Lasho, Terra L.

Abstract Background : In essential thrombocythemia (ET) , ̴ 85% of patients harbor one of three "driver" mutations, with mutational frequencies of approximately 58%, 23% and 4%, for JAK2, CALR and MPL, respectively; ̴ 15% are wild type for all three mutations and are operationally referred to as "triple negative" (Blood. 2014;124:2507). In one of the original descriptions on CALR mutations, CALR -mutated patients with ET, compared to their JAK2-mutated counterparts, were reported to have better survival (NEJM. 2013;369:2379). However, this observation was not supported by subsequent studies while other reports suggested differential prognostic effect from distinct CALR variants in myelofibrosis (Blood. 2014;124:2465). In this study, we sought to clarify the impact of all three mutations, and CALR variants, on overall (OS), myelofibrosis-free (MFS) and leukemia-free (LFS) survival. Methods: Patientswere selected from our institutional database of myeloproliferative neoplasms, based on availability of mutational status inforomation. ET diagnosis was according to WHO criteria (Blood. 2009;114:937). 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 diagnosis to date of death or last contact. MFS and LFS calculations considered fibrotic or leukemic transformation events as uncensored variables, respectively. Cox proportional hazard regression model was used for multivariable analysis. Results : A total of 502 patients (median age 59 year; 61% females) met study eligibility criteria. Median levels of hemoglobin, platelet count and leukocyte counts were 13.7 g/dL, 893 x 10 (9)/L and 8.8 x 10(9)/L, respectively. All patients were annotated for JAK2/CALR/MPL mutations as well as CALR variants; 324 harbored JAK2, 111 CALR and 13 MPL mutations; 54 patients were triple-negative. The 111 CALR-mutated patients included type 1 (n=55), type 2 (n=41) or other (n=15) CALR variants. At a median follow-up time of 9.9 years, 172 (34.3%) deaths, 42 (8.4%) fibrotic progressions, 15 (3%) blast transformations and 12 (2.4%) polycythemic conversions were documented. In univariate analysis, survival data appeared significantly better in "triple negative" patients (median not reached) and inferior in MPL-mutated cases (median 8.5 years) whereas median survival times were similar for JAK2 (18.5 years) and CALR (22.1 years) mutated cases (Figure 1; p=0.0006). However, the difference in survival was no longer apparent (p=0.60) during multivariable analysis that included age and sex, which are known to differentially cluster with specific driver mutations; in the current study, median age/sex distributions for "triple-negative", CALR, JAK2 and MPL mutated cases were 44 years/72% females, 48 years/46% females, 60 years/65% females, 70 years/46% females, respectively (p=<0.0001/0.0007). Of note, both age and sex were independently predictive of shortened survival. OS data remained unchanged when CALR-mutated patients were further stratified into type 1 vs type 2 vs other CALR variants, with similar survival data between the three CALR mutation groups (p=0.98). In univariate analysis, MPL-mutated patients were significantly more prone to fibrotic progression (Figure 2; p=0.0083). The prognostic relevance of MPL mutations to MFS remained significant when age and sex were included in multivariable analysis (p=0.008). In the current cohort, univariate analysis identified lower hemoglobin and lower platelet count as the only other risk factors for fibrotic progression. Multivariable analysis confirmed the independent prognostic relevance of MPL mutations (p=0.003), lower hemoglobin level (p=0.0009) and lower platelet count (p=0.0094) for MFS. There was no significant difference in LFS among the four driver mutational categories (p=0.9): 9 events in JAK2, 6 in CALR, none in triple negative and none in MPL mutated cases. Among the 6 leukemic transformations in CALR-mutated cases, three were type 1, two type 2, and one other CALR variants. Conclusions : Age- and sex-adjusted survival is similar among ET patients with JAK2 vs CALR vs MPL vs "triple-negative" mutational status. Survival is also similar between patients with distinct CALR variants. MPL -mutated patients with ET might be at a higher risk of fibrotic progression. 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 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 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 Background : Gene mutations are common (~90%) in patients with chronic myelomonocytic leukemia (CMML) and involve epigenetic regulators (TET2 ~ 60%, ASXL1 ~40%), spliceosome components (SRSF2 ~40%) and signal pathways (RAS ~30%). Of these, thus far, only ASXL1 mutations have been shown to adversely impact overall survival (OS). In the current study, we used a 27-gene panel assay to identify additional prognostically-relevant mutations in CMML and to also determine if number of mutations carries prognostic relevance. Methods : 175 patients with WHO-defined CMML were included in the study. All patients had bone marrow (BM) biopsies and cytogenetics performed at diagnosis. Targeted capture assays were carried out on BM DNA specimens obtained at diagnosis 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 NEBNext Ultra Library prep protocol on the Agilent Bravo liquid handler. Capture libraries were assembled according to Nimblegen standard library protocol. Base-calling was performed using Illumina's RTA version 1.17.21.3. Genesifter software was utilized to analyze targeted sequence data. Specific variants were deemed as mutations if they were associated with a hematological malignancy (as identified by COSMIC database), or if they were not associated with a dbSNP Results: Among the 175 study patients, 66% were males and median age was 70 years. 146 (83%) patients were subclassified as CMML-1. At a median follow-up of 23 months, 146 (83%) deaths and 25 (14%) leukemic transformations were documented. Median survivals were 24 months for CMML-1 and 16 months for CMML-2 (p=0.38). Mutational frequencies were; TET2 46%, ASXL1 45%, SRSF2 45%, SETBP1 19%, CBL 14%, RUNX1 14%, NRAS 12%, U2AF1 8%, SF3B1 6%, ZRSR2 6%, Tp53 5%, DNMT3A 5%, IDH2 5%, PTPN11 5%, SH2B3 5%, JAK2 4%, NPM1 3%, CSF3R 2%, IDH1 2%, EZH2 1%, SUZ12 1%, KIT 1%, FLT3 1%, CALR 1%. 172 patients (98%) had at least one mutation, 21 (12%) had 2, 24 (14%) had 3, 20 (11%) had 4, 9 (5%) had 5, while one (1%) patient had 6 concurrent mutations. Risk stratification was based on Mayo prognostic model: 25% high, 32% intermediate and 43 % low risk. In univariate analysis, presence of ASXL1 mutations (p=0.01), absence of TET2 mutations (p=0.005) and presence of DNMT3A mutations (p=0.02) were associated with inferior survival; in multivariable analysis, ASXL1 (p=0.01) and TET2 (p=0.03) mutations remained significant. In order to determine prognostic interaction between these two mutations, patients were stratified into four mutational categories: ASXL1wt/TET2wt (n =56), ASXL1mut/TET2wt (n =31), ASXL1mut/TET2mut (n =50) and ASXL1wt/TET2mut (n =38). Survival data in these four groups showed significant difference in favor of ASXL1wt/TET2mut (median survival 38 months; p=0.016), compared to those with ASXL1wt/TET2wt (19 months), ASXL1mut/TET2wt (31 months)and ASXL1mut/TET2mut (16 months); there was no significant difference in survival among the latter three groups (p=0.3) (Figure). The number of mutations per patient did not affect outcome (p=0.3). In multivariable analysis, presence of ASXL1 mutations (P=0.01) and absence of TET2 mutations (p=0.003) remained significant when risk factors used in the Mayo prognostic model (HB <10 gm/dl, AMC >10 x 10(9)/L, platelets <100 x 10(9)/L, circulating IMC) were added to the model; the same was true for ASXL1wt/TET2mut (p=0.036). In a separate multivariable analysis that included the Mayo prognostic model as a single variable along with presence of ASXL1 and absence of TET2 mutations; or absence of ASXL1wt/TET2mut mutational status, the respective hazard ratios were 1.4 (95% CI 1.07-2.1; p=0.012), 1.5 (95% CI 1.07-2.1; p=0.03) and 1.8 (95% CI 1.2-2.7; p=0.001). Leukemia-free survival was worse in ZRSR2 -mutated cases (p=0.03). Conclusions: Almost 100% of patients with CMML express one or more myeloid neoplasm-relevant mutations. The current study suggests a favorable prognostic impact from TET2 mutations, unless accompanied by ASXL1 mutations. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 1280 Poster Board I-302 Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF) are defined as clonal hematopoietic stem cell disorders. These disorders show an inherent tendency for transformation into leukemia (MPN-blast phase) which is hypothesized to be accompanied by acquisition of additional genomic lesions. We, therefore, obtained a comprehensive profile of genomic alterations associated with leukemic transformation by using single-nucleotide polymorphism (SNP) array in 88 MPN patients, as well as 71 cases with MPN-blast phase, and correlated these findings with their clinical parameters. A relatively high number of genomic alterations was found in MPN after leukemic transformation with 4.6 ± 0.6 abnormalities per sample compared to only 1.4 ± 0.2 changes per patient in chronic phase (p<0.001). Compared to the cytogenetic data, SNP-chip analysis detected about 47% additional chromosomal changes in the MPN samples, and 31% more in the MPN-blast phase cases, whereas SNP-array allelokaryotyping practically captured all cytogenetic abnormalities in our study population. Several additionally altered regions were detected in patients with MPN-blast phase compared to chronic phase, including both deletion and copy-number neutral-loss of heterozygosity (CNN-LOH) on chromosome 12p (9%) and 21q (9%), involving ETV6 and RUNX1. Notably, deletion and CNN-LOH on 17p involving TP53 were diagnosed in 18% of MPN-blast phase samples, which was highly associated with preceding treatment with alkylating agents (p=0.016). Moreover, trisomy 8, as well as amplification of 8q24.21 involving the MYC gene, were detected in 13% of patients with MPN-blast phase who were almost exclusively negative for the JAK2V617F mutation. Genome-wide inspection of further critical regions with promising new candidate genes involved in the evolution to the MPN-leukemic phase included deletion and CNN-LOH on 7q22.1 (SH2B2) in 18%, duplication/amplification on 19p13.2 (PIN1, ICAM1, CDC37) in 13% and 21q22.2 (ERG) in 9% of MPN patients with blast crisis. In contrast, we detected a decreased frequency of JAK2V617F in MPN-blast phase samples (52%) compared to chronic phase (71%). Also, the percentage of patients with homozygous mutant JAK2 as a result of CNN-LOH was lower in the MPN-blast phase (43%) compared to the chronic phase (53%). Taken together, the data suggest that gain-of-function mutation of JAK2 is not a perquisite for leukemic transformation. Remarkably, CNN-LOH on either 7q or 9p was related to decreased survival after leukemic transformation (p=0.02 and p=0.012, respectively). Given the variety of allelic imbalances, our data suggest that MPN-blast phase appears to be a heterogeneous disease prone to have evolved multiple mechanisms to provide a proliferative advantage to the abnormal leukemic clone. Our analysis of MPN genomes in the chronic compared to the leukemic stage provided new prognostic insights, as well as novel causative genes which might be involved in the transformation to MPN-blast phase. Disclosures No relevant conflicts of interest to declare.

Abstract Background Imetelstat is a 13-mer lipid-conjugated oligonucleotide that targets the RNA template of human telomerase reverse transcriptase. A pilot study of imetelstat therapy in myelofibrosis showed the induction of complete (CR) or partial remissions, as well as molecular remissions, in a subset of patients (Blood. 2014;124:634). In the particular study, CR was significantly higher in patients with SF3B1 or U2AF1 spliceosome mutations, which provided the rationale for the current study; spliceosome mutations are closely associated with refractory anemia with ring sideroblasts with (RARS-T) or without (RARS) thrombocytosis (Leukemia. 2013;27:1826). Methods Diagnosis of RARS and RARS-T was according to WHO criteria (Blood. 2009;114:937). Study patients were treated with a 2-hour intravenous infusion of 7.5 mg/kg imetelstat (Janssen Biotech Inc., Horsham, PA, USA) every 4 weeks. Drug activity was monitored by both formal response criteria (Blood. 2006;108:419) and effect on spleen size, thrombocytosis and leukocytosis. Adverse events (AEs) were monitored by Common Terminology Criteria for Adverse Events (Version 4.03). Laboratory correlative studies included analysis of mutations. Results Accrual to this signal study is now complete and median time from study registration, as of 5/10/2015, was 17.1 months (range 16.1-20.1). Among the 9 study patients, 5 had RARS-T, 3 had RARS and one had "MDS/MPN overlap". Median age was 70 years (78% males). 8 (89%) patients were transfusion-dependent. One patient (RARS-T) had marked splenomegaly (palpable at 16 cm). Three patients each had leukocytosis and thrombocytosis. Seven (78%) patients had received prior treatments, including erythropoiesis stimulating agents in 6 (67%). Baseline international MDS prognostic scoring system scores were intermediate-1 (n=7) and intermediate-2 (n=2). Karyotype was normal in all but one patient with loss of Y chromosome. Drug activity Median duration of treatment with imetelstat was 13.7 months (range 6.6-17.9). Three (38%) patients achieved transfusion-independence (TI) during treatment, defined as not requiring transfusions for at least 8 weeks; 2 had RARS and one RARS-T. The median time to TI was 11 weeks (range 9-14) and the median TI duration was 28 weeks (range 9-37). One of the 3 "TI" patients also had resolution of leukocytosis and thrombocytosis. A 4th patient experienced >50% decrease in palpable spleen size as well as a decrease in transfusion need. In addition, imetelstat induced normalization of counts in two additional patients with thrombocytosis and leukocytosis. As of 5/10/2015, 4 (44%) patients remain on active treatment and one death had been recorded (deemed unrelated to imetelstat). The 5 treatment discontinuations were: death during active therapy, discovery of second malignancy, progression into acute leukemia and insufficient response (n=2). Safety Treatment-emergent grade 4 neutropenia and thrombocytopenia were seen in 2 (22%) and 1 (11%) patients, respectively; neutropenia, and thrombocytopenia were seen in 4 (44%), and 2 (22%) patients. Grade 3 anemia was seen in 6 (67%) of the patients, however baseline grade 2 anemia was noted in 2 (22%) and grade 3 in 3 (30%) patients. None of the grade ≥3 hematologic toxicities lasted ≥4 weeks.. The following grade 3 non-hematologic events occurred in 1 subject: aspiration, fatigue, and lipase increase and a patient with pre-existing cardiovascular disease history experienced a grade 5 heart failure. Treatment-emergent liver function test abnormalities affected: ALT (grade 1 56%/grade 2 11%), AST (grade 1 56%/grade 2 11%), ALP (grade 1 33%), and total bilirubin (grade 2 11%). For patients that discontinued treatment with imetelstat, these events were mostly reversible. Mutation studies Patients were screened for mutations involving JAK2, SF3B1, U2AF1 and SRSF2. Three patients were mutated for JAK2, 7 for SF3B1 (4 K700E, 2 H662Q, one K666N) and one for U2AF1 (Q157P). All three patients with "TI" (H662Q, K700E, K666N) and the 4th patient with spleen response (K700E) were SF3B1mutated. Post-treatment analysis showed no effect of imetelstat on mutations. Conclusions Treatment with imetelstat may offer clinical benefit in patients with RARS or RARS-T with generally acceptable safety profile and warrants further clinical investigation. Disclosures Al-Kali: Celgene: Research Funding. Wan:Janssen Research & Development: Employment, Equity Ownership.

Abstract Background The Dynamic International Prognostic Scoring System-plus (DIPSS-plus) uses eight adverse factors to predict survival in primary myelofibrosis (PMF): age >65 years, hemoglobin <10 g/dL, leukocytes >25 x 109/L, circulating blasts 31%, constitutional symptoms, red cell transfusion need, platelets <100 x 109/L and unfavorable karyotype (J Clin Oncol. 2011;29:392). The latter two also predicted leukemic transformation. Most recently, an international study of 879 PMF patients identified ASXL1, SRSF2, EZH2 and IDH1/2 mutations as inter-independent predictors of shortened overall or leukemia-free survival (Leukemia 2013Apr 26 doi: 101038/leu2013119. 2013). Molecular prognostication is now well established for acute myeloid leukemia with normal karyotype (NK). There is currently little information on clinical or molecular prognostication in NK-PMF. Methods Diagnosis of PMF was according to WHO criteria. The study population included all referrals to the Mayo Clinic, Rochester, MN, USA or the University of Florence, Florence, Italy. Information on clinical and laboratory parameters and karyotype was available in all study patients, at time of referral, which coincided with time of sample collection for mutation screening. The current study considered only those patients with NK-PMF. ASXL1, EZH2, SRSF2, IDH1/2 and JAK2 mutations were performed on variable number of patients depending on bone marrow or granulocyte DNA availability. Clinical parameters examined for prognostic relevance included those listed for DIPSS-plus, less unfavorable karyotype. Results A total of 690 patients with NK-PMF patients were studied. Median age was 65 years (14-89). The percentage of patients with age >65 years was 48%, red cell transfusion dependent 35%, hemoglobin <10 g/dL 49%, platelets <100 x 10(9)/L 19%, leukocyte count >25 x 10(9)/L 15%, circulating blasts ≥1% 49% and constitutional symptoms 33%. At a median follow-up of 29 months, 351 (51%) deaths and 39 (6%) leukemic transformations were recorded. Mutational frequencies and clinical correlates The respective frequencies (mutated/number of patients studied) of JAK2V617F, ASXL1, EZH2, SRSF2 and IDH1/2 mutations were 60% (284/473), 34% (72/214), 8% (14/179), 15% (37/249) and 5% (14/262). There was no significant association between JAK2V617F and any one of the aforementioned mutations. Inter-mutation association was evident only between SRSF2 and IDH1/2 (p=0.0005). Each one of the DIPSS-plus risk parameters was examined for correlation with a specific mutation: ASXL1 was associated with leukocyte count >25 x 10(9)/L (p<0.0001) and circulating blasts ≥1% (p=0.0005); EZH2 with leukocyte count >25 x 10(9)/L (p=0.008); and SRSF2 with age >65 years (p=0.0007), transfusion need (p=0.04) and hemoglobin <10 g/dL (p=0.03). JAK2V617F was associated with age>65 years (p=0.003) and leukocyte count >25 x 10(9)/L (p=0.02). Predictors of overall and leukemia-free survival In univariate analysis, all 7 DIPSS-plus parameters, as well as ASXL1, EZH2, SRSF2 and IDH1/2 mutations showed significant association with shortened survival: When each of these analyses was adjusted for age, all except constitutional symptoms and SRSF2 mutations retained their significance. Multivariable analysis of the six age-independent DIPSS-plus variables identified all but transfusion-need as independent predictors of inferior survival. A similar analysis restricted to mutations identified ASXL1 and EZH2 as being independently adverse. When both mutations and DIPSS-plus variables were included in the multivariable model, only four parameters remained significant: age >65 years (HR 4.2; p<0.0001), platelets <100 x 10(9)/L (HR 3.4; p<0.0001), ASXL1 mutations (HR 2.2; p=0.0001) and EZH2 mutations (HR 2.7; p=0.001). A similar analysis identified SRSF2 mutations (HR 5.9; p=0.0003) and platelets <100 x 10(9)/L (HR 4.3; p=0.005) as the only predictors of leukemia-free survival. Conclusions In NK-PMF, molecular markers might be prognostically more useful than conventional models that rely on clinical features. In the current study, thrombocytopenia was the only clinical variable, other than age, with additional value to molecular prediction of survival and leukemic transformation. Disclosures: No relevant conflicts of interest to declare.