Tefferi, Ayalew

Abstract Background: Over the last decade there have been three FDA-approved agents available for the treatment of myelodysplastic syndromes (MDS); azacitidine was approved in 2004 for all subtypes of MDS; lenalidomide in 2005 for MDS with del(5q); and decitabine in 2006 for intermediate/high risk MDS. However the ability of these drugs to improve survival outside of clinical trials remains controversial (Neukirchen J, Leuk Res 2015, Bernal T, Leukemia 2015). Objectives : i) Evaluate trends in overall survival (OS) and leukemic transformation (LT) rate amongst primary MDS patients (pts) by year of diagnosis and, ii) Evaluate trends in OS by treatment received. Methods : The Mayo Clinic database was used to identify pts with primary MDS in whom bone marrow histologic and cytogenetic information was obtained at the time of diagnosis. WHO criteria were used for MDS diagnosis and LT. A comparative analysis was performed based on year of diagnosis commensurate with the approval of the aforementioned drugs (Group 1- diagnosis prior to the year 2000, Group 2- year 2001-2004, Group 3- year 2005-2009, and Group 4- year 2010-2014). Results : i) Patient characteristics: A total of 1000 pts met the above-stipulated criteria. 85% of pts were above 60 years of age (median 72 years) with 69% being males. The distribution of pts by year of diagnosis was as follows: Group 1 (n=281)(28%), Group 2 (n=250)(25%), Group 3 (n=264)(26%), and Group 4 (n=205 (21%). Median follow-up of our cohort was 27 months (range; 0-300 months) during which time 808 (81%) deaths and 129 (13%) LT were documented. ii) Comparison of patient characteristics by year of diagnosis: Pts in group 1 and 2 compared with groups 3 and 4 were more likely to present with anemia defined as hemoglobin < 10 g/dl (61%/59% vs 50%/55%)(P =.04). In addition, groups 1 and 2 displayed a higher incidence of RA (5%/4% vs 1% each), and RARS (17%/16% vs 9%/8%), compared to groups 3 and 4 that had a higher incidence of RCMD (37%/44% vs 17%/28%) (P <.001). The IPSS-R risk distribution was not significantly different; 17% very low, 36% low, 21% intermediate, 15% high and 11% very high risk with median survivals of 72, 43, 24, 18 and 7 months, respectively (P <.001). As expected, a higher proportion of pts in groups 3 and 4 (41% and 57% respectively) received "disease-modifying" therapy including allogeneic transplant and hypomethylating agents as opposed to only 6% and 22% in groups 1 and 2 respectively (P <.001). iii) Trends in OS and LT rate by year of diagnosis: The median OS of the entire cohort was 30 months, with median OS and LT rates being similar amongst groups 1 through 4 at 31 vs 33 vs 30 vs 27 months (P =.79) (Figure) and 10% vs 16% vs 12% vs 15% (P =.25), respectively. iv) Trends in OS by treatment received: In univariate analysis survival was significantly better in pts who underwent allogeneic transplant (n=65) with median survival of 55 months vs 26 months for non-transplant pts (P<. 001); and among non-transplant lenalidomide-treated pts (n=44) with median survival of 54 months vs 26 months for the remainder of pts (P =.02). However, these results lost significance on multivariable analysis with the addition of age as a co-variate for transplant pts (P =.28), and IPSS-R as a co-variate for lenalidomide treated pts (P =.10). Excluding transplant pts, pts that received hypomethylating agents (n=158) had similar survival to pts not treated with hypomethylating agents (27 vs 29 months; P =.19, age-adjusted P =.11). In addition, the 54 pts who received other chemotherapeutic agents had similar survival to pts not treated with these agents (33 vs 26 months; P =.57, age-adjusted P =.80). Supportive care alone was utilized in 702 pts that had comparable survival to the 298 pts that received "disease-modifying" therapy (27 months vs 34 months; P =.05, age-adjusted P =.11). Conclusions : In this single center analysis of 1000 pts with primary MDS, stratified by year of diagnosis, the poor outcome of these pts has not improved over the last two decades, in spite of the significantly higher utilization of "disease-modifying" therapy, including hypomethylating agents since 2005. The lack of improvement in survival with hypomethylating therapy is consistent with recently published results from the Spanish MDS registry (Bernal T, Leukemia 2015). However, our retrospective study is not designed to detect marginal survival benefit, which has thus far been reported in only one clinical trial. Figure 1. Figure 1. Disclosures Al-Kali: Novartis: Research Funding. Pardanani:Stemline: Research Funding.

Abstract Background : Hydroxyurea has been the mainstay of cytoreductive therapy in essential thrombocythemia (ET) for over four decades. In 1988, anagrelide was first reported as an effective drug in ET (Silverstein et al. NEJM 1988;318:1292) and hundreds of patients were enrolled in clinical trials, which subsequently led to FDA approval in 1997. As a result, an increasing number of patients with ET were being treated with anagrelide until a subsequent controlled study suggested an inferior treatment outcome with anagrelide compared to hydroxyurea (Harrison et al. NEJM 2005;353:33). In the current study, we examined the impact of the corresponding practice changes on overall, myelofibrosis-free and leukemia-free survival, by comparing patients with ET diagnosed before 1988, between 1988 and 1997, and between 1998 and 2005 Methods: More than 45,000 patient charts with "thrombocytosis" were reviewed to identify those who met either PVSG (older patient cohort) or WHO criteria for diagnosis of ET. Survival was calculated from time of initial diagnosis to the time of last follow-up or death. For leukemia- or fibrosis-free survival, the time of the transformation events was considered as the uncensored variable. Conventional statistics was utilized for all analyses. Patients were divided into three groups commensurate with changes in treatment practices, corresponding to the aforementioned publications; Group 1 patients were diagnosed prior to 1988 (n =222), Group 2 1988 to 1997 (n =253), and Group 3 1998 to 2005 (n =264). Results : A total of 739 patients met the above-stipulated criteria (median age 57 years; 65% females). At a median follow-up of 12.2 years (range: 0.01-43.6 years), 368 (50%) deaths, 48 (6.5%) fibrotic transformations and 23 (3%) leukemic transformations were documented. Median overall survival (OS) was 18.6 years. On multivariable analysis, older age (P <0.001), increased leukocyte count (P <0.001), thrombosis history (P =0.04), and male sex (P<0.0001) were identified as predictors of inferior OS. Myelofibrosis-free survival (MFS) was predicted by platelet count <100 x 10(9)/L and lower hemoglobin level (multivariate P values 0.03 and <0.001, respectively). Leukemia-free survival (LFS) was predicted by increased leukocyte count, on multivariable analysis (P =0.002). Comparison of presenting features and outcome by period of diagnosis: Significant differences, in presenting features, between groups 1, 2, and 3 included age (median 55, 56, and 59 years, respectively; P =0.003), female preponderance (68, 69, and 58%, respectively; P =0.02), platelet count (1080, 995, and 890 x 10(9)/L, respectively; P <0.001), and leukocyte count (10, 9.6, and 8.4 x 10(9)/L, respectively; P <0.001). Median survival was 20.7 years, 19 years and 14.3 years, in groups 1, 2 and 3, respectively (P =0.02) (Figure 1). However, the difference in survival was no longer apparent during multivariable analysis that included age as a covariate (P=0.88). In univariate analysis, MFS was significantly shorter in group 3 patients (P=0.02; Figure 2); borderline significance was retained during multivariable analysis (p=0.13). LFS was similar among the three groups (P = 0.27). Conclusions : The results from the current retrospective study suggest increased rate of progression to myelofibrosis in ET patients diagnosed in the anagrelide era, an observation that is consistent with the results from a previous controlled study (Harrison et al. NEJM 2005;353:33). Regardless, survival in ET in the last four decades remains unchanged. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Pardanani: Stemline: Research Funding.

Abstract Abstract 3070 Background: EZH2 and ASXL1 mutations were recently described in a spectrum of myeloid malignancies; mutational analysis of small patient cohorts has suggested the highest mutational frequency in myelofibrosis (MF) and chronic myelomonocytic leukemia (CMML). The current study seeks to determine i) EZH2 and ASXL1 mutational frequencies in WHO-defined subcategories of MF, CMML and blast-phase myeloproliferative neoplasm (MPN), ii) if these mutations are mutually exclusive of TET2, IDH, JAK2 and MPL mutations and iii) clinical correlates of ASXL1 and EZH2 mutations in primary MF (PMF) and CMML. Methods: The study population included 94 patients: 46 PMF, 22 post-polycythemia vera/essential thrombocythemia MF (post-PV/ET MF), 11 blast-phase MPN and 15 CMML (10 CMML-1 and 5 CMML-2). High throughput DNA resequencing was used to screen archived bone marrow for EZH2, ASXL1, TET2, IDH, JAK2 and MPL mutations. Results: ASXL1 mutations were identified in all disease categories, including PMF (13%), post-PV/ET MF (23%), blast phase MPN (18%), and CMML (20%). We identified somatic mutations in TET2 in 15%, 14%, 18%, and 13% of PMF, post-PV/ET MF, blast phase MPN, and CMML, respectively. By contrast, mutations in EZH2 and IDH1/2 were less frequent. EZH2 mutations were seen in 3 out of 46 PMF patients (7%) and were not observed in patients with post-PV/ET MF or blast phase MPN. Mutations in IDH1/2 were restricted to blast-phase MPN (36%) and PMF (7%). No mutations in EZH2 or IDH1/2 were seen in CMML. Although we identified frequent TET2 and ASXL1 mutations, we only identified one patient with concurrent mutations in both genes. Three ASXL1 mutation-positive patients also had mutations in EZH2 or IDH and one patient had concurrent ASXL1, TET2 and IDH mutations. In addition, 7 ASXL1, 7 TET2, and 1 IDH mutated patients were JAK2V617F-positive. MPL mutations were also documented in all three mutation categories. All EZH2- and ASXL1-mutated PMF patients displayed normal karyotype and none underwent leukemic transformation during follow-up. Furthermore, mutated versus unmutated patients, in both instances, were not significantly different in age and sex distribution or clinical characteristics. The 3 EZH2-mutated PMF patients died after 29, 48 and 67 months from the time of mutation analysis. In univariate analysis, the presence of mutant ASXL1 in PMF was associated with worse survival (p=0.06) but the borderline significance was lost during multivariable analysis that included risk stratification according to DIPSS (Passamonti et al. Blood 2010; 115: 1703–1708). The 3 ASXL1 mutated CMML cases were alive after 40, 34 and 12 months from time of mutation analysis and none of them had progressed to acute leukemia; karyotype was normal in two of the patients and showed isolated trisomy 8 in one. Conclusions: ASXL1 mutations are as frequent as TET2 mutations in MF and CMML. In contrast, EZH2 mutations are infrequent and cluster with PMF. ASXL1 and EZH2 mutations are not mutually exclusive events, seem to be associated with normal karyotype and do not appear to be leukemogenic or prognostically detrimental in PMF or CMML. Disclosures: No relevant conflicts of interest to declare.

Abstract Background : In primary myelofibrosis (PMF), ̴ 88% of patients harbor JAK2, CALR or MPL, with respective mutational frequencies of approximately 60%, 22% and 6%; ̴ 12% are wild type for all three driver mutations and are labelled as being "triple negative" (Blood. 2014;124:2507). It is now well-established that CALR-mutated patients with PMF display significantly better survival, compared to all the other mutational categories; in addition, preliminary data have suggested inferior survival for "triple-negative" cases (Leukemia. 2014;28:1472; Blood. 2014;124:1062) and the possibility that the favorable impact of CALR mutations might be restricted to CALR type 1 or type 1-like variants (Blood. 2014;124:2465). In the current study, we sought to clarify the prognostic impact of driver mutations, and CALR variants, on overall (OS) and leukemia-free (LFS) survival, in a two-center study involving 1118 patients. Methods: A total of 1118 patients with PMF from the Mayo Clinic (n =722) and University of Florence, Italy (n =396) were included in the current study. Study patients were selected based on availability of mutation information. PMF diagnosis was according to World Health Organization criteria (Blood. 2009;114:937). Previously published methods were used for CALR, JAK2 and MPL mutation analyses and determination of CALR variants (Blood. 2014;124:2465). Kaplan-Meier survival analysis was considered from the date of first referral for the Mayo cohort and date of diagnosis for the Florence cohort. Leukemic transformation events replaced death as the uncensored variable during LFS analysis and Cox proportional hazard regression model was used for multivariable analysis. Results : Analysis was first conducted on the Mayo cohort of 722 patients (median age 64 years; 64% males). DIPSS-plus risk distributions were 14% low, 17% intermediate-1, 37% intermediate-2 and 33% high. All patients were annotated for JAK2/CALR/MPL mutations as well as CALR variants; 477 harbored JAK2, 139 CALR and 41 MPL mutations; 65 patients were triple-negative. The 139 CALR -mutated patients included type 1/type 1-like (n =115) and type 2/type 2-like (n =24). At a median follow-up time of 3.1 years, 439 (61%) deaths and 63 (8.7%) blast transformations were documented. In univariate analysis, survival in patients with CALR type 1/type 1-like mutations was significantly longer than every other mutational category: HR (95% CI) were for triple-negative 2.7 (1.8-4.0), JAK2 2.7 (2.0-3.7), CALR type 2/type 2-like 2.3 (1.3-4.2) and MPL 1.9 (1.1-3.1) (Figure 1); these differences were sustained during multivariable analysis that included ASXL1 mutations (n =480) and DIPSS-plus, and were also apparent in the Florence cohort of 396 cases that included 251 JAK2, 53 CALR type 1/type 1-like, 21 CALR type 2/type 2-like, 50 triple-negative and 21 MPL mutated cases (Figure 2). There was no difference in survival among the Mayo cohort across mutational categories not including CALR type 1/type 1-like variants (p=0.33). Because specific driver mutations in PMF differentially cluster with age and sex, multivariable analysis including these two parameters was also performed and confirmed the significant survival advantage of CALR type 1/type 1-like over triple-negative, JAK2 and CALR type 2/type 2-like mutated cases. Based on the above information, we divided driver mutational categories into "type 1/type 1-like" (median survival 10.3 years) and "all other mutational categories" (median survival 3.9 years; p<0.01). The difference in survival between these two groups remained significant (HR 2.1, 95% CI 1.5-2.9) during multivariable analysis that included both DIPSS-plus (P<0.01) and ASXL1 mutation (HR 1.8, 95% CI 1.4-2.2). LFS was similar between these two groups (p=0.4) but it was inferior in triple-negative cases compared to CALR type 1/type 1-like variants (HR 2.8, 95% CI 1.3-6.2), MPL (HR 4.6; 95% CI 1.04-20.4) and JAK2 (HR 2.5, 95% CI 1.3-4.8) but not CALR type 2/type 2-like variants (p=0.56). There was no difference in LFS among mutational categories not including triple-negative cases (p=0.33). Conclusions : In PMF, driver mutations might be classified into two prognostically distinct categories: "favorable" (type 1/type 1-like) and "unfavorable" (all other mutational categories). In addition, triple-negative cases might be at a higher risk of leukemic transformation. Disclosures Pardanani: Stemline: Research Funding. Vannucchi:Novartis: Other: Research Funding paid to institution (University of Florence), Research Funding; Shire: Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract Abstract 5053 Background: The Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF) is a concise instrument of patient reported outcomes (PRO) designed to assess the unique spectrum of symptoms present in the majority of patients (Mesa et. al. Cancer 2007). We sought to validate the Swedish Translation of the MPN-SAF which addresses 19 separate symptoms reported by MPN patients. Methods: We utilized the standard practice of PRO translation in which 3 independent translations are created by 3 independent translators fluent in both languages. A fourth translator then discussed the translations with the other translators and a consensus translation was obtained. Patients self completed the MPN-SAF: Swedish at the time of a physician office visit and the Swedish EORTC-QLQ-C30 was co-administered for validation purposes. Results: Patients and Symptomatic Burden: 114 patients were prospectively enrolled (ET (N=53; 47%), PV (N=53; 47%) and MF (N=8; 6%)) a median of 6 years (range:0-42) from their diagnosis. Patients were of a median age (68; range 27–88 years) and gender (53% females) typical of the disease. 78% (N=88) had received some form of non-aspirin medical therapy for their disease, and 75% were on therapy at the time of completing the questionnaire. Patients frequently had a history of either thrombotic events (26%) and/or hemorrhagic events (10%). The MPN-SAF measured 19 items in the enrolled patients (data summarized in Table 1). Validation Analysis: EORTC-QLQ-C30 Consistent with our experience with the MPN-SAF:English Pearson correlations between MPN-SAF-Swedish individual symptom scores and the Swedish EORTC-QLQ C30 showed excellent correlations with co-validation questions including fatigue, pain, headache, insomnia, early satiety, and sad mood (all p<0.001). Correlations with EORTC-QLQ-C30 subscales demonstrated excellent correlations between MPN-SAF measurements and corresponding subscales. Comparison with Mpn-Saf:English: Comparison with 102 patients prospectively completing the MPN-SAF: English (ET=20, PV=N=23, MF=59) showed (when adjusting for MPN subtype) excellent correlation with all but 3 items in which the prevalence of the symptoms was similar but the severity less in the Swedish cohort (early satiety, headache and impaired quality of life). Additionally, the ranking of the prevalence of symptoms was very well correlated between both cohorts when controlling for disease type. Conclusions: The MPN-SAF: Swedish is an easy to administer, clear, 19 item inventory of patient reported outcomes that is specific to MPNs. Additionally, the instrument is validated by 1) comparison to previously validated Swedish Instruments and 2) the correlation with the MPN-SAF:English. Utilization of the instrument in Swedish MPN clinical trials will allow for useful comparison to patients completing the MPN-SAF in other countries. Disclosures: Samuelsson: Roche Sweden: Mesa: SBio: Research Funding; Novartis: Research Funding; Celgene: Research Funding; Incyte: Research Funding; Roche: Research Funding; eisai: Research Funding; telik: Research Funding.

Abstract Background: Addition of sex combs-like 1 (ASXL1) is frequently mutated (mutational frequency 14-29%) and is prognostically relevant in myelodysplastic syndromes (MDS) (Bejar, NEJM 2011, Thol, JCO 2011). The prevalence and prognostic impact of ASXL1 mutation in MDS with ring sideroblasts (MDS-RS) is not known. MDS-RS is defined by the presence of ≥15% RS in bone marrow (BM), with refractory anemia with ring sideroblasts (RARS) being the prototype but may also be seen in other categories. Methods : Our institutional database was reviewed to identify patients with WHO-defined primary MDS with ≥1% BM RS. Pathology slides, including iron stains, were reviewed to accurately quantify BM RS percentage and confirm WHO morphologic categories. All patients were annotated for their mutational status including ASXL1, JAK2, MPL and IDH with a subset for SF3B1 by PCR sequencing performed on BM specimens obtained at diagnosis. Results : i) Patient characteristics: A total of 76 MDS patients displayed ≥1% BM RS (median age 72 years; 76% males); 51 (67%) patients had ≥15% BM RS. IPSS-R risk categories for the entire 76 study patients were 30% very low, 37% low, 14% intermediate, 11% high and 8% very high; IPSS-R karyotype was normal in 63%, very good/good risk 17%, intermediate risk 12%, and poor/very poor in 8%; 3% had monosomal karyotype. ii) Prevalence of ASXL1 mutations: Twenty-one (28%) of the 76 study patients were ASXL1 mutated; ASXL1 mutational frequencies were 25% (16/63 patients) in the absence and 38% (5/13 patients) in the presence of excess blasts (P =0.34). When considering only those patients with ≥15% BM RS (n =51), ASXL1 mutations were detected in 12 (24%) patients with mutational frequencies of 24% (11/46 patients) in the absence and 20% (1/5 patients) in the presence of excess blasts (P =0.84); ASXL1 mutational frequencies were 13% (3/23 patients) in RARS and 37% (7/19 patients) in RCMD-RS (P=0.07). In terms of other mutations, all 76 study patients were wild-type for JAK2 and MPL, whereas IDH2 R140Q mutations were present in 4 (5%) patients, including 3 with concomitant ASXL1 and IDH2 mutations. IDH1 mutation was seen in only 1 patient. 25 of 43 (58%) patients screened were mutated for SF3B1, including 4 (9%) who were mutated for both ASXL1 and SF3B1. Significant associations were evident between ASXL1 and IDH2 mutations (P =0.02) but not between ASXL1 and SF3B1 mutations (P =0.61). iii) Clinical correlates of ASXL1 mutations: Among all 76 study patients, presence of ASXL1 mutation did not correlate with age (P =0.30), hemoglobin level (P =0.17), platelet count (P =0.53), BM blast percentage (P =0.17), WHO morphologic category (P =0.34), transfusion dependence (P =0.84), IPSS-R cytogenetic categories (P =0.93), or IPSS-R risk group (P =0.33). The results were unchanged when analyzing the 51 patients with MDS-RS (i.e. ≥15% BM RS). Amongst the ASXL1 mutated patients (n =21) IPSS-R cytogenetic categories were as follows: 13 patients with normal karyotype (62%), 2 patients with trisomy 8 (10%), 1 patient each with -Y, del(11q), del(5q), del(20q), isochromosome 17 and monosomy 7 (5% each). iv) Prognostic impact of ASXL1 mutations: Median follow-up was 42.5 months, during which time 69 (91%) deaths and 8 (11%) leukemic transformations were documented. ASXL1 mutated patients had a median survival of 29 months, compared to 45 months in ASXL1 wild-type patients (P =0.04). However, the difference in median survival was no longer significant during multivariable analysis, which instead identified only IPSS-R and transfusion need as being independent predictors of inferior survival. Amongst those patients without excess blasts (n =63), presence of ASXL1 mutation predicted inferior survival in univariate analysis (P =.04) and significance was sustained during multivariable analysis that included IPSS-R cytogenetic categories (P =.04) but became borderline when WHO morphologic category was included (P =0.06); ASXL1 mutated patients had a shortened median survival of 43 months compared to 66 months in ASXL1 wild-type patients (P =.04). In the presence of ≥15% BM RS, ASXL1 mutations did not affect survival (P =0.48); the results were the same for RARS (P =0.9) and RCMD-RS (P =0.64). Conclusions : ASXL1 mutations might not affect survival in MDS patients with ≥15% BM RS, including those with RARS or RCMD-RS. Furthermore, an apparent survival disadvantage seen in ASXL1 -mutated MDS patients with ≥1% BM RS was accounted for by IPSS-R. Disclosures Pardanani: Stemline: Research Funding.

Abstract Background The median survival (OS) of patients with primary myelofibrosis (PMF; 6.5y) is significantly shortened compared to reference population (Cervantes et al, JCO 2012; 30:2981). OS is predicted by the four risk categories of IPSS, Dynamic-IPSS and DIPSS-plus score, nevertheless pts heterogeneity persists within these categories, necessitating improved risk stratification. We recently reported that pts harboring mutations in any one of the prognostically relevant ASXL1, EZH2, IDH1/2 and SRSF2 genes constitute a IPSS-and DIPPS-plus independent Molecular High Risk category (MHR+) characterized by significant reduction of OS and leukemia free survival (LFS) (Vannucchi et al, Leukemia 2013). The aim of this work was to analyze the impact of the number of prognostically relevant mutated genes on OS and LFS in PMF. Patients and methods Two independent cohorts are included, a “test cohort” from Europe, analyzed at diagnosis, and a “validation cohort” from Mayo Clinic, analyzed at any time after diagnosis. Mutation analysis was performed in DNA from whole blood or granulocytes using RTQ-PCR, HRM and direct sequencing; all mutations were confirmed at least twice. The prognostic value of the molecular variables with regard to OS and LFS was analyzed by Cox regression. Test cohort It included 490 pts (median age 61y; males = 301) risk stratified by IPSS into high (n=74, 15%), intermediate-2 (n=93, 19% ), intermediate-1 (n=147, 30%) and low (n=176, 36%). The median follow-up was 3.63y (95% CI, 0.06-28.33); 161 pts died (33.0%), of whom 76 (15.6%) had progressed to acute leukemia after a median of 3.4y (0.04-28.3) from diagnosis. One hundred forty-six pts (29.8%) presented at least one of the four aforementioned mutated genes and were classified as MHR+. The OS of MHR+ pts was significantly reduced compared to patients with no mutations (n=344): 80.7 vs 148.9 mo (HR 2.2, CI95% 1.6-3.03). One hundred twelve (22.8%) pts had 1 mutation and 34 (6.9%) had 2 or more mutations. In univariate analysis presence of 2 or more mutated genes was significantly more detrimental for OS (29.5mo; HR 4.12, IC95% 2.6-6.4) than having 1 mutated gene (84.2mo; HR 1.8, IC95% 1.2-2.5) compared with having no mutations (148.9mo). Multivariate analysis results adjusted for IPSS indicated that having two or more mutations was an independent prognostic factor for OS (HR 2.9, 95% CI 1.8-4.5). Notably, the prognostic relevance of harboring two or more mutations involved both lower (low and intermediate-1; HR 1.87, 95% CI 1.3-2.6) and higher (intermediate-2 and high; HR 1.6, 95% CI 1.2-2.1) categories of IPSS. Also LFS resulted significantly shorter (129mo; HR 3.1, 95%CI 1.9-4.8) in MHR+ pts compared with pts with no adverse mutations (323mo). Having 2 or more mutations correlated with greater reduction of LFS (79.6mo; HR 7.02, CI95% 3.9-12.6) than having one mutation only (133.9mo; HR 2.2, IC95% 1.3-3.7) as compared with no mutations (323.2mo). Multivariate analysis showed that IPSS high risk category (HR 4.5; 95% CI 2.3-8.8) and two or more mutated genes (HR 5.3; 95% CI 2.9-9.8) predicted independently for a significant reduction in LFS. The negative impact on LFS of having 2 or more mutated genes compared to one or no mutations was maintained in the lower (HR 6.4, 95% CI 2.6-15.2) and higher (HR 5.5, 95% CI 2.3-12.8) risk categories. Validation cohort Validation cohort included 262 patients (median age 64 years; males = 167) risk stratified by DIPSS-plus into high (n=89, 34%), intermediate-2 (n=92, 35%), intermediate-1 (n=46, 18%) and low (n=34, 13%). One hundred forty-six pts (56%) displayed none of the four aforementioned mutations, 93 (36%) harbored one mutation whereas 23 (9%) harbored two or more mutations. In univariate analysis, having two or more mutations (HR 3.7; 95% CI 2.2-6.1) was significantly more detrimental for OS than having no mutations, which is more favorable than having one mutation (HR 1.9; 95% CI 1.4-2.7). When adjusted for DIPSS-plus, the presence of two or more mutations retained its significance (HR 2.1; 95% CI 1.3-3.6) and outperformed ASXL1 mutation alone in its prognostic relevance Conclusions Overall, these results show that the number of prognostically relevant mutated genes correlate with OS and LFS in pts with PMF, suggesting that screening for these mutations might help to improve risk stratification. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: CD123 is the α-subunit of the high-affinity receptor for interleukin-3; its expression in normal bone marrow (BM) is highly restricted, however CD123 is highly expressed in certain hematolymphoid malignancies. In a pilot study of 11 systemic mastocytosis (SM) patients (Lasho et al., Leukemia, 2015), we made the following novel observations (i) CD123 was aberrantly expressed on neoplastic mast cells (MC); and (ii) there was focal proliferation of plasmacytoid dendritic cells (PDC) around MC aggregates, suggesting a tumor promoting role for PDC. Objectives: To study MC CD123 expression by IHC in a larger cohort of SM patients to ascertain: (i) whether there is a significant difference in CD123 expression between SM subtypes; (ii) whether focal PDC proliferation around MC aggregates is correlated with CD123 expression; and (iii) whether CD123 expression has prognostic value for overall survival (OS). Methods: The current study was approved by the Mayo Clinic institutional review board. SM was classified as per standard criteria. Archived BM slides were stained for tryptase and CD123 as previously described (Lasho et al., Leukemia, 2015). Slides were reviewed independently by 2 hematopathologists who were blinded with regards to SM subgroups and scored on the following pre-defined parameters: (i) CD123 expression yes/no; (ii) CD123 expression strong/weak; (iii) focal PDC proliferation yes/no; and (iv) BM MC%. Results: We studied 58 SM patients and 3 controls. Of the former, 23 had indolent SM (ISM), 10 aggressive SM (ASM), 23 SM with associated hematological disease (SM-AHD), and 2 had mast cell leukemia (MCL). Of the SM-AHD patients, 10 (43%) had CMML, 5 (22%) myeloproliferative neoplasm-unclassified (MPN-u), 3 (13%) PV, 2 (9%) acute myeloid leukemia, and 1 each had lymphoma, myeloma and myelodysplastic syndrome. The median follow up from diagnosis was 32 months (range 0.1 to 162). Over this period, 3 patients (5%) had leukemic transformation and 30 patients (52%) died. Concordance rates between hematopathologists for CD123 positive/negative, CD123 strong/weak, focal PDC proliferation yes/no and MC% were 97%, 94%, 98% and 100%, respectively. MC CD123 expression was absent in control cases but demonstrable in 37 (64%) SM patients; expression rates were 100%, 61%, 57%, and 0% in ASM, ISM, SM-AHD and MCL, respectively (p=0.02). Similar to MCL, both cases with SM-AML showed absent MC CD123 expression. Strong MC CD123 expression (comparable to background interstitial PDC) was observed in 23 patients (62%), including 85% with SM-AHD, 60% with ASM, and 43% with ISM (p=0.08). Focal PDC proliferation around MC aggregates, regardless of CD123 expression status, was noted in 44 patients (76%), including 90% with ASM, 87% with ISM, 65% with SM-AHD and 0% with MCL (p=0.02). Focal PDC proliferation was significantly higher in CD123-positive versus -negative patients (87% versus 50%, p=0.005). OS was predicted by SM subtype; the median OS for ISM, ASM, SM-AHD and MCL patients was not reached, 30 months, 25 months and 3 months, respectively (p=0.005). CD123 expression status did not have prognostic value for OS overall, however, in SM-AHD patients, CD123 expression was associated with significantly inferior survival (p=0.04), independent of the associated disease (ie, CMML vs AML vs MPN vs other). Survival outcome of SM-CMML patients expressing MC CD123 (n=3, median OS=11 months) was significantly inferior as compared to patients who did not (n=7, median OS=44 months), independent of age and absolute monocyte count (p=0.01). CD123 staining intensity had borderline significance for OS in the overall cohort (p=0.08). When restricted to non-indolent SM cases, strong CD123 expression was associated with significantly worse overall survival (15 versus 88 months, p=0.04). Conclusions: In this cohort, aberrant MC CD123 expression was observed in the majority of SM patients with highest prevalence in ASM patients and uniformly absent expression in leukemic phase SM (MCL or SM-AML). Focal PDC proliferation around MC aggregates was significantly higher in CD123-positive cases, indicating a potential functional interaction. CD123 expression identified SM-AHD patients with inferior OS, especially those with SM-CMML. Strong CD123 staining intensity may be associated with inferior OS in non-indolent SM patients. CD123 expression on MC and PDC constitutes a novel therapeutic target in non-leukemic SM. Disclosures Pardanani: Stemline: Research Funding. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

Abstract We recently reported that the induction of polyploidization of malignant megakaryocytes shows great promise as a new therapy for acute leukemia. Polyploidization inducers such as dimethylfasudil (diMF) and MLN8237, both of which target Aurora A kinase (AURKA), induce proliferation arrest, polyploidization, expression of megakaryocyte differentiation markers and apoptosis of leukemic megakaryocytes in vitro and in vivo. Since megakaryocytes in primary myelofibrosis (PMF) show impaired polyploidization and maturation, and likely directly contribute to the disease, we predicted that polyploidization inducers would provide a new therapeutic strategy. To determine the effect of these compounds on the growth of MPN cells, we first treated the JAK2 V617F mutant megakaryocytic SET2 cell line with varying doses of MLN8237 and diMF. Both compounds effectively and dose dependently inhibited proliferation, induced polyploidization and upregulation of lineage specific markers CD41 and CD42, and increased apoptosis. Furthermore, MLN8237 synergized with ruxolitinib to induce apoptosis of the SET2 cells and also potently induced growth arrest of JAK2 inhibitor persistent SET2 cells. We observed a similar polyploidization and differentiating activity of MLN8237 and diMF on megakaryocytes derived from primary human PMF progenitors. The ability of these agents to induce polyploidization was specific, as the non-megakaryocyte fractions of the cultures were not affected. Next, we assayed the activity of polyploidization inducers on progression of MPNs in two mouse models: JAK2V617F conditional knockin mice and mice engrafted with MPLW515L expressing bone marrow progenitors. Of note, spleens from both mouse models displayed a robust increase in both total and phosphorylated forms of AURKA relative to control animals, further suggesting that AURKA is a rational target in this disease. We first assayed the activities of MLN8237 and diMF in the MPLW515L bone marrow transplantation model. Recipient mice develop a rapid MPN characterized by leukocytosis, thrombocytosis and bone marrow fibrosis. Both MLN8237 and diMF reduced the disease burden, as evidenced by significant reductions in the liver and spleen weights, white cell counts and platelet counts. Both compounds also led to a significant decrease of fibrosis in the bone marrow, diminished infiltration of megakaryocytes and granulocytes in the liver, and a profound reduction in the numbers of megakaryocytes within the spleen. Moreover, plasma levels of TGF-β a known myelofibrogenic cytokine, were decreased by more than 3-fold by the drug treatment. Both diMF and MLN8237 led to selective polyploidization of megakaryocytes in the spleen as well as marked reductions in the levels of p-AURKA. Of note, neither agent affected the extent of phosphorylation of STAT3 or STAT5. Therefore, we tested whether the combined use of a JAK inhibitor and a polyploidy inducer would show enhanced activity in vivo. Indeed, the combination of MLN8237 and ruxolitinib led to greater reductions in tumor burden in the MPLW515L mouse model than either agent alone. Similar results were obtained using the JAK2V617F knock-in model. To further validate our conclusion that AURKA is a target in PMF, we infected Aurkafl/fl floxed bone marrow progenitors with MPLW515L and transplanted the cells to irradiated recipients. Excision of both alleles of Aurka by Cre mediated recombination completely resolved the disease, while heterozygous deletion of Aurka significantly reduced the disease burden, in a manner similar to treatment with MLN8237. Given that heterozygous deletion of Aurka does not alter normal hematopoiesis in mice, the fact that a 50% reduction in kinase expression was associated with a significant decrease in disease burden suggests that there is an effective therapeutic window in which AURKA inhibitors will be effective against MPN while sparing normal hematopoiesis. Although JAK inhibitors provide symptomatic relief, it is becoming clear that they are not curative. Thus, there is an urgent need to develop new agents to use in combination with JAK inhibitors. Our data reveal that inducing polyploidization and differentiation of dysplastic megakaryocytes in PMF ameliorates features of the disease both in vitro and in vivo. These results support the initiation of clinical studies that combine a JAK inhibitor with an AURKA inhibitor. Disclosures: Crispino: Sanofi: Research Funding.

Abstract Abstract 3069 Background: Monosomal karyotype (MK) is defined as the presence of two or more distinct autosomal chromosome monosomies or a single autosomal monosomy associated with at least one structural abnormality (Breems DA et al. J Clin Oncol 2008; 26: 4791). In acute myeloid leukemia (AML), MK has been shown to be prognostically worse than complex or other unfavorable karyotype (Breems DA et al. J Clin Oncol 2008; 26: 4791). In primary myelofibrosis (PMF), complex karyotype or isolated trisomy 8 predicts inferior survival (Hussein K et al. Blood 2010; 115: 496). Objective: To determine if MK in PMF is prognostically distinct from previously defined poor cytogenetic risk categories including complex karyotype and isolated trisomy 8. Methods: The Mayo Clinic database for PMF was used to identify consecutive patients with unfavorable karyotype including complex karyotype and sole trisomy 8. WHO criteria were used for PMF diagnosis and leukemic transformation (Vardiman JW et al. Blood 2009; 114: 937). Results: Among 793 PMF patients with cytogenetic information at the time of their first time referral to the Mayo Clinic, 452 displayed a normal karyotype and 341 (43%) an abnormal karyotype. Of the latter, 41 (12%) displayed complex karyotype and 21 (6%) sole trisomy 8. Among the 41 patients with complex karyotype, 17 (42%) met the criteria for MK and 24 (58%) displayed complex karyotype without monosomies. Overall survival was significantly inferior in patients with MK compared to those with either complex karyotype without monosomies (p=0.02; HR 2.3, 95% CI 1.1–4.8) or trisomy 8 (p=0.02; HR 2.4, 95% CI 1.2–5.1) (Fig. 1). Prognosis among all three groups was significantly worse than patients with normal karyotype (Fig. 1). Leukemia-free survival was also significantly inferior in patients with MK compared to those with either complex karyotype without monosomies (p=0.02; HR 6.9, 95% CI 1.3–37.3) or trisomy 8 (p=0.02; HR 14.8, 95% CI 1.7–130.8) (Fig. 2). LFS in patients with normal karyotype was similar to those with either complex karyotype without monosomies (p=0.31) or trisomy 8 (p=0.86) (Figure 2). Conclusions: Monosomal karyotype in PMF is distinctly associated with extremely poor overall and leukemia-free survivals that are significantly worse than those seen in PMF patients with other unfavorable karyotype including complex karyotype without monosomies and sole trisomy 8 abnormalities. Disclosures: No relevant conflicts of interest to declare.