1638 PV cases diagnosed by Polycythemia Vera Study Group (PVSG) criteria, risk factors were age over 65 years and prior thrombotic events. After the JAK2V617F discovery, diagnosis has undergone substantial revision by the World Health Organization (WHO). Consequently, current risk factors determination should be described in patients diagnosed by the most recent recommendations and requires a large sample size with reliable and sufficiently long clinical course documentation. Accordingly, the current study includes 1545 patients with strictly WHO-defined PV diagnosed and followed-up in seven centers from Italy, Austria and the United States, belonging to the International Working Group for Myeloproliferative Neoplasm (MPN) Research and Treatment (IWG-MRT). Objective and design 1) Describe the rate of thrombosis in this population; 2) Report the sites and frequency of recurrences after a first arterial or venous event; 3) Identify separately risk factors for arterial versus venous thrombosis and develop practically useful prognostic models. Results 1. Presenting features The characteristics of the cohort recruited reflected well the heterogeneity of epidemiologic and clinical features that are found in the routine clinical practice of PV. Arterial and venous thrombosis history before or at diagnosis was documented in 246 (16%) and 114 (7.4%) patients, respectively. The frequencies of arterial and venous thrombosis were lower than those recorded in the ECLAP4 (27% and 11% respectively) but comparable to those reported in the recent Cyto-PV2 (arterial 17%, venous 12%) studies. The discrepancy might be partly related to earlier diagnosis in the post-ECLAP JAK2mutation era. 2. Thrombotic events after diagnosis Median follow-up was 6.9 years and treatment included aspirin (84%) and cytoreductive agents (73%), in addition to phlebotomy. Post-diagnosis total major thrombosis rate was 2.62% pt/yr, lower than reported in the ECLAP trial (4.4% pt/yr) but comparable to the Cyto-PV study (2.7% pt/yr) where management of cardiovascular risk factors was more intensive than in ECLAP. Arterial or venous thrombosis occurred in 184 (12%, rate: 1.59 % pts/yr) and 137 (9%, rate: 1.05 % pts/yr) patients, respectively. In addition, prior arterial events were associated in 75% of cases with subsequent arterial events and in the remaining 25% with venous thrombosis including splanchnic vein events. Moreover, patients with prior venous events had recurrences in venous district in 61% of cases and in the arterial district in 39%. 3. Risk factors for arterial versus venous thrombosis and prognostic models In multivariable analysis, arterial thrombosis was predicted by previous arterial event (HR 1.7, 95% CI 1.2-2.4), and hypertension (HR 1.6, 95% CI 1.2-2.2) while predictors of venous events were previous venous thrombosis (HR 2.6, 95% CI 1.5-4.4), and age ≥ 65 years (HR 1.7, 95% CI 1.2-2.5). We used the above-mentioned risk factors in order to develop prognostic models allowing to identify patients at different probability to develop arterial or venous thrombosis. For arterial thrombosis, the absence of previous arterial thrombosis and hypertension defined lower-risk (rate 1.27 % pts/yr) while the presence of one or both identified higher-risk patients (rate 2.04 % pts/yr). Previous venous event and/or age ≥ 65 years identified patients at higher risk of venous (rate 1.62 % pts/yr) thrombosis and, if absent, patients at lower risk (rate 0.78 % pts/yr). Conclusion The overall rate of thrombosis was half than in ECLAP and should be considered in future clinical trials. Of note, patients with a prior venous thrombosis had a more frequent subsequent recurrence in the venous district but a proportion of them (40%) had also arterial events; conversely, recurrences after previous arterial event were also registered in the leg and non-leg veins in 30 % of cases, indicating that in PV, venous and arterial thrombosis may share some common pathogenetic mechanisms. These findings may have practical implications and suggests antiplatelet therapy and anticoagulant therapy as the favorite choice to prevent arterial and venous thrombosis, respectively, but also suggests studies of antithrombotic drug combination. Disclosures No relevant conflicts of interest to declare.

Abstract Background : Prognosis in chronic myelomonocytic leukemia (CMML) is dire and blast transformation (BT) is a main cause of death. Current risk factors and prognostic models are mostly designed to predict overall (OS) and not leukemia-free (LFS) survival. Established risk factors for OS in CMML include anemia, thrombocytopenia, leukocytosis, monocytosis, presence of circulating immature myeloid cells (IMC), peripheral blood (PB) and bone marrow (BM) blasts, lymphocytosis, ASXL1 mutations, high risk abnormal karyotype and advanced age. In the current study, we examined risk factors for BT in CMML and survival outcome after BT. Methods : All CMML patients seen at our institution and with complete presentation data were considered. Clinical and laboratory variables, including cytogenetic information, at time of CMML diagnosis and also at time of BT were collected. DNA analysis for SRSF2, SF3B1, U2AF1, ASXL1 and SETBP1 mutations was carried out on specimens obtained at time of CMML diagnosis. We used both conventional and revised (see accompanying ASH 2014 abstract) cytogenetic risk models to query their influence in the current study population. Results: Clinical and laboratory features at time of CMML diagnosis: 277 consecutive patients (median age 71 years, range 18-91; 67% males) with WHO-defined CMML were considered. 236 (85%) patients had CMML-1 and the remainder CMML-2. According to the molecular Mayo risk model, 86 (33%) were high, 77 (29%) were intermediate-2, 77 (29%) were intermediate-1 and 24 (9%) were low risk. Mutational frequencies were 40% for ASXL1 (n evaluable =264), 44% for SRSF2 (n evaluable =263), 5% for SETBP1 (n evaluable =263). According to the revised Mayo-French consortium cytogenetic risk model, karyotype was high risk in 5%, intermediate in 21% and low risk in 74%. Risk factors for blast transformation (BT) : At a median follow-up of 16.2 months for all 277 patients from time of CMML diagnosis, 204 (74%) deaths and 38 (14%) BTs were documented. Median time from CMML diagnosis to BT was 13.3 months. In univariate analysis, female sex, younger age, lower hemoglobin, higher leukocyte count, higher absolute monocyte count, higher lymphocyte count, presence of circulating immature myeloid cells (IMC), increased peripheral blood (PB) blast %, increased bone marrow (BM) blast %, BM blasts ≥10%, CMML-2, abnormal karyotype, high risk karyotype per the Spanish or Mayo-French consortium, and higher risk disease per Mayo, MDACC or Dusseldorf models were all associated with higher risk of BT. In contrast, ASXL1 (p=0.84), SRSF2 (p=0.28) or SETBP1 (p=0.97) mutations were not found to be significant. In multivariable analysis, only PB blast % (p<0.0001), presence of PB IMC (p=0.004) and younger age (p=0.03) remained significant. Outcome of blast transformation : At time of BT, karyotype was abnormal in 58% and clonal evolution from time of CMML diagnosis was documented in 13 (36%) of 36 evaluable patients. Among the 38 patients with BT, induction chemotherapy was documented in 12 patients and allogeneic stem cell transplant (ASCT) in 6. At a median follow-up of 4.2 months from time of BT, 34 (89%) of the 38 patients with BT had died. Among the four patients who were alive at the time of this writing, 3 had received induction chemotherapy and two ASCT. Median survival of patients with BT, from time of disease transformation, was 4.5 months with 1, 2 and 3 year survival rates of approximately 30%, 16% and 3%, respectively. In univariate analysis, higher leukocyte count (p=0.01) and not receiving induction chemotherapy (p=0.03) were associated with shortened survival. Neither ASXL1 nor SRSF2 mutations affected survival after BT. Conclusions : Blast transformation in CMML occurs in approximately 14% of patients followed for a median of less than 1.5 years. Presence of circulating immature cells and percentage of peripheral blood blasts are the major determinants of BT in CMML. Genetic risk factors for overall survival did not appear to influence leukemia-free survival. Prognosis of post-CMML BT is dismal with median survival of less than 6 months and a 3-year survival of less than 5%. Outcome was better in patients receiving induction chemotherapy followed by ASCT. Disclosures No relevant conflicts of interest to declare.

Abstract BACKGROUND: Our group has periodically analyzed (Silverstein et. al. JAMA1974;227(4):424 to Tefferi et. al. Blood2000;95(7):226) presentation, peri-operative and long-term outcomes from splenectomy for myelofibrosis with myeloid metaplasia (MMM) in an attempt to improve patient selection and management. We now report on an additional 91 patients (pts) to see if control of post splenectomy thrombocytosis, and modern techniques, have diminshed morbidity and mortality. Additionally, we provide long-term follow-up on prior cohorts across a twenty-eight year period. METHODS: A comprehensive retrospective analysis of presentation, peri-operative and long-term outcomes in a single institution experience with splenectomy in pts with MMM from 1976–2004. RESULTS: Presplenectomy : 314 MMM pts (171 men 54%; 208 (66%) with agnogenic myeloid metaplasia) were included. Presplenectomy 55% of pts presented with a Lille MMM prognostic score of ≥1, 90 pts (29%) failed a prior therapy for splenomegaly and pre-operatively had median spleen size of 20cm below the left costal margin (95%CI 18.7–20.4). Perioperative Outcomes : Pts were splenectomized (1976–1985 (Decade 1(D1)) (n=90 (29%)); 1986–1996 (D2) (n=133 (42%)); 1997–2004 (D3) (n=91 (29%)) a median of 27 months (range 0–389 months) after the diagnosis of MMM (age at diagnosis (median 65 (range 19–83)) with a median splenic mass 2700 gm (range 300–11750)). Primary indication for surgery included mechanical symptoms n=156 (49%), anemia n=78 (25%)), portal hypertension n=47 (15%), or thrombocytopenia n=33 (11%). Peri-operative complications occured in 103 pts (33%) and included infection (n=30), thrombosis (n=34), and bleeding (n=48; 37 required a second laparotomy) of which 28 (9% of all pts) were fatal (30 day mortality (D1=10%, D2=9%, D3=7.7% (p=NS)). Overall length of hospital stay (D3) was median of 8 days (range 3–63), during which 6 pts (7%) required platelet pheresis (1–8 times; for platelet counts of 870–2400 x 109/L) and/or acute platelet lowering agents (n=12; 13%), respectively. Additionally, median post-splenectomy increases in leukocyte and platelet count were 8 x 109/L (range −65 to 190.2) and 127 x 109/L (range −270 to 1221), respectively. Long-Term Outcomes and Survival : Amongst the recent cohort (n=91) 69 pts (76%) experienced a palliative benefit for their primary surgical indication for a median of 12 months (range 1–91). Post splenectomy rates of thrombocytosis (21%), accelerated hepatomegaly (8%), and leukemic transformation (11%) did not vary across the three decades analyzed. Overall survival (n=263 (84%) have expired)) from the diagnosis of MMM and splenectomy was 62 months (95%CI 54–66) and 19 months (95%CI 14–22), respectively. Survival after splenectomy was not significantly different by type of MMM, surgical indication, Lille MMM prognostic score, leukemic transformation or decade performed. However, patients with preoperative thrombocytopenia (<50 x 109//L (p<0.0001) or <100 x 109/L (P=0.006)) clearly had a decrease in survival, although this was independent of the development of peri-operative complications. CONCLUSIONS: Splenectomy for MMM remains a procedure with significant peri-operative risks, but potential for palliating symptoms. Decreased survival in thrombocytopenic pts probably arises from advanced disease as opposed to increased complications. The lack of improvement in overall post-splenectomy survival across the study period may not be attributable to peri-operative issues but a reflection on the failure of medical therapy to improve survival in MMM.

Abstract Abstract 430 Background: The Dynamic International Prognostic Scoring System (DIPSS-plus) uses eight risk factors to predict overall survival (OS) in primary myelofibrosis (PMF): unfavorable karyotype, peripheral blood (PB) blast count ≥1%, platelet count <100 × 109/L, white blood cell count (WBC) >25 × 109/L, hemoglobin level <10 g/dL, red blood cell transfusion need, constitutional symptoms, and age >65 years (JCO 2011;29:392). Among these risk factors, karyotype holds a dominant role in predicting both overall (OS) and leukemia-free (LFS) survival (Blood 2011;118:4595). More recently, certain PMF-associated somatic mutations (IDH, EZH2, ASXL1, SRSF2) have been reported (in print or at recent meetings) to carry prognostic relevance for OS or LFS in PMF while other mutations (JAK2, MPL, TET2, SF3B1) did not. The purpose of the current study was to examine the individual and combined prognostic relevance of the former in the context of cytogenetic risk stratification and DIPSS-plus. Methods: An updated Mayo Clinic database of 1008 karyotypically- and DIPSS-plus-annotated patients with PMF was used to identify study patients in whom DNA at time of referral was available for mutation analysis. OS and LFS were calculated from the time of referral, which was also the time of study sample collection. Results: In an ongoing project, a total of 397 PMF patients have so far been studied for IDH1/2 (n=367), SRSF2 (n=355), ASXL1 (n=260) and EZH2 (n=43) mutations. The corresponding mutational frequencies were 5.4% (20/367), 14.4% (51/355), 31.2% (81/260) and 7% (3/43). These mutations were not exclusive of either each other or JAK2 or MPL mutations and a significant clustering was noted between SRSF2 and IDH mutations; IDH mutational frequency was 21% (10/48) in SRSF2-mutated versus 3% (9/279) in unmutated cases (P<0.0001). Mutational hotspots were P95 in exon 1 for SRSF2 and exons 1, 2 and 5 for ASXL1. OS and LFS effect have so far been examined for ASXL1, SRSF2 and IDH 1/2 mutations; in univariate analysis, all three mutations significantly affected OS (ASXL1 RR 1.9, 95% CI 1.4–2.7; SRSF2 RR 1.7, 95% CI 1.2–2.5; IDH 1/2 RR 1.8, 95% CI 1.1–3.1) whereas only IDH 1/2 (RR 5.5, 95% CI 2.5–11.8) and SRSF2 (RR 3.5, 95% CI 1.7–7.0) but not ASXL1 (p=0.29) mutations affected LFS. The observed prognostic relevance for OS was sustained during multivariable analysis that included ASXL1 with either SRSF2 or IDH mutations, as co-variates, whereas both IDH and SRSF2 mutations remained significant for LFS. Based on the above observations, study patients were classified into two groups: i) unmutated for ASXL1, SRSF2 and IDH1/2 (all unmutated; n=146) and ii) mutated for at least one of the three mutations (one or more mutated; n=130); the latter group constituted 62%, 52%, 18% and 27% of DIPSS-plus high, intermediate-2, intermediate-1and low risk patients, respectively (p<0.0001). The corresponding rates for patients with favorable and unfavorable karyotype were 47% and 43% (p=0.7) and for normal vs. abnormal karyotype were 53% and 36% (p=0.008). The “all unmutated group”, compared to the “one or more mutated group” enjoyed a significantly superior OS (RR 0.5, 95% CI 0.4–0.7) and LFS (RR 0.3, 95% CI 0.2–0.7) that was DIPSS-plus-independent for OS (p=0.03) and karyotype-independent for LFS (p=0.004). The prognostic value of distinguishing “all unmutated” from “one or more mutated” patient groups was most evident for DIPSS-plus intermediate-2 or lower risk groups (p=0.002) and its added value for high-risk group was limited (p=0.23) (Figure). Conclusions: A concomitant analysis strategy reveals that SRSF2, ASXL1 and IDH mutations carry inter-independent prognostic relevance for overall or leukemia-free survival in PMF. Any one of these three mutations is more likely to occur in patients with normal karyotype and such an event predicts shortened survival that is not accounted for by DIPSS-plus and a higher risk of leukemic transformation that is independent of karyotype. The current study suggests that such information might be useful in treatment decisions involving intermediate-2 or lower risk patients. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 755 Background: TG101348 is a potent, orally bioavailable, JAK2-selective small molecule inhibitor, that is currently being evaluated in a Phase I study for the treatment of myelofibrosis. Data from the dose escalation cohort (n=28; 30-800mg administered as a single daily dose) showed dose-linear plasma exposure, with mean elimination T1/2 at steady state ranging from 20 to 52 hours. The dose-limiting toxicity was asymptomatic grade 3 or 4 amylasemia/lipasemia that was reversible, and the maximum tolerated dose (MTD) was 680mg. The most frequent non-hematological toxicities were mild nausea, vomiting, and/or diarrhea that were easily controlled or resolved spontaneously. Grade 3/4 neutropenia and thrombocytopenia were observed in 14% and 25% of patients, respectively. TG101348 had activity in reducing spleen size, leukocyte count, and JAK2V617F (VF) allele burden. Here, we present updated results with a focus on data from the dose confirmation cohort who initiated treatment at a dose of 680mg/day. Results: Fifty nine patients (median age=66 years; range 43-86) have been treated – 28 in the dose escalation phase, and 31 in the dose confirmation phase. Overall, 44 patients had PMF, 12 post-PV MF, and 3 post-ET MF; 86% were VF-positive. Median palpable spleen size was 18cm and 22 patients were RBC transfusion-requiring at study enrollment. After a median follow-up of 12 weeks (range <1-76), 18 (31%) patients have discontinued treatment due to toxicity (n=7; thrombocytopenia=3, neutropenia=1), comorbidities (n=5), withdrawal of consent (n=4), or non-compliance/lack of response (1 each). The remaining 41 patients are currently at the following dose levels: 680mg (n=14), 520-600mg (n=16), 360-440mg (n=10), and 240mg (n=1). The cumulative drug exposure to date is 362 patient-months; exposure at or above MTD (≥680mg) is 154 patient-months. Forty patients (68%) started treatment at ≥680mg. Toxicity: TG101348 is well tolerated. Of the patients who started at ≥680mg, Gr3/4 neutropenia was observed in 15/0% and Gr3/4 thrombocytopenia in 20/10%. Twenty four (60%) patients did not require RBC transfusions at baseline (median Hgb=9.6g/dL; range 7.4-13.1); of these, 42% and 8% of patients developed Gr3 and Gr4 anemia, respectively. The majority of patients who started at ≥680mg developed mild nausea (1 Gr3), vomiting (1 Gr3), and/or diarrhea (3 Gr3) that were self-limited or easily controlled. Other non-hematological toxicities included Gr1/2 transaminitis (38%), Gr1/2 serum creatinine elevation (38%), and asymptomatic hyperlipasemia (33%). Efficacy: Thirty three patients who started at ≥680mg have completed at least 3 cycles of treatment; at 3 months, reduction in palpable spleen size (baseline median=18cms; range 6-32) was at least 50% in 22 (67%) patients; the spleen became non-palpable in 9 (27%) patients. All 21 patients with leukocytosis at baseline (WBC range 11 to 203 ×109/L) who started at ≥680mg have experienced a marked reduction in their WBC count (range 4 to 90); 70% had a normal WBC count at their last follow-up visit. Overall, 48 of the 51 VF-positive patients completed at least 1 cycle and were evaluable for response in VF allele burden; at last available follow-up, the median decrease in granulocyte mutant allele burden was 48%; 21 (44%) patients have had a ≥50% reduction, and in the group who started treatment at ≥680mg, 48% have had a ≥50% reduction. Of those evaluable, there was clinically significant benefit or resolution of constitutional symptoms, including early satiety, fatigue, cough, pruritus, and night sweats. Conclusions: TG101348 continues to be well tolerated in patients with myelofibrosis. Spleen and leukocyte responses are frequent, observed early, and produce substantial clinical benefit for patients. These responses are associated with significant decrease in VF allele burden and point to activity of TG101348 against the malignant clone in myelofibrosis. Disclosures: Pardanani: TargeGen: Research Funding; Cytopia: Research Funding. Off Label Use: Data from ongoing clinical trial will be presented. Gotlib:TargeGen: Research Funding. Jamieson:Merck: Research Funding; Pfizer: Research Funding; Wintherix: Consultancy; TargeGen: Research Funding; Celgene: Research Funding. Cortes:Targegen: Research Funding. Stone:Cephalon: ad hoc advisory board. Silverman:TargeGen: Consultancy. Shorr:TargeGen: Employment, Equity Ownership. Gilliland:Merck: Employment. Tefferi:TargeGen: Research Funding.

Abstract Abstract 424 Background: Azacitidine for injection has been shown to prolong overall survival in patients (pts) with higher-risk myelodysplastic syndromes (MDS) compared with conventional care regimens (CCR) (Lancet Oncol, 2009). An oral formulation of azacitidine (CC-486) is in development. Oral azacitidine may maximize convenience, eliminate injection-site reactions, and if administered in extended dosing schedules, may enhance and prolong the therapeutic effects of azacitidine. Oral azacitidine administered once-daily (QD) for 7 days (d) of repeated 28d cycles has been shown to be bioavailable, biologically and clinically active, and well-tolerated in pts with MDS and acute myeloid leukemia (Garcia-Manero, J Clin Oncol, 2011). Preliminary evidence suggests that extending oral azacitidine dosing to 14d or 21d of the 28d cycle may enhance pharmacodynamic and epigenetic activity (Laille, Leuk Res, 2011). Purpose: To evaluate hematologic response and safety associated with extended dosing regimens of oral azacitidine in pts with lower-risk MDS. Methods: This ongoing, multicenter, phase 1 study, enrolled pts with lower-risk (IPSS Low or INT-1) MDS who were RBC transfusion dependent (TD) and/or thrombocytopenic (average platelet count ≤50,000 within 56d prior to the first dose) at baseline. Pts were sequentially assigned to receive oral azacitidine 300mg QD for either 14d or 21d of repeated 28d cycles. Hematologic assessments were made every 2 weeks. Hematologic response was assessed using IWG 2006 criteria (Cheson, Blood, 2006). Adverse events (AEs) were graded using NCI-CTCAE version 3.0. Results: At data cut-off (May 18, 2012), 53 pts with lower-risk MDS had enrolled (300mg oral azacitidine QDx14d, n=26; QDx21d, n=27). Demographic and disease characteristics at baseline were similar in the 14d and 21d treatment cohorts (Table 1). Median (range) hematology counts at baseline were Hgb 8.7 g/L (6.0–13.0), ANC 1.6×109/L (0–30.3), and platelets 56.0×109/L (6.0–564.0). At study entry, 40% of pts had received no prior MDS treatment (except transfusions), 45% had received erythropoiesis-stimulating agents, and 15% had received WBC growth factors. The number of oral azacitidine treatment cycles received ranged from 1 to 12 (median numbers of oral azacitidine cycles were 6 in the QDx14d and 4 in the QDx21d cohorts). Four pts in the 21d cohort and 1 pt in the 14d cohort received reduced oral azacitidine doses (200mg QD). Overall, 10 pts discontinued the study, including 6 pts (3 pts in each cohort) who discontinued due to AEs that may have been treatment-related (gastrointestinal [n=2] or intracranial [n=1] hemorrhage, febrile neutropenia [n=1], pneumonia [n=1], thrombocytopenia [n=1]). Overall response rates (ORR), which included complete (CR) and partial remission (PR), any hematologic improvement (HI), and transfusion independence (TI), ranged from 38.5% in the QDx14d cohort to 29.6% in the QDx21d cohort, and RBC TI was achieved by 47% and 33%, respectively, of pts who were RBC TD at baseline (Table 2). For pts who received at least 4 cycles of oral azacitidine (14d, n=19; 21d, n=14), ORR was 47.4% in the 14d and 50.0% in the 21d cohorts, and RBC TI rates in RBC TD pts (n=16) were 67% in the 14d and 57% in the 21d cohorts. The most frequent (≥5%) grade 3/4 hematologic AEs in the QDx14d cohort were anemia (11.5%), thrombocytopenia (11.5%), and neutropenia (7.7%); and in the QDx21d cohort were neutropenia (14.8%), anemia (7.4%), and febrile neutropenia (7.4%). Most frequent grade 3/4 non-hematologic AEs were gastrointestinal, including vomiting (7.7%) in the QDx14d cohort, and diarrhea (11.1%) and vomiting (7.4%) in the QDx21d cohort. Conclusions: Oral azacitidine 300mg QD administered in extended dosing schedules of 14d or 21d of repeated 28d cycles was effective and well-tolerated in these pts with lower-risk MDS. Beside hematologic AEs, the most frequently observed AEs with oral azacitidine were gastrointestinal and were manageable. Efficacy and safety outcomes with 300mg QD oral azacitidine were generally comparable between the 14d and 21d extended dosing regimens. Based on these data, oral azacitidine administered once-daily in extended dosing schedules is active and well-tolerated and warrants further investigation in randomized, controlled trials. Disclosures: Garcia-Manero: Celgene: Research Funding, Speakers Bureau. Gore:Celgene Corporation: Consultancy, Research Funding. Scott:Celgene Corporation: Honoraria, Research Funding, Speakers Bureau. Hetzer:Celgene Corporation: Employment, Equity Ownership. Kumar:Celgene Corporation: Employment, Equity Ownership. Skikne:Celgene: Employment, Equity Ownership.

Abstract Abstract 423 Background: Cytogenetic abnormalities at diagnosis are seen in 20–70% of patients with myelodysplastic syndromes (MDS). The International Prognostic Scoring System -revised (IPSS-R) classifies these cytogenetic abnormalities into five prognostic groups: very good (del(11q), ΠY with overall survival (OS) of 61 months); good (normal, del(20q), del(5q), del(12p) with OS 49 months); intermediate (+8,7q-, i(17q),+19,+21 with OS 26 months); poor (der(3)q21/q26, −7, complex with 3 abnormalities with OS 16 months); and very poor (complex with >3 abnormalities with OS of 6 months) (Schanz J, JCO 2012, Greenberg P, Blood 2012). Monosomal karyotype (MK) is defined as the presence of two or more autosomal monosomies or a single monosomy associated with a structural abnormality. We have previously shown that MK identifies a prognostically worse subgroup of MDS patients with a complex karyotype (Patnaik M, Leukemia 2010). Objectives: Methods: The Mayo Clinic database for MDS was used to identify patients in whom bone marrow histologic and cytogenetic information was obtained at the time of diagnosis. WHO criteria were used for MDS diagnosis and leukemic transformation. Results: i) Patient characteristics: A total of 1,014 patients met the above-stipulated criteria. Median age of the cohort was 71 years (range: 18–98 years) with 684 (67%) males. 819 patients (81%) had primary MDS with 195 cases (19%) of therapy-related MDS. 759 patients (75%) are dead at a median follow-up of 18 months (range: 0–223 months) with leukemic transformation documented in 125 cases (12%). IPSS-R risk distribution was as follows: very low (n=149, 15%), good (n=261, 26%), intermediate (n=221, 22%), high (n=131, 13%), and very high (n=252, 25%). ii) IPSS-R karyotype (Entire cohort; n=1,014): The five cytogenetic groups by IPSS-R were as follows: very good (n=43, 4%), good (n=577, 57%), intermediate (n=187, 18%), poor (n=83, 8%), and very poor (n=124, 12%). We evaluated the prognostic impact of the above groups by Kaplan Meier and Cox proportional hazards analysis. OS for the five cytogenetic groups was as follows: good (36 months), very good (28 months), intermediate (20 months), poor (13 months), and very poor (6 months) (Figure 1). OS of patients with very good karyotype was significantly better than patients with very poor and poor karyotype (p<0.0001, RR 3.2 and p=0.04, RR 1.6 respectively) but similar to patients with intermediate and good karyotype (p=0.22 and 0.28 respectively). iii) IPSS-R karyotype (Primary MDS; n=819): Cytogenetic risk groups by IPSS-R were as follows: very good (n=38, 5%), good (n=512, 63%), intermediate (n=153, 19%), poor (n=46, 6%), and very poor (n=70, 9%). OS of the five groups was as follows: good (39 months), very good (23 months), intermediate (21.5 months), poor (14.5 months), and very poor (6.5 months). OS of patients with very good karyotype was significantly better than patients with very poor karyotype (p=0.0002, RR 2.5) but similar to patients with poor, intermediate and good karyotype (p=0.23, 0.26 and 0.18 respectively). iv) Monosomal karyotype: MK was identified in 153 (15%) cases of which 4 cases were intermediate, 42 poor, and 107 very poor by IPSS-R. OS of patients with MK was similar to those with very poor karyotype (6 months). In addition, OS of patients with very poor without MK was akin to patients with poor without MK (p=0.21). Furthermore, among primary MDS we identified 83 (8%) cases of MK of which 4 were intermediate, 21 were poor, and 58 very poor by IPSS-R. We confirmed that OS of patients with MK was similar to patients with very poor karyotype (6.5 months). Moreover, survival of patients with very poor without MK was similar to patients with poor without MK (p=0.51). Conclusions: In this independent analysis of 1,014 patients with MDS, IPSS-R cytogenetic risk characterization failed to discriminate between very good, good and intermediate prognostic groups. In addition, patients with very poor without MK have a similar prognosis to patients with poor without MK and should be included in the poor category whereas the presence of MK should warrant inclusion into the very poor risk category. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 925 Diffuse large B-cell lymphomas (DLBCL) consists of biologically distinct subtypes: Germinal Center B-cell (GCB)–like and Activated B-cell (ABC)-like. Studies to date suggest that patients with tumors that have ABC features have an inferior survival; therefore, it is important to develop new therapies specifically for ABC-type DLBCL based on mechanistic differences. It has been shown that the ABC-DLBCL has high levels of total and phosphorylated STAT3 (pSTAT3) protein and this STAT3 activity may play a role in the survival of ABC-DLBCL cells. Indeed, when ABC and GCB cell were incubated with the novel JAK2 inhibitor TG101348 (TargeGen, San Diego, CA) we found potent cytotoxicity (LD50 2μM) in ABC DLBCL but not in GCB DLBCL. This provides further rationale to study the regulation of STAT3 in the survival of ABC type DLBCL cells. In studies of HDAC expression in ABC DLBCL cells, we found over-expression of the class 1 histone deacetylases, specifically HDAC1 and HDAC3. We then performed co-immunoprecipitation experiments to learn the functional interaction between STAT3 and HDACs. We found that STAT3 formed complexes with HDAC1 to a greater extent than HDAC3. We also showed that STAT3 did co-precipitate with HDAC1 suggesting a reciprocal relationship between these two proteins. These results suggested that HDAC1 (but not HDAC3) might be involved in the regulation of STAT3. The HDAC inhibitor LBH589 (LBH, Novartis Pharm) was more cytotoxic to ABC (LD50, 10-20nM) than GCB (LD50, 50-100nM) and was shown to dephosphorylate STAT3. These results led to experiments designed to determine the effect of LBH on STAT3 acetylation. When ABC cells were incubated with LBH, co-immunoprecipitation data showed a dose-dependent increased in acetylation of STAT3 without significant change on the levels of STAT3 immunoprecipitated. Next, we asked that how LBH is dephosphorylating STAT3. One potential mechanism is activation of a protein phosphatase. Immunoprecipitation of protein phosphatase 2A (PP2A) in the lysates of LBH-treated cells followed by western blotting of STAT3 demonstrated a positive correlation between LBH dose and amount of STAT3 co-immunoprecipitated with PP2A. Our data show that when LBH is incubated with ABC cells, the LBH increases binding of PP2A to STAT3 and the increased phosphatase activity turns off STAT3 signaling. Since both HDAC inhibitor (LBH) and the JAK2 inhibitor (TG) had selective cytotoxic effects on ABC cells, and since both agents dephosphorylate STAT3, we combined the two drugs. We found that the combination exhibited enhanced inhibitory effects on the growth of ABC cells and was able to alter the association of STAT3 with HDAC1 and subsequently dephosphorylate STAT3 to greater extent than either agent alone. Furthermore, we knocked down HDAC1 expression (through siRNA) in OCILy10 (ABC) cells and showed that inhibition of HDAC1 inhibits constitutively activated STAT3. This data suggests that inhibiting HDAC1 expression results in STAT3 dephosphorylation. In summary, we have demonstrated that a key consequence of HDAC expression in ABC cells is continuous activation of STAT3. These data indicate that HDACs activate STAT3 and that PP2A may be important in maintaining STAT3 in the activated state in the ABC cells. This study describes a new mechanism by which STAT3 mediates ABC cell survival and provides the rationale for targeting ABC type DLBCL by simultaneously inhibiting HDAC activity and STAT3 activation by pharmacologic grade inhibitors such as LBH589 and TG101348, which are currently in clinical trial. A phase 1 and 2 trial of JAK2 inhibitor, TG101348 for ABC/GCB type DLBCL is planned. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3790 Background: Chronic myelomonocytic leukemia (CMML) is a clonal stem cell disorder characterized by: persistent peripheral blood (PB) monocytosis (>1 × 10(9)/L), absence of BCR-ABL1 fusion, absence of rearrangement of the PDGFRA/B genes, <20% bone marrow (BM) & PB blasts, and dysplasia involving one or more myeloid cell lines. It is clinically considered to be an overlapping syndrome with myelodysplastic and myeloproliferative features. The natural history and prognostic features of CMML are not well defined with the MD Anderson prognostic score (MDAPS) and the Spanish risk stratification by cytogenetics being the two major prognostic tools currently being used in clinical practice. Methods: 227 patients with WHO defined CMML were seen at the Mayo Clinic from 1997 through 2007. All patients underwent bone marrow (BM) examination and cytogenetic evaluation at diagnosis. We evaluated the prognostic relevance of several clinical and laboratory parameters including those previously identified by the MDAPS (Blood 2002;99:840) and the Spanish cytogenetic risk stratification (Haematologica 2011;96:375). Results: Among the 227 study patients, 153 (67%) were males and median age was 71 years (range, 17–90 years). There were 192 (85%) patients with CMML-1 & the remainder had CMML-2. At a median follow-up of 15 months, 166 (73%) deaths and 33 (14.5%) leukemic transformations were documented. Median survivals were 22 months for CMML-1 and 14 months for CMML-2. In univariate analysis, significant risk factors for survival included decreased hemoglobin level, decreased platelet count and increased levels of white blood cells (WBC), absolute neutrophils (ANC), absolute monocytes (AMC), absolute lymphocytes (ALC), PB blasts, BM blasts and presence of circulating immature myeloid cells (IMC; inclusive of PB blasts). However, on multivariable analysis that included the aforementioned Spanish cytogenetic risk stratification, only increased AMC (>10 × 10(9)/L, RR 2.5, 95% CI 1.7–3.8), presence of circulating IMC (RR 2.0, 95% CI 1.4–2.7), decreased hemoglobin (<10 g/dL; RR 1.6, 99% CI 1.2–2.2), and decreased platelet count (<100 × 10(9)/L; RR 1.4, 99% CI 1.0–1.9) retained significance. Using these four independent risk factors, we prepared a new prognostic risk model that performed better than both the MDAPS and the Spanish cytogenetic risk models (Figure). The Mayo risk model was also predictive of leukemic transformation: high risk RR 4.9 (95% CI 1.9–12.8) and intermediate risk RR 2.6 (1.1–5.9). Individual parameters of independent significance for leukemic transformation included PB blast count and AMC >10 × 10(9)/L. Conclusions: Absolute monocyte count is the strongest predictor of survival in CMML. Other independent risk factors include circulating immature myeloid cells, anemia and thrombocytopenia. A risk model based on these four risk factors is effective in predicting both overall and leukemia-free survival and outperforms both the MDAPS and risk stratification by cytogenetics. Disclosures: No relevant conflicts of interest to declare.