Tefferi, Ayalew

Abstract Introduction: CMML is a heterogeneous malignancy characterized by peripheral monocytosis and a propensity for AML transformation. Given the low incidence of CMML and its broad range complexity, a large disease-specific data set is necessary to discern independent co-variates predictive of disease behavior. Several prognostic models derived from smaller data sets have been developed to stratify CMML patients into distinct groups that are predictive for OS. However, the validity of these models in a large international data set has never been investigated. Here, we present the largest International CMML data base established to date and report key baseline characteristics predictive for disease outcome. We externally validate and perform a detailed statistical comparison between the International Prognostic Scoring System (IPSS), Revised (R)-IPSS, Global MD Anderson Scoring System (MDASC), MD Anderson Prognostic Score (MDAPS), Dusseldorf Score (DS), Mayo, and Spanish Scoring Systems (CPSS). Methods: Data were abstracted by each contributing institution and deposited for central data review at the Moffitt Cancer Center. Manual review of cases was performed to ensure data quality prior to analysis. Only WHO defined CMML was included. The primary objective of this study was to establish an international CMML data set and validate the above models calculated at the time of presentation to each center. All prognostic models were calculated as previously described. The Kaplan–Meier (KM) method was used to estimate median OS and the log rank test was used to compare KM survival estimates with SPSS version 21.0. Random Forest Survival (RSF) and ROC analysis was done with R. Results: Between July 1981 and June 2014, 1832 CMML patients were captured in the International CMML database. Median age at diagnosis was 70 (16-93) years with a male (67%) predominance. By the WHO classification, the majority of patients had CMML-1 (79.9% vs. 20.1%) and most patients were evenly subcategorized as MPN-CMML (49.8%) versus MDS-CMML (50.2%) by FAB criteria. Splenomegaly was demonstrable in 25%. Most patients had favorable cytogenetics by both IPSS (70%) and CPSS (67.6%) classification schemas. Mean BM blast % was 5.6 and mean monocyte count was 4.85x103/dL. Median OS of the entire data set was 31.6 months. All tested prognostic models were valid and able to predict OS (p<0.0001). To compare relative model performance, 1013 complete cases with sufficient data to calculate all risk models were compared using time dependent receiver operator characteristic (ROC) curves and their area under the curves (AUC). ROC curves were calculated for OS at 36 months. The R-IPSS model had the highest AUC (0.694) while the DS model had the lowest (0.635). The difference in AUC between the R-IPSS and DS models was statistically significant (p = 0.003) whereas there was no significant difference from other models tested. To determine which models were most vulnerable to reclassification from low-risk to higher risk, we calculated a vulnerability score defined by the number of models able to up-stage low-risk disease in more than 15% of cases. Using this metric, the Mayo and MDASC scores were least vulnerable to up-staging by other models. Lastly, we used an orthogonal method for determination of variable importance (RSF for OS at 36 months). RSF is an ensemble method of classification and regression tree methodology. Ranking of variables by their weighted importance determined by this procedure identified the top five variables were hemoglobin, circulating blasts, platelet count, number of cytopenias, and karyotype. A new risk model was developed using the results of the RSF with an AUC of 0.720, which was comparable but not significantly different than other modern scoring systems. Conclusions: This represents the largest international CMML-specific data set derived from eight unique centers of excellence. All modern prognostic models were valid and performed comparably. Despite novel strategies for variable importance discovery, an improved model could not be constructed. This data suggest that the power of clinical variables has reached an asymptote and highlights the need for incorporation of molecular and other novel biological features. This data set is currently being populated with molecular data and is being leveraged to test published CMML molecular prognostic models which will be disclosed at the time of presentation. Disclosures Garcia-Manero: Epizyme, Inc: Research Funding. Fenaux:Celgene, Janssenm, Novartis: Research Funding. Kantarjian:ARIAD, Pfizer, Amgen: Research Funding. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen Corp: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity's Board of Directors or advisory committees.

Abstract Activating mutations in colony stimulating factor 3 receptor gene (CSF3R) have recently been identified as specific driver mutations in chronic neutrophilic leukemia (CNL)(NEJM 2013;368:1781). We sought to outline the clinical and laboratory characteristics and natural history of this molecularly defined entity. Methods : An IRB- approved retrospective analysis of all cases of CSF3R-mutated WHO-defined CNL was undertaken. Methods of mutation analysis have been previously published. (Leukemia 2013; 27: 1870) Results : From 10/1995 to 3/2013, fourteen patients (median age 67 years; 57% male) with CSF3R-mutated CNL were evaluated at the Mayo Clinic. Most of the CSF3R mutations identified occurred in isolation: T618I (n=11), M696T (n=2) and I598I SYN (n=1), although one T618I mutant patient also carried a cytoplasmic domain mutation (2341_2342insC ). All patients were BCR/ABL negative. Five carried a SETBP1 mutation: G870D (n=2), D868N (n=2) and G872R (n=1), all of whom carried the T618I mutation. Only one of 10 tested carried a CALR mutation (E398D) and none of 13 the JAK2 V617F mutation. Apart from fatigue, most patients were asymptomatic at diagnosis and leukocytosis had been mostly an incidental laboratory finding. Ten (71%) patients had preceding chronic leukocytosis, median duration 12.5 months (range 5-84), 5 reported weight loss, which was accompanied by fevers/ sweats in 3. Five (36%) patients had prior exposures to adjuvant chest wall irradiation for breast cancer (n=2), radioiodine for thyroid disease (n=2) and methotrexate/cyclophosphamide for autoimmune disease (n=2). Five (36%) patients had palpable splenomegaly. Peripheral blood (PB) parameters, median (range) at diagnosis were: Hb 10.8 g/dl (8.5-16.0), WBC: 54 x 10(9)/L (21.7-176.2), platelets: 201 x 10(9)/L (25-476), neutrophils: 85% (78-94), immature cells (myelocytes + metamyelocytes): 6% (0-11) and monocytes: 3% (0-10). Neutrophils had toxic granulation and Dohle Bodies in 71% of cases. LDH was elevated in all patients: 354 U/L (225-496). None had a monoclonal protein. The bone marrow (BM) was hypercellular in all patients: 95% (80-100%) and granulocytic precursors were markedly increased with left-shifted (n=11) or normal maturation. BM blasts were < 5%. Reticulin fibrosis was absent (n=5), 1+ (n=5) or 1-2+ (n=1) in those with staining. Cytogenetics were normal in all but one at diagnosis. One patient was lost to follow up after diagnosis. Hydroxyurea (HU) was the most frequently used therapy (initial therapy in 10 patients) and was ultimately received by all. Although initially effective at controlling leukocytosis, clinical course of most was defined by progressive refractory mature neutrophilia with the development of red cell (n=6) and platelet transfusion (n=3) dependancy. Other therapies included IFN or PEG/IFN (n=4), hypomethylating agents (n=4), ruxolitinib (n=2), thalidomide (n=2), cladribine (n=2) and imatinib (n=1). None of these additional drugs, induced remission and they provided primarily palliative and transient benefit. One patient transformed into AML at 17 months, one received induction chemotherapy for suspected AML (PB and BM blasts at 11-13%) at 19 months and two evolved into WHO-defined CMML, at 20 and 27 months, respectively. Both patients with documented/suspected AML were both T618I and SETBP1 positive. Of the cases progressing to CMML (one with T618I, one with M696T) neither was SETBP1 positive. At last follow up, eight patients had died and median survival was 22.5 months. Progressive refractory neutrophilic leukocytosis often preceded and contributed to death. Four (29%) patients are currently alive after a median follow-up of 61 months (14.3- 69.4). Conclusion : CSF3R-mutated CNL is an aggressive disease (median survival <2 years) characterized by progressive neutrophilic leukocytosis which is only partially controlled with current drugs, including the putative targeted therapy ruxolitinib (both ruxolitinib-treated patients progressed on treatment). Patients with CNL could progress into AML or CMML; however, most succumb from complications associated with progressive treatment-refractory leukocytosis. The observation that both patients with documented/suspected AML were SETBP1-mutation positive suggests potential pathogenetic link. Disclosures Off Label Use: Ruxolitinib, cladribine, thalidomideand inteferon. These drugs were used in a historical account of CNL and I report on these in a retrospective chart review only..

Abstract Introduction Pruritus, often aquagenic, is a common and disabling feature of myeloproliferative neoplasms. It's prevalence in both polycythemia vera and primary myelofibrosis (PMF) has been well documented. Historically, a number of medications have been used to treat pruritus in PMF, including hydroxyzine, anti-histamines, hydroxyurea, and selective serotonin re-uptake inhibitors (SSRI's). Recent clinical trials with JAK or mammalian target of rapamycin (mTOR) inhibitors in PMF have identified pruritus as one of the most treatment-responsive disease traits. However, there has not been a comprehensive study comparing treatments for pruritus, response rate, and time to response in a large cohort of patients with PMF. Aims 1) Describe treatment strategies for pruritus in a large cohort of patients with PMF. II) Compare effectiveness of different treatments used to alleviate pruritus in PMF. Methods The current study population was comprised of 88 patients taken from a larger primary cohort of patients seen at the Mayo Clinic, Rochester between the years 2000 and 2010 with biopsy proven diagnosis of PMF (n=566) who reported pruritus at any time during their care at Mayo Clinic. Patients with pruritus secondary to underlying skin disorder confirmed by Mayo Clinic Department of Dermatology were excluded from evaluation. Clinical records were reviewed and data relating to pruritus was collected in the following domains: treatment type, response rate, and time to response. Treatment was stratified into medication class, and mean response rate and time to response was calculated. Results Eighty eight patients (16% of primary cohort) reported pruritus that was not attributable to an underlying skin disorder. Forty nine of these patients (56%) reported severe pruritus and required treatment. The most commonly used medications to treat pruritus were anti-histamines (n=30), anti-depressants (n=22), topical corticosteroids (n=19), topical non steroids (n=13), hydroxyzine (n=14), JAK inhibitors (n=13), and hydroxyurea (n=8). Other medications included thalidomides (n=6), pain relievers (n=6), prednisone (n=5), UVB phototherapy (n=3), alpha interferon (n=2), gabapentin (n=1), and imatinib (n=1). Among the 49 patients who received treatment for pruritus, response to therapy was documented in 35 patients. Eight three percent of patients (n=29) reported resolution of pruritus; 17% (n=6) had no improvement. Highest response rates were observed in patients treated with a JAK inhibitors (92%), thalidomide (83%), or pain relievers (83%). More traditional therapies including hydroxyzine, anti-histamines, and anti-depressants were moderately effective at relieving pruritus (43%, 37%, and 32% response rates respectively). The average time to pruritus alleviation in the entire cohort was 2 months (range: 1 day-25 months). See table 1 for response rates and average time to response for all treatment modalities. Conclusions Pruritus is a treatment responsive feature of PMF. Over 80% of patients with PMF and pruritus in our cohort experienced resolution of pruritus after treatment. JAK inhibitors had the highest response rates for alleviating pruritus. As further molecular correlates for pruritus and PMF are discovered, additional targeted treatments like JAK inhibitors will hopefully emerge. Disclosures: No relevant conflicts of interest to declare.

Abstract Background Increased serum levels of lactate dehydrogenase (LDH) typically accompany primary myelofibrosis (PMF) and might be linked to increased cell turnover from clonal myeloproliferation, including leukocytosis, low grade hemolysis and extra-medullary hematopoiesis in the liver. Despite the fact that serum LDH has been extensively utilized as a prognostic marker in both lymphoid neoplasms and solid tumors, there are limited studies in PMF that have systematically examined the clinical significance of the degree of serum LDH elevation in PMF. Methods Study patients fulfilled the 2016 WHO criteria for the diagnosis of PMF, including both pre-PMF and overt PMF (Blood. 2016;127:2391). Additional selection criteria included the availability of serum LDH at time of referral. Marked elevation of serum LDH was defined as a value of ≥1000 U/L (i.e. over 4-fold increase from the upper limit of the normal range for our institution, which was 122-222 U/L), based on preliminary analysis of the threshold for prognostic effect. Targeted next generation sequencing was used to screen for prognostically-relevant mutations (Blood 2015126:354). Statistical analyses considered clinical and laboratory parameters obtained at time of initial referral to the Mayo Clinic. Results LDH values in overt PMF versus pre-PMF: The entire study population consisted of 357 patients, including 311 with overt PMF and 46 with pre-PMF. The median serum LDH level was 514 U/L (range 136-2263): overt PMF 532 U/L (range 136-2263); pre-PMF 401 U/L (range 180-1237) (p=0.0003). Accordingly, in order to avoid the confounding effect of the difference in serum LDH level between overt PMF and pre-PMF, and considering the small number of patients with pre-PMF, further analysis was limited to the 311 patients with overt PMF. Patient characteristics: The 311 patients (median age 64 years; 66% males) with overt PMF included 205 (66%) JAK2, 49 (16%) type 1/like CALR, 13 (4%) type 2/like CALR, 16 (5%) MPL mutated cases and 28 (9%) were "triple-negative". DIPSS-plus risk distribution was 31% high, 43% intermediate-2, 15% intermediate-1 and 12% low; 30% displayed red cell transfusion-dependency and 37% abnormal karyotype, including 14% with unfavorable karyotype. 184 patients were screened for ASXL1 mutations with 42% mutated and 205 for SRSF2 mutations with 16% mutated. Clinical correlates of markedly elevated LDH (≥1000 U/L): Among all 311 study patients with overt PMF, 37 (12%) displayed marked elevation of LDH (≥1000/L). Patients with marked elevation of LDH displayed significantly higher leukocyte count (p=0.005; R-squared = 0.05), circulating blast percentage (p=0.03; R-squared = 0.07) and SRSF2 mutational frequency (44% vs 12%; p<0.0001). Survival analysis: After a median follow up of 3 years, 199 (64%) deaths and 31 (10%) leukemic transformations were documented. In univariate analysis, increased serum LDH level was associated with inferior survival, both as a continuous variable (p=0.002) and as a categorical variable with the cutoff level of 1000 U/L (HR 2.02, 95% CI 1.3-3.1; p=0.001); the survival effect LDH ≥1000/L was independent of DIPSS-plus (HR 1.6, 95% CI 1.1-2.5). Other variables that were significantly associated with shortened survival, on univariate analysis, included all 8 DIPSS-plus variables (p≤0.01 in all instances), absence of CALR type 1/type 1-like (p<0.0001) and presence of ASXL1 (p<0.0001) or SRSF2 (p=0.0006) mutations. In multivariable analysis that included only genetic risk factors, serum LDH retained its significance (HR 2.2, 95% CI 1.3-3.6), along with absence of CALR type 1/type 1-like, or presence of ASXL1 or SRSF2 mutations or unfavorable karyotype. In multivariable analysis that included only clinical variables, serum LDH ≥1000/L was again independently predictive of poor survival (HR 1.7, 95% CI 1.1-2.6), along with age >65 years, hemoglobin <10 g/dL, platelets <100 x 10(9)/L, leukocyte count >25 x 10(9)/L and constitutional symptoms. Patients with marked LDH elevation were also more likely to undergo leukemic transformation (HR 3.1, 95% CI 1.2-7.6). Conclusion The current study suggests that marked elevation of serum LDH in PMF indicates aggressive tumor biology that is currently not accounted for by established clinical or genetic risk factors; the low R-squared values seen in relation to leukocyte count and circulating blasts are consistent with this assumption. Disclosures No relevant conflicts of interest to declare.

Background: Although the association of acquired isolated factor V (FV) deficiency with myeloproliferative neoplasms (MPN) has been described, it has not been well characterized. Objective: To characterize the clinical and laboratory features of patients with MPN and isolated FV deficiency. Methods: After IRB approval, patients with MPN who underwent coagulation profile diagnostic testing from January 1980 to December 2012 were identified via the Mayo Clinic Special Coagulation Laboratory Registry. Medical records of those with MPN and concurrent isolated FV deficiency or those with mild coagulopathy who had at least 20% discordantly lower FV activity were reviewed. Excluded were patients with known inherited bleeding diathesis, clinically significant liver disease, or on concurrent warfarin treatment. Data on patient demographics, clinical features and laboratory data were abstracted. Results: Thirty three patients met our study criteria (median age 67 years; 55% males; median BMI 25 kg/m2). Twenty one (64%) patients had either primary or post-polycythemia vera (PV) or post-essential thrombocythemia (ET) myelofibrosis; five (15%) had PV; three (9%) had unclassified MPN; two (6%) had ET; and one (3%) each had chronic myeloid leukemia and chronic myelomonocytic leukemia. Majority of these patients had splenomegaly (85%), with a median palpable spleen size of 14.3 cm below the left costal margin. Nearly 40% had hepatomegaly (median 4 cm below the right costal margin). Twelve (36%) patients were being treated with hydroxyurea; nine (27%) with aspirin and seven (21%) with an investigational agent. Nearly two-thirds (n=21; 64%) of these individuals had bleeding episodes, nine (43%) of which were provoked either by a surgical intervention or trauma. Majority of the bleeding episodes (n=15; 71%) were clinically significant/major bleed, as defined by the standard International Society of Thrombosis Haemostasis criteria, of whom eight required red cell transfusion support. Most of the bleeding were from muco-cutaneous surfaces (45%), followed by excessive bleeding from surgical site (24%). Median time from MPN diagnosis to bleeding was 5.4 years (range: 0 – 25). Of note, 42% (n=14) of all patients had thrombotic events (5 ischemic stroke/ TIA; 4 peripheral artery disease; 1 myocardial infarction; 1 splenic artery thrombosis; 3 leg DVT; and 3 splanchnic vein thrombosis) prior to their bleeding episode. Median time from MPN diagnosis to thrombosis was 1.4 years (range: 0.1 – 19.7). The median (range) leukocyte and platelet count were 17.8 (3.6 – 212.7) x 1000/ mm3 and 315 (16 – 1284) x 1000/ mm3, respectively. The median (range) PT [reference range: 10.3 – 12.8 sec] and aPTT [reference range: 26 – 36 sec.] were 13.3 sec (10.1 – 18.7) and 38 sec (29 – 66), respectively. The median FV activity [reference range: 70 – 165%] was 44% (range: 18% - 60%). Seven of the 18 individuals (39%) tested for von Willebrand syndrome had ristocetin cofactor activity (RCoA)/von Willebrand antigen (vWF Ag) ratio of <0.7, although their vWF Ag and RCoA were within the normal reference range. Seventeen (52%) patients had platelet aggregation studies; all had at least one aggregation abnormality. Abnormal or absent response to epinephrine was the commonest noted abnormality (n=15), followed by an abnormal response to collagen (n=6). On univariate analysis, there was no significant correlation of FV activity with age, sex, BMI, myelofibrosis, hepatomegaly, splenomegaly, WBC count, platelet count, or use of hydroxyurea or investigational drugs. Further, on univariate analysis, neither the aforementioned covariates, nor aspirin use, WBC count, platelet count, FV activity, abnormal platelet aggregation, RCoA/vWF Ag ratio < 0.7 were predictive of either overall bleeding, provoked bleeding or clinically significant/major bleed. Conclusions: Isolated FV deficiency associated with MPN often co-exists with platelet dysfunction and/or an acquired vWF abnormality, which together may contribute to a clinically significant bleeding diathesis. Our experience did not lead to identification of specific risk factors for MPN-associated isolated FV deficiency. Disclosures No relevant conflicts of interest to declare.

Abstract BACKGROUND: Disease manifestations in myelofibrosis with myeloid metaplasia (MMM) that require therapy include anemia, thrombocytopenia, hepatosplenomegaly, and constitutional symptoms. We have previously reported on the therapeutic value of low-dose thalidomide in combination with prednisone for the treatment of both anemia and thrombocytopenia in MMM (Blood2003;101:2534) and of etanercept (tumor necrosis factor-alpha antagonist) for alleviation of constitutional symptoms (Blood2002;99(6):2252). In the current study, we investigated the value of combining all three drugs (PET). 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 daily, etanercept 25 mg subcutaneously twice-a-week, 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 and etanercept. RESULTS: To date 12 patients have been enrolled to the trial (median age 67 years, range, 54–77; 11 males). At enrollment, all patients were severely anemic (6 were red cell transfusion dependent), 6 were at least moderately thrombocytopenic (platelet count < 100 x 109//L), and 4 had severe constitutional symptoms. The responses to date included resolution of constitutional symptoms in all 4 pertinent patients, a 12–326% improvement in thrombocytopenia in 4 of 6 pertinent patients, and anemia response in 4 patients including one patient who became red cell transfusion independent. A more than 50% decrease in splenomegaly was documented in 3 patients. Only one patient experienced greater or equal to grade 3 toxicity (i.e. elevated bilirubin and infection). Follow-up bone marrow examination, when available, did not show post-treatment histological changes. CONCLUSIONS: The preliminary findings from the current ongoing phase II study of combination therapy with thalidomide-prednisone-etanercept in patients with MMM confirms the previously recognized value of etanercept in alleviating constitutional symptoms. However, at least preliminarily the PET regimen does not appear to be superior to thalidomide-prednisone combination in terms of therapeutic value for anemia, thrombocytopenia, or splenomegaly.

Abstract Background We have previously reported on the significant, but heterogeneous baseline MPN symptom burden among an international sample of MPN patients (including essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF)) utilizing the MPN Symptom Assessment Form (MPN-SAF) and the derivative Total Symptom Score (MPN-SAF TSS). Recent clinical trials have sought to determine optimal MPN symptom response criteria, such as absolute 10 point improvement in MPN SAF TSS for ET/PV (ELN Criteria, Barosi et. al. Blood 2013) and 50% reduction in MPN-SAF TSS for MF (IWG-MRT, Tefferi et. al. Blood 2013). We sought to determine the role of improvement in MPN-SAF TSS quartiles as potential thresholds to assess symptomatic response to therapy. Methods Utilizing prospectively gathered MPN-SAF TSS (Emanuel et. al. JCO 2012) in patients we assessed potential thresholds of response by evaluating quartile thresholds for severity of symptom burden. The MPN-SAF TSS was scored as the average of 10 symptoms (individual symptoms scores of 0-10, with a total score of 0 (best) to 100 (worst)). MPN-SAF TSS quartiles were identified by the percentage of scores between 0-24% (quartile 1 (Q1)), 25-49% (quartile 2 (Q2)), 50-74% (quartile 3 (Q3)), 75-100% (quartile 4 (Q4)). Results MPN-SAF TSS Quartiles: MPN-SAF TSS quartiles were identified among 1858 MPN patients (ET N=775, PV N=654, and MF N=423). Overall MPN-SAF TSS scores of 0 - 7 were designated as Q1, 8 - 17 as Q2, 18 - 31 as Q3, and ≥ 32 was as Q4. MPN-SAF TSS scores were significantly different between clusters (p<0.001). Associations Between Quartiles and Demographic/ Disease Factors: As quartiles increased, the proportion of PV and ET patients diminished and MF increased (Table 1, p<0.001). Cytopenias and transfusion dependence increased in prevalence in the higher quartiles (p<0.001). A history of prior thrombosis was also significantly more prevalent in the quartiles with highest symptom burden (p<0.001). The prevalence of women was significantly higher among the more symptomatic quartiles females 48.9% Q1, 49.4% Q2, 58.4% Q3, and 60.1% Q4; p<0.001). Associations Between Individual Symptoms and MPN-SAF TSS Quartiles: All individual symptoms measured in the MPN-SAF TSS were significantly worse in quartiles as they increased (p<0.0001). Evaluation of Prognostic Scoring and MPN-SAF TSS Quartiles: Comparison of each patients individual risk score (IPSET, PV, DIPSS for MF) and worsening symptom quartile showed the highest correlation with MF patients (DIPSS) (Table 1). However, ET and PV risk scores were not surrogates for symptom burden by quartile. Conclusions Distribution of MPN patient symptomatic burden by MPN-SAF TSS quartiles provides an easy-to-calculate method to cluster and analyze MPN patients of similar burden. Although MF patients are most prevalent in the most severe quartile of MPN symptomatology it is notable that Q4 has many patients with PV and ET. Future prospective efforts are ongoing to assess the potential of using changes in quartile (i.e. improving from Q3 to Q1) as potential symptomatic response thresholds. Disclosures: Etienne: novartis: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Pfizer: Membership on an entity’s Board of Directors or advisory committees; Ariad: Membership on an entity’s Board of Directors or advisory committees. Roy:Novartis, BMS: Honoraria. Harrison:Gilead: Honoraria, Membership on an entity’s Board of Directors or advisory committees; S Bio: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Shire: Speakers Bureau; Celgene: Honoraria; YM Bioscience: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau. Vannucchi:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Birgegard:Vifor Pharma: Honoraria.

Abstract Background We previously reported that patients with MF enrolled in a randomized Phase II study of fedratinib (SAR302503) (ARD11936; NCT01420770) had clinically meaningful reductions in splenomegaly and improvements in MF-associated constitutional symptoms after 24 weeks of treatment (Haematologica 2013;98:S1113). Here, we report updated efficacy and safety results from this study after 48 weeks of treatment (end of Cycle 12). Methods Patients with intermediate risk-2 or high-risk MF were randomized to receive once-daily fedratinib at doses of 300 mg, 400 mg, or 500 mg, for consecutive 4-weekly cycles, until disease progression or unacceptable toxicity. Eligible patients were aged ≥18 years, with palpable splenomegaly (5 cm below costal margin), and a platelet count ≥50 × 109/L. The primary measure for this study was percent change in spleen volume from baseline at the end of Cycle 3 (Blood 2012:120;Abstract 2837. Haematologica 2013;98:S1113). Endpoints for the current analysis included spleen response (≥35% reduction in spleen volume from baseline, assessed by a blinded independent central review by MRI), safety, and changes in bone marrow fibrosis (BMF). Results A total of 31 patients were randomized and treated: median age 63 years, 52% male, 58% primary MF, 58% high-risk MF, 90% JAK2V617F positive. The median numbers of treatment cycles were 12, 14, and 13 in the 300 mg, 400 mg and 500 mg dose groups, respectively, with median durations of exposure of 48.2, 56.2, and 52.4 weeks. At the cut-off date for this analysis, 21 patients (68%) remained on treatment; the most common reasons for treatment discontinuation were adverse events (AEs) (n=5) and withdrawal of consent (n=2). Overall, 58% (18/31) of patients achieved a spleen response at any time during treatment. The median spleen response duration was >35 weeks at all doses (Table). At Week 48, a spleen response was achieved by 30% (3/10), 80% (8/10), and 45% (5/11) of patients in the 300 mg, 400 mg, and 500 mg groups, respectively. Responses were generally maintained across all treatment groups. From Week 24 to Week 48 two additional patients achieved a spleen response (both in the 400 mg group), while one patient in the 500 mg group did not maintain a response (this patient had a fedratinib dose reduction to 200 mg). Changes in BMF up to Week 48 are being evaluated. The most common non-hematologic AE was diarrhea, with a Grade 3 rate of 13% (4/31 patients) but no Grade 4 cases were recorded. The rates of diarrhea decreased after the first cycle of treatment; from Cycle 2, the incidence of diarrhea (any grade) did not exceed 16% (5/31) at any cycle, and only one case of diarrhea was reported at Week 48 (end of Cycle 12). Anemia was the most-common hematologic toxicity, with a Grade 3 rate of 58% (18/31); no Grade 4 cases were reported. All Grades thrombocytopenia occurred in 55% (17/31) of patients, Grade 3 in three patients, and Grade 4 in two patients. Discontinuation of treatment due to AEs occurred in five patients over the 48 weeks (300 mg [n=2]; 400 mg [n=2]; 500 mg [n=1]), with two cases reported after Week 24 (dyspnea and leukocytosis [400 mg]; anemia and thrombocytopenia [500 mg]). There were 2 deaths (one in the 300 mg group due to unknown reasons [85 days after fedratinib discontinuation] and one in the 500 mg group due to disease progression [36 days after fedratinib discontinuation]). No cases of leukemic transformation were reported. Conclusions This updated analysis of the ARD11936 Phase II trial shows that treatment with fedratinib results in durable reductions in splenomegaly in patients with MF. No additional safety signals were observed with prolonged exposure to fedratinib. This study was sponsored by Sanofi. Disclosures: Pardanani: Sanofi, Bristol Myers Squibb, PharmaMar and JW Pharma: Clinical trial support Other. Jamieson:J&J, Roche: Research Funding; Sanofi: Membership on an entity’s Board of Directors or advisory committees. Lebedinsky:Sanofi: Employment. Gao:Sanofi: Employment. Talpaz:Novartis, Bristol-Myers Squibb, Ariad, Deciphera: Research Funding; Novartis, Bristol-Myers Squibb, Ariad, Deciphera: Speakers Bureau.

Abstract Primary myelofibrosis (PMF) is a clonal hematologic malignancy resulting from the transformation of a pluripotent hematopoietic progenitor cell leading to increased hematopoiesis and overproduction of abnormal blood cells. PMF is characterized by bone marrow (BM) 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 BM and spleen. Numerous mutations are associated with PMF including JAK2, CALR, MPL, TET2, CBL, ASXL1, and IDH. The presence of abnormal Mks in the BM of PMF patients is an important characteristic of the disease, as these cells are believed to contribute to the fibrosis.Here, we performed an analysis of Mk differentiation from individuals with PMF compared to healthy individuals as a way to better understand the mechanism leading to their defects. CD34+ cells obtained from PMF and healthy donors were cultivated in presence of thrombopoietin to induce Mks differentiation. Under these conditions, we observed that the proliferative capacity of CD34+ cells from PMF patients was increased as compared to normal CD34+ cells. At the same time, the percentage of mature Mks obtained was reduced. Semi-solid culture conditions confirmed the proliferative advantage of the PMF CD34+ cells. To investigate the mechanism responsible for this phenotype, we performed genome wide expression analysis of sorted Mks from patients versus controls. Nine PMF patients with different mutations (5 JAK2V617F, 1 MPLW515L, 2 CALR and 1 triple negative) and 4 healthy donors were included in the study. Gene Set Enrichment Analysis (GSEA) analysis revealed that the top scoring pathways included the RPS14 signature, ribosome biogenesis, rRNA metabolic process, and rRNA processing. We first studied the RPS14 geneset, which consists of a group of genes that were up-regulated in CD34+ cells after knock-down of RPS14, a component of the small ribosome subunit. Using shRNA against RPS14, we discovered that its knock down leads to a significant reduction in the development of mature Mks from normal CD34+ cells. The microarray data also revealed changes in the expression of several myeloid genes. CEBPa and GFI-1 were over-expressed in PMF Mks, whereas GFI-1b and PF4, which are associated with Mk differentiation, were down-regulated. Surprisingly the expression of GATA1, a master regulator of megakaryopoiesis, was not changed. However, consistent with previous report, we discovered that GATA1 protein level was reduced in PMF Mks. GATA1 deficient Mks are impaired in their maturation, so we reasoned that the reduction in GATA1 contributes to the defects in PMF. Indeed, we found that overexpression of GATA1 in PMF CD34+ cells rescued Mk differentiation. To understand how GATA1 protein but not mRNA, is reduced, we knocked down RPS14 in the JAK2 mutant SET2 megakaryoblastic cell line. As predicted, we observed a reduction in GATA1 protein, but not mRNA. By ribosomal profiling, we saw that the shRPS14 led to an abnormal ribosomal profile with a reduction of the 40s peak. Finally, by RT-PCR analysis of the ribosomal and polysomal fractions, we found that GATA1 mRNA was less abundant in the polysomal fraction in cells expressing the shRPS14. This finding is consistent with recent data showing that alterations in ribosomal genes cause a decrease in GATA1 protein in Diamond-Blackfan Anemia. Although we identified the RPS14 gene signature, we did not observe changes in RPS14 expression or in any of the ribosomal subunits. Since our GSEA analysis also identified deficiencies in three ribosomal biogenesis pathways, we looked for genes present in all three signatures. NOP58, a ribonucleoprotein required for ribosome biogenesis, was significantly reduced in all samples of PMF Mks. To link the reduction in NOP58 expression with PMF associated mutations, we expressed MPLW515L or the CALR type II mutant in hematopoietic progenitor cells and detected a reduction in NOP58 expression. Our findings suggest that the activation of JAK/STAT signaling leads to a reduction in NOP58, which contributes to defects in ribosome assembly, decreased GATA1 expression and subsequent impairment in Mk differentiation. This work reveals, for the first time, that defective ribosomes contribute to the pathogenesis of PMF and suggests new avenues for therapeutic intervention. Disclosures Stein: Incyte Corporation: Honoraria, Speakers Bureau; Sanofi Oncology: Honoraria.

Abstract Background : Controversy persists regarding diagnostic accuracy as well as added value for quantitative assessment of neutrophil PRV-1 expression in polycythemia vera (Klippel et al. Blood2003;102:3569; Tefferi et al. Blood2004;103:3547). This is partly because of limited data regarding the relationship between neutrophil PRV-1 transcript level and other laboratory parameters in polycythemia vera (PV). More information is also needed on the PRV-1 expression profile in other myeloproliferative disorders (MPD), including essential thrombocythemia (ET) and agnogenic (AMM), post-polycythemic (PPMM), and post-thrombocythemic (PTMM) myeloid metaplasia. Methods : The current prospective study involves 141 subjects, including 49 with PV, 40 with secondary or spurious polycythemia (SP), 23 with ET, 11 with AMM, 4 with PPMM, 3 with PTMM, and 11 normal controls. Peripheral blood neutrophil PRV-1 expression was quantified by real-time PCR according to published methods (Klippel et al. Blood 2003). The results were first compared among the different disease categories and controls. Subsequently, within the specific disease categories of both PV and ET, PRV-1 expression was correlated with several laboratory [leukocyte count, absolute neutrophil count (ANC), leukocyte alkaline phosphatase score (LAP) score, CD34 count, platelet count, hematocrit, serum EPO level, MCV, serum ferritin, reticulin fibrosis] and clinical (age, sex, disease duration, spleen size, thrombosis history, and presence of treatment with cytoreductive agent, aspirin, or phlebotomy) parameters. Results : The incidence of increased neutrophil PRV-1 expression was the highest in PV and PPMM (p<0.0001) while the results in the other disease categories (ET, SP, AMM, PTMM) were similar among each other and controls ( p=0.89). Table 1 Parameters Cont. (n=11) SP (n=40) PV ( n=49) ET (n=23) AMM (n=11) PPMM (n=4) PTMM (n=3) Age in years median NA 51 58 46 60 63 47 range NA 19–78 18–83 18–81 41–80 56–71 47–72 Females NA 20% 55% 65% 18% 25% 33% PRV-1/GAPDH median 1.25 1.25 1.01 1.24 1.26 1.04 1.25 range 1.14–1.43 0.99–1.41 0.83–1.28 0.9–1.44 1.08–1.43 0.93–1.11 1.23–1.27 PRV-1 ↑ 9% 15% 76% 17% 18% 100% 0% In both PV and ET, neutrophil PRV-1 expression strongly correlated with LAP score (p=0.003 and 0.04, respectively). In addition, in PV, univariate analysis revealed significant correlations between PRV-1 expression and leukocyte count, ANC, and reticulin fibrosis. On multivariate analysis, only LAP score retained its significance. Figure Figure Conclusion :The current study confirms the previously recognized close association between increased neutrophil PRV-1 expression and PV as well as suggests a similarly strong association with PPMM but not with ET, MMM, or PTMM. The study also underscores the suboptimal diagnostic accuracy of the neutophil PRV-1 assay in distinguishing PV from SP. Most importantly, the demonstrated tight correlation between PRV-1 transcript level and LAP score raises the issue of added value to currently available diagnostic tests.