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Determining the underlying cause of persistent eosinophilia is important for effective clinical management but remains a diagnostic challenge in many cases. We identified STAT5B N642H, an established oncogenic mutation, in 27/1715 (1.6%) cases referred for investigation of eosinophilia. Of the 27 mutated cases, a working diagnosis of hypereosinophilic syndrome (HES; n = 7) or a myeloid neoplasm with eosinophilia (n = 20) had been made prior to the detection of STAT5B N642H. Myeloid panel analysis identified a median of 2 additional mutated genes (range 0–4) with 4 cases having STAT5B N642H as a sole abnormality. STAT5B N642H was absent in cultured T cells of 4/4 positive cases. Individuals with SF3B1 mutations (9/27; 33%) or STAT5B N642H as a sole abnormality had a markedly better overall survival compared to cases with other additional mutations (median 65 months vs. 14 months; hazard ratio = 8.1; P

Objectives To introduce a new sonographic marker of intrathoracic liver herniation in fetuses with left-sided congenital diaphragmatic hernia (CDH). Methods In a consecutive series of fetuses with isolated CDH, an ultrasound volume of the fetal abdomen was acquired. On this volume, offline calculation of the angle formed by the midline of the abdomen (joining the center of the vertebral body to the abdominal insertion of the umbilical cord) and a second line joining the center of the vertebral body to the intra-abdominal convexity of the umbilical vein was carried out to give the umbilical vein deviation angle (UVDA). The UVDA was measured in a group of normal fetuses selected as controls. At follow-up, the presence of liver herniation was investigated in all cases of CDH. UVDA values were compared between the CDH group and controls, and between CDH ‘liver-up’ vs ‘liver-down’ cases. A receiver–operating characteristics (ROC) curve was constructed to identify a cut-off value of the UVDA with the highest accuracy in predicting liver herniation in the CDH group. Results Between 2009 and 2015, 22 cases of left-sided CDH were included in the study group, of which nine cases had liver herniation. Eighty-eight normal fetuses were recruited as controls. The UVDA was significantly higher in the cases vs controls (15.25 ± 7.91° vs 7.68 ± 1.55°; P

Splanchnic vein thrombosis (SVT) is one of the vascular complications of myeloproliferative neoplasms (MPN). We designed a phase 2 clinical trial to evaluate safety and efficacy of ruxolitinib in reducing splenomegaly and improving disease-related symptoms in patients with MPN-associated SVT. Patients diagnosed with myelofibrosis (12 cases), polycythemia vera (5 cases) and essential thrombocythemia (4 cases) received ruxolitinib for 24 weeks in the core study period. Spleen volume was assessed by magnetic resonance imaging (MRI) and splanchnic vein circulation by echo-Doppler analysis. Nineteen patients carried JAK2V617F, one had MPLW515L, and one CALRL367fs*46 mutation. Eighteen patients had spleno-portal-mesenteric thrombosis, two had Budd-Chiari syndrome, and one had both sites involved; 16 patients had esophageal varices. Ruxolitinib was well tolerated with hematological toxicities consistent with those of patients without SVT and no hemorrhagic adverse events were recorded. After 24 weeks of treatment, spleen volume reduction ≥35% by MRI was achieved by 6/21 (29%) patients, and a ≥50% spleen length reduction by palpation at any time up to week 24 was obtained by 13/21 (62%) patients. At week 72, 8 of the 13 (62%) patients maintained the spleen response by palpation. No significant effect of treatment on esophageal varices or in splanchnic circulation was observed. MPN-related symptoms, evaluated by MPN-symptom assessment form (SAF) TSS questionnaire, improved significantly during the first 4 weeks and remained stable up to week 24. In conclusion, this trial shows that ruxolitinib is safe in patients with MPN-associated SVT, and effective in reducing spleen size and disease-related symptoms.

The prognostic significance of bone marrow (BM) fibrosis grade in patients with primary myelofibrosis (PMF) is still debated. A fibrosis grade greater than 1 was shown to associate with higher risk of death, and addition of fibrosis grade to IPSS score resulted in a more accurate prediction of survival. The aim of this study was to analyze the prognostic impact of BM fibrosis in 490 patients with PMF, evaluated at diagnosis, molecularly annotated and with extensive follow-up information. We found that fibrosis grade 2 and greater on a 0-3 scale was associated with clinical characteristics indicative of a more advanced disease, such as anemia, leukopenia, thrombocytopenia, constitutional symptoms, larger splenomegaly and a higher IPSS risk category. Patients with higher grade of fibrosis were also more likely to have additional somatic mutations in ASXL1 and EZH2, that are prognostically adverse. Median survival was significantly reduced in patients with grade 2 and 3 fibrosis as compared with grade 1; this effect was maintained when analysis was restricted to younger patients. In multivariate analysis, fibrosis grade independently predicted for survival regardless of IPSS variables and mutational status; the adverse impact of fibrosis was noticeable especially in lower IPSS risk categories. Overall, results indicate that higher grades of fibrosis correlate with unique clinical and molecular aspects and represent an independent adverse variable in patients with PMF; these observations deserve confirmation in prospectively designed series of patients. Am. J. Hematol. 91:918-922, 2016. © 2016 Wiley Periodicals, Inc.

Systemic mastocytosis is a rare heterogeneous myeloproliferative neoplasm characterized by abnormal proliferation and activation of mast cells. We describe a large multicentre series of 460 adult patients with systemic mastocytosis, with a diagnosis based on WHO 2008 criteria, in a "real-life" setting of ten Italian centers with dedicated multidisciplinary programs. We included indolent forms with (n = 255) and without (n = 165) skin lesions, smouldering (n = 20), aggressive (n = 28), associated with other hematological diseases mastocytosis (n = 21) and mast cell leukemia (n = 1). This series was uniquely characterized by a substantial proportion of patients with low burden of neoplastic mast cells; notably, 38% of cases were diagnosed using only minor diagnostic criteria according to WHO 2008 classification, underlying the feasibility of early diagnosis where all diagnostic approaches are made available. This has particular clinical relevance for prevention of anaphylaxis manifestations, that were typically associated with indolent forms. In multivariate analysis, the most important features associated with shortened overall survival were disease subtype and age at diagnosis >60 years. Disease progression was correlated with mastocytosis subtype and thrombocytopenia. As many as 32% of patients with aggressive mastocytosis suffered from early evolution into acute leukemia. Overall, this study provides novel information about diagnostic approaches and current presentation of patients with SM and underlines the importance of networks and specialized centers to facilitate early diagnosis and prevent disease-associated manifestations. Am. J. Hematol. 91:692-699, 2016. © 2016 Wiley Periodicals, Inc.

Transformation to secondary myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PPV-MF) and essential thrombocythemia (PET-MF). Although primary (PMF) and secondary MF are considered similar diseases and managed similarly, there are few studies specifically focused on the latter. The aim of this study was to characterize the mutation landscape, and describe the main clinical correlates and prognostic implications of mutations, in a series of 359 patients with PPV-MF and PET-MF. Compared with PV and ET, the JAK2V617F and CALR mutated allele burden was significantly higher in PPV-MF and/or PET-MF, indicating a role for accumulation of mutated alleles in the process of transformation to MF. However, neither the allele burden nor the type of driver mutation influenced overall survival (OS), while absence of any driver mutation (triple negativity) was associated with significant reduction of OS in PET-MF, similar to PMF. Of the five interrogated subclonal mutations (ASXL1, EZH2, SRSF2, IDH1, and IDH2), that comprise a prognostically detrimental high molecular risk (HMR) category in PMF, only SRSF2 mutations were associated with reduced survival in PET-MF, and no additional mutation profile with prognostic relevance was highlighted. Overall, these data indicate that the molecular landscape of secondary forms of MF is different from PMF, suggesting that unknown mutational events might contribute to the progression from chronic phase disease to myelofibrosis. These findings also support more extended genotyping approaches aimed at identifying novel molecular abnormalities with prognostic relevance for patients with PPV-MF and PET-MF. Am. J. Hematol. 91:681-686, 2016. © 2016 Wiley Periodicals, Inc.

Refractoriness to ruxolitinib in patients with myelofibrosis (MF) was associated with clonal evolution; however, whether genetic instability is promoted by ruxolitinib remains unsettled. We evaluated the mutation landscape in 71 MF patients receiving ruxolitinib (n = 46) and hydroxyurea (n = 25) and correlated with response. A spleen volume response (SVR) was obtained in 57% and 12%, respectively. Highly heterogenous patterns of mutation acquisition/loss and/or changes of variant allele frequency (VAF) were observed in the 2 patient groups without remarkable differences. In patients receiving ruxolitinib, driver mutation type and high-molecular risk profile (HMR) at baseline did not impact on response rate, while HMR and sole ASXL1 mutations predicted for SVR loss at 3 years. In patients with SVR, a decrease of ≥ 20% of JAK2V617F VAF predicted for SVR duration. VAF increase of non-driver mutations and clonal progression at follow-up correlated with SVR loss and treatment discontinuation, and clonal progression also predicted for shorter survival. These data indicate that (i) ruxolitinib does not appreciably promote clonal evolution compared with hydroxyurea, (ii) VAF increase of pre-existing and/or (ii) acquisition of new mutations while on treatment correlated with higher rate of discontinuation and/or death, and (iv) reduction of JAK2V617F VAF associated with SVR duration.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hyperplastic megakaryopoiesis and myelofibrosis. We recently described the upregulation of MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) in PMF CD34+ hematopoietic progenitor cells (HPCs) compared to healthy donor. Here we demonstrated that MAF is also upregulated in PMF compared with the essential thrombocytemia (ET) and polycytemia vera (PV) HPCs. MAF overexpression and knockdown experiments shed some light into the role of MAF in PMF pathogenesis, by demonstrating that MAF favors the megakaryocyte and monocyte/macrophage commitment of HPCs and leads to the increased expression of proinflammatory and profibrotic mediators. Among them, we focused our further studies on SPP1 and LGALS3. We assessed SPP1 and LGALS3 protein levels in 115 PMF, 47 ET and 24 PV patients plasma samples and we found that SPP1 plasma levels are significantly higher in PMF compared with ET and PV patients. Furthermore, in vitro assays demonstrated that SPP1 promotes fibroblasts and mesenchymal stromal cells proliferation and collagen production. Strikingly, clinical correlation analyses uncovered that higher SPP1 plasma levels in PMF patients correlate with a more severe fibrosis degree and a shorter overall survival. Collectively our data unveil that MAF overexpression contributes to PMF pathogenesis by driving the deranged production of the profibrotic mediator SPP1.

Polycythemia vera (PV) and essential thrombocythemia (ET) are Philadelphia-negative myeloproliferative neoplasms (MPNs) characterized by erythrocytosis and thrombocytosis, respectively. Approximately 95% of PV and 50–70% of ET patients harbor the V617F mutation in the exon 14 of JAK2 gene, while about 20–30% of ET patients carry CALRins5 or CALRdel52 mutations. These ET CALR-mutated subjects show higher platelet count and lower thrombotic risk compared to JAK2-mutated patients. Here, we showed that CALR-mutated and JAK2V617F-positive CD34+ cells display different gene and miRNA expression profiles. Indeed, we highlighted several pathways differentially activated between JAK2V617F- and CALR-mutated progenitors, i.e., mTOR, MAPK/PI3K, and MYC pathways. Furthermore, we unveiled that the expression of several genes involved in DNA repair, chromatin remodeling, splicing, and chromatid cohesion are decreased in CALR-mutated cells. According to the low risk of thrombosis in CALR-mutated patients, we also found the downregulation of several genes involved in thrombin signaling and platelet activation. As a whole, these data support the model that CALR-mutated ET could be considered as a distinct disease entity from JAK2V617F-positive MPNs and may provide the molecular basis supporting the different clinical features of these patients.

The 2013 discovery of calreticulin (CALR) mutations in myeloproliferative neoplasms was attended by their association with longer survival in primary myelofibrosis (PMF). Subsequent studies have suggested prognostic distinction between type 1/like and type 2/like CALR mutations and detrimental effect from triple-negative mutational status. Among 709 Mayo Clinic patients with PMF, 467 (66%) harbored JAK2, 112 (16%) CALR type 1/like, 24 (3.4%) CALR type 2/like, 38 (5.4%) MPL mutations and 68 (10%) were triple-negative. Survival was longer with type 1/like CALR, compared to JAK2 (HR 2.6, 95% CI 1.9-3.5), type 2/like CALR (HR 2.5, 95% CI 1.4-4.5), MPL (HR 1.8, 95% CI 1.1-2.9) and triple-negative mutational status (HR 2.4, 95% CI 1.6-3.6), but otherwise similar between the non-type 1/like CALR mutational states (P = .41). In multivariable analysis, the absence of type 1/like CALR (P

With the intent of dissecting the molecular complexity of Philadelphia-negative myeloproliferative neoplasms (MPN), we designed a target enrichment panel to explore, using next-generation sequencing (NGS), the mutational status of an extensive list of 2000 cancer-associated genes and microRNAs. The genomic DNA of granulocytes and in vitro-expanded CD3+T-lymphocytes, as a germline control, was target-enriched and sequenced in a learning cohort of 20 MPN patients using Roche 454 technology. We identified 141 genuine somatic mutations, most of which were not previously described. To test the frequency of the identified variants, a larger validation cohort of 189 MPN patients was additionally screened for these mutations using Ion Torrent AmpliSeq NGS. Excluding the genes already described in MPN, for 8 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, POLG and NRAS), we demonstrated a mutation frequency between 3 and 8%. We also found that mutations at codon 12 of NRAS (NRASG12V and NRASG12D) were significantly associated, for primary myelofibrosis (PMF), with highest dynamic international prognostic scoring system (DIPSS)-plus score categories. This association was then confirmed in 66 additional PMF patients composing a final dataset of 168 PMF showing a NRAS mutation frequency of 4.7%, which was associated with a worse outcome, as defined by the DIPSS plus score.

The aim of the study was to evaluate the feasibility of obtaining the three-vessel and trachea view (3VTV) in an unselected population undergoing first trimester screening for aneuploidy, and to investigate its role in the early detection of congenital heart defects (CHD).Cardiac examination was performed by expert sonographers. Abnormal findings of 3VTV were classified in three different subgroups: number, size and spatial relationship of the vessels.We enrolled 6350 consecutive singleton pregnancies and included 5343 cases. Examination of 3VTV was feasible in 94% of cases. Fifty-seven (1%) CHD were present in the study period; 24 cases were excluded because parents opted for termination of pregnancy. Of the remaining 33 cases, 25 were suspected at the first trimester and eight were detected only at the mid-trimester. An abnormal 3VTV was suspected in 22 cases, and it was confirmed in 21. Five cases that were erroneously classified in the subgroup of abnormal vessel number were actually characterized by a diminutive size of one of the great arteries. The detection rate for CHD, including 4-CV and 3VTV, was 75.8%.Our study demonstrates that 3VTV is an easy plane to obtain by expert sonographers in an unselected population during first trimester. Typical suspicions include detection of abnormal number, size or spatial relationship of the vessels. © 2017 John WileySons, Ltd.

The 2016 revision of the World Health Organization (WHO) classification of myeloproliferative neoplasms defines 2 stages of primary myelofibrosis (PMF): prefibrotic/early (pre-PMF) and overt fibrotic (overt PMF) phase. In this work, we studied the clinical and molecular features of patients belonging to these categories of PMF. The diagnosis of 661 PMF patients with a bone marrow biopsy at presentation was revised according to modern criteria; clinical information and annotation of somatic mutations in both driver and selected nondriver myeloid genes were available for all patients. Compared with pre-PMF, overt PMF was enriched in patients with anemia, thrombocytopenia, leukopenia, higher blast count, symptoms, large splenomegaly, and unfavorable karyotype. The different types of driver mutations were similarly distributed between the 2 categories, whereas selected mutations comprising the high mutation risk (HMR) category (any mutations in ASXL1, SRSF2, IDH1/2, EZH2) were more represented in overt PMF. More patients with overt PMF were in higher International Prognostic Scoring System risk categories at diagnosis, and the frequency increased during follow-up, suggesting greater propensity to disease progression compared with pre-PMF. Median survival was significantly shortened in overt PMF (7.2 vs 17.6 years), with triple negativity for driver mutations and presence of HMR mutations representing independent predictors of unfavorable outcome. The findings of this "real-life" study indicate that adherence to 2016 WHO criteria allows for identification of 2 distinct categories of patients with PMF where increased grades of fibrosis are associated with more pronounced disease manifestations, adverse mutation profile, and worse outcome, overall suggesting they might represent a phenotypic continuum.

Despite many highly effective methods of contraception are available nowadays, many pregnancies are unintended. Emergency contraception (EC) is the use of drug or device after unprotected intercourse to prevent an unwanted pregnancy. It is a woman's last chance to prevent unintended pregnancy. Nevertheless the confusion about mechanisms of action, side effects, clinical efficacy and controindications makes the intervention underused in every setting investigated. So far levonorgestrel (LNG) has been considered the gold standard for oral EC. Today, a new type of second generation progesterone receptor modulator, ulipristal acetate (UPA) has been proposed as a more effective drug than LNG in prevention of unwanted pregnancies by delaying or inhibiting ovulation; even if many other devices are disposable in commerce. We revised the literature to concern most of the data available on the role of EC and moreover clarifying the available methods, the action windows of the accessible devices, the adverse events and the controindications.

We genotyped 370 subjects with primary myelofibrosis (PMF) and 148 with postpolycythemia vera/postessential thrombocythemia (PPV/PET) MF for mutations of EZH2. Mutational status at diagnosis was correlated with hematologic parameters, clinical manifestations, and outcome. A total of 25 different EZH2 mutations were detected in 5.9% of PMF, 1.2% of PPV-MF, and 9.4% of PET-MF patients; most were exonic heterozygous missense changes. EZH2 mutation coexisted with JAK2V617F or ASXL1 mutation in 12 of 29 (41.4%) and 6 of 27 (22.2%) evaluated patients; TET2 and CBL mutations were found in 2 and 1 patients, respectively. EZH2-mutated PMF patients had significantly higher leukocyte counts, blast-cell counts, and larger spleens at diagnosis, and most of them (52.6%) were in the high-risk International Prognostic Score System (IPSS) category. After a median follow-up of 39 months, 128 patients (25.9%) died, 81 (63.3%) because of leukemia. Leukemia-free survival (LFS) and overall survival (OS) were significantly reduced in EZH2-mutated PMF patients (P = .028 and P < .001, respectively); no such impact was seen for PPV/PET-MF patients, possibly due to the low number of mutated cases. In multivariate analysis, survival of PMF patients was predicted by IPSS high-risk category, a < 25% JAK2V617F allele burden, and EZH2 mutation status. We conclude that EZH2 mutations are independently associated with shorter survival in patients with PMF.

Low birth weight, caused either by preterm birth or by intrauterine growth restriction, has recently been associated with increased rates of adult renal and cardiovascular disease. Since aortic intima–media thickening is a noninvasive marker of preclinical vascular disease, we compared abdominal aortic intima–media thickness among intrauterine growth restricted and equivalent gestational age fetuses in utero and at 18 months of age. The relationship between intrauterine growth restriction, fetal aortic thickening, and glomerular function during infancy was measured by enrolling 44 mothers with single-fetus pregnancies at 32 weeks gestation: 23 growth restricted and 21 of appropriate gestational age as controls. Abdominal aortic intima–media thickness was measured by ultrasound at enrollment and again at 18 months of age. Fetuses with intrauterine growth restriction had significantly higher abdominal aortic intima–media thickness compared with age controls when measured both in utero and at 18 months. At 18 months, the median urinary microalbumin and median albumin–creatinine ratio were significantly higher in those infants who experienced intrauterine growth restriction compared to the controls. Our results show that intrauterine growth restriction is associated with persistent aortic wall thickening and significantly higher microalbuminuria during infancy.

Background. Progression to acute myeloid leukemia (sAML) occurs in 20% of myelofibrosis (MF) and 10% of polycythemia vera (PV) or essential thrombocythemia (ET). sAML has dismal outcome with median survival of Aim. To study clonal heterogeneity and clonal progression in the leukemic transformation of MPN we performed whole exome (WES) and trascriptome (RNAseq) sequencing of paired samples (chronic (CP)/blast phase(BP). Patients and Methods. WES and RNAseq was done on CD34+ or blast cells of 12 paired samples from 5 PMF, 3 PET-MF, 1 PPV-MF, 3 ET pts who transformed to sAML. In addition, a myeloid neoplasm-relevant 34-gene panel was used for target NGS sequencing of paired samples plus an additional 23 samples collected from MPN pts at leukemic phase (15 PMF, 3 PET-MF, 3 ET, 2 PV). Results. In the entire series, 20 pts (57%) were JAK2 V617F mutated at CP, 8 CALR (23%; 6 type-1 and 2 type-2) and 7 (8.6%) MPL mut, while 4 (11.4%) were triple-negative. JAK2 variant allele frequency (VAF) declined by ≥50% (n=3; 15%) or became undetectable (n=4; 20%) whereas 3 heterozygous pts (15%) became homozygous at the time of BP. One of the 8 CALR mut cases halved its VAF, whereas no meaningful VAF changes were observed in MPL mut pts. The most frequent mutated genes detected by NGS at BP, other than driver mutations, were ASXL1 (51.4%), RUNX1 (37.1%), TET2 (17%), SRSF2 (16%), IDH1 (14%), TP53 (14%), NRAS (14%), FLT3 (14%), U2AF1 (11%) and KRAS (11%); three (8%) cases were mutated for EZH2DNMT3A, CBL and PTPN11, 2 cases for IDH2, SH2B3, SF3B1 IKZF1, SETBP1 and ZRSR2, and 1 case for ABL1, ATV6, BRAF and ARID1A. There were also 5 CEBPA-mutated pts, 3 of which, unlike none of 27 CEBPA-mutated de novo AML, showed an in-frame 6-bp duplication polymorphism (p. P196_197insHP) reported in 20-40% of all de novo AML and By WES analysis, on average 60.000 variants per paired sample unique to the BP compared with CP were identified. However, no recurrent abnormalities were found in the 12 paired samples outside the above listed mutations found by target sequencing. For RNAseq analysis, transcripts were annotated according to UCSC hg19 for a total of 25,369 genes. We found large transcriptional differences between CP and BP with 129 transcripts differentially expressed (23 up- and 106 down-regulated) and 4,155 isoforms (2,120 up- and 2,035 down-regulated). Among the most abnormally expressed transcripts we selected 8 genes (5 down-regulated: LCN2, PDGFB, PRTG, CRISP3, PF4; 3 up-regulated CDKN2, SH2D1A and LIN28) for validation by QRT-PCR based on the extent of differential expression. PF4 and CDKN2 were confirmed to be down-regulated and over-expressed in BP, respectively (P Conclusions. Comprehensive comparative analysis of genomic and RNA abnormalities acquired in the transition from CP to BP in MPN has not been previously reported. Our data indicate that BP is associated with significant changes in mutation complexity and RNA expression, overall affecting different intracellular pathways whose further characterization might help to identifying potential targets for therapy. Disclosures No relevant conflicts of interest to declare.

The prognostic significance of bone marrow (BM) fibrosis grade in patients with primary myelofibrosis (PMF) is still debated. A fibrosis grade greater than 1 was shown to associate with higher risk of death, and addition of fibrosis grade to IPSS score resulted in a more accurate prediction of survival. The aim of this study was to analyze the prognostic impact of BM fibrosis in 490 patients with PMF, evaluated at diagnosis, molecularly annotated and with extensive follow-up information. We found that fibrosis grade 2 and greater on a 0-3 scale was associated with clinical characteristics indicative of a more advanced disease, such as anemia, leukopenia, thrombocytopenia, constitutional symptoms, larger splenomegaly and a higher IPSS risk category. Patients with higher grade of fibrosis were also more likely to have additional somatic mutations in ASXL1 and EZH2, that are prognostically adverse. Median survival was significantly reduced in patients with grade 2 and 3 fibrosis as compared with grade 1; this effect was maintained when analysis was restricted to younger patients. In multivariate analysis, fibrosis grade independently predicted for survival regardless of IPSS variables and mutational status; the adverse impact of fibrosis was noticeable especially in lower IPSS risk categories. Overall, results indicate that higher grades of fibrosis correlate with unique clinical and molecular aspects and represent an independent adverse variable in patients with PMF; these observations deserve confirmation in prospectively designed series of patients. Am. J. Hematol. 91:918-922, 2016. © 2016 Wiley Periodicals, Inc.

Primary myelofibrosis (PMF) is a Myeloproliferative Neoplasm (MPN) characterized by megakaryocyte hyperplasia, progressive bone marrow fibrosis, extramedullary hematopoiesis and transformation to Acute Myeloid Leukemia (AML). A number of phenotypic driver (JAK2, CALR, MPL) and additional subclonal mutations have been described in PMF, pointing to a complex genomic landscape. To discover novel genomic lesions that can contribute to disease phenotype and/or development, we integrated gene expression and copy number (CN) signals and identified several genomic abnormalities leading to a concordant alteration in gene expression levels. In particular, copy number gain in the polyamine oxidase (PAOX) gene locus is accompanied by a coordinated transcriptional up-regulation in PMF patients. To assess the impact of PAOX deregulation on the survival of PMF and normal primary hematopoietic cells, PMF and normal donors cells were incubated with increasing doses of the PAOX inhibitor MDL-72,572. PAOX inhibition resulted in rapid cell death of PMF progenitor cells (5,7±0,9% of late apoptotic cells in NT sample vs 20,1±3,2% in 150μM treated-sample, p Moreover, our integrative analysis of gene expression and CN data pointed out the concomitant copy number loss and transcriptional down-regulation in PMF patients of the chromatin modifier HMGXB4. Interestingly, silencing of HMGXB4 in CD34+ stem/progenitor cells induced megakaryocyte differentiation, as demonstrated by increased expression of CD41 (36±1,9% vs 26,1±2,9% at day 12, 52,5±4,9% vs 33±1% at day 14 of serum free liquid culture, p In conclusion, our work sheds light on the influence of genomic abnormalities on gene expression regulation in PMF CD34+ cells and on their impact to features typical of PMF, such as a hyperplastic megakaryopoiesis and resistance to apoptosis, and therefore potentially contributing to the development of the myeloproliferative disease. Disclosures 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.

We recently defined a high-molecular risk category (HMR) in primary myelofibrosis (PMF), based on the presence of at least one of the five 'prognostically detrimental' mutated genes (ASXL1, EZH2, SRSF2 and IDH1/2). Herein, we evaluate the additional prognostic value of the 'number' of mutated genes. A total of 797 patients were recruited from Europe (n=537) and the Mayo Clinic (n=260). In the European cohort, 167 (31%) patients were HMR: 127 (23.6%) had one and 40 (7.4%) had two or more mutated genes. The presence of two or more mutations predicted the worst survival: median 2.6 years (hazard ratio (HR) 3.8, 95% confidence interval (CI) 2.6-5.7) vs. 7.0 years (HR 1.9, 95% CI 1.4-2.6) for one mutation vs 12.3 years for no mutations. The results were validated in the Mayo cohort and prognostic significance in both cohorts was independent of International Prognostic Scoring System (IPSS; HR 2.4, 95% CI 1.6-3.6) and dynamic IPSS (DIPSS)-plus (HR 1.9, 95% CI 1.2-3.1), respectively. Two or more mutations were also associated with shortened leukemia-free survival (HR 6.2, 95% CI 3.5-10.7), also Mayo validated. Calreticulin mutations favorably affected survival, independently of both number of mutations and IPSS/DIPSS-plus. We conclude that the 'number' of prognostically detrimental mutations provides added value in the combined molecular and clinical prognostication of PMF.

Mutations in the calreticulin (CALR) gene were recently discovered in patients with essential thrombocythemia (ET) lacking the JAK2V617F and MPLW515 mutations, but no information is available on the clinical correlates. In this series, CALR mutations were found in 15.5% of 576 World Health Organization-defined ET patients, accounting for 48.9% of JAK2 and MPL wild-type (wt) patients. CALR-mutated patients were preferentially male and showed higher platelet count and lower hemoglobin and leukocyte count compared with JAK2- and MPL-mutated patients. Patients carrying the CALR mutation had a lower risk of thrombosis than JAK2- and MPL-mutated patients; of interest, their risk was superimposable to patients who were wt for the above mutations. CALR mutation had no impact on survival or transformation to post-ET myelofibrosis. Genotyping for CALR mutations represents a novel useful tool for establishing a clonal myeloproliferative disorder in JAK2 and MPL wt patients with thrombocytosis and may have prognostic and therapeutic relevance.

Objective To examine cardiac function in appropriately grown, small for gestational age and intrauterine growth restricted fetuses and investigate the relationship between cardiac function and fetal arterial and venous Doppler parameters. Study design Myocardial performance index, isovolumetric contraction time, isovolumetric relaxation time, ejection time, and umbilical artery, middle cerebral artery and ductus venosus Doppler pulsatility index were measured for women between 24 and 32 weeks with small for gestational age and intrauterine growth restricted fetuses. Forty-eight appropriately grown, 11 small for gestational age and 12 intrauterine growth restricted cases were included. The relationship between cardiovascular parameters and gestation was defined and Doppler values converted to Z -scores in relation to gestational age. Results In small for gestational age fetuses and fetuses with intrauterine growth restriction the myocardial performance index was 0.66 (0.63–0.7) and 0.64 (0.60–0.67), respectively, and compared to appropriately grown fetuses, at 0.45 (0.43–0.47), was significantly increased ( p = 0.001). No relationship was found between the myocardial performance index and arterial and venous Doppler Z -score. Conclusion Small for gestational age and intrauterine growth restricted fetuses demonstrate altered cardiac function in the late second and early third trimester of pregnancy. Importantly, the myocardial performance index is raised in small for gestational age fetuses before the arterial and venous Doppler abnormalities that characterize hypoxia are evident.

Introduction: Systemic mastocytosis (SM) is an heterogeneous myeloproliferative neoplasm characterized by uncontrolled growth of cKIT mutated clonal mast cells, with clinical manifestations ranging from indolent forms (ISM) with only skin lesions and/or anaphylactic shock to smouldering SM (SSM) with evidence of organ involvement to aggressive forms (ASM) with extensive organ damage. SM with an associated hematological neoplasm (SM-AHN) is a subtype with adverse prognosis possibly related to the associated neoplasm rather than SM. Recent data indicate that mutations in genes associated to myeloid malignancies correlate with survival and disease progression but information are still scanty and not completely concordant, and mutations apparently did not correlate with disease manifestations; furthermore, most patients analyzed were SM-AHN, therefore the contribution of mutations associated with the non-mast cell clone could not be ascertained clearly. The aim of this study was to address the prognostic relevance of mutations at diagnosis in a series of 70 pts with SM, excluding SM-AHD. Methods: There were 58 ISM, 4 SSM, 7 ASM and 1 mast cell leukemia (MCL) pts. cKITD816V mutation was assessed by RTQ-PCR. Mutations in myeloid-neoplasm associated genes were assessed by next generation sequencing (NGS) with Ion Torrent PGM platform; analyzed genes included ASXL1, CBL, cKIT, ETNK1, EZH2, IDH1, IDH2, SRSF2, RUNX1, TET2. We also evaluated the cKITD816V variant allele frequency (VAF) by NGS. Kruskal-Wallis test and Chi-squared test were used for analysis of continuous and categorical variables, respectively. Cox regression model was used for survival analysis; variables included were WHO subgroup, cKITD816V >2% VAF, mutations in additional genes, age at diagnosis >60 years, serum tryptase >200 ng/mL, alkaline phosphatase and white blood cells greater than upper normal limit. We did not considered as single entities the features included in B and/or C findings. Results: Median age at diagnosis was 45 years (y)(range 17-76) for ISM, 67 y (53-74) for SSM, 73 (41-81) for ASM (p At last follow up (median 2 years, range 0.2-14) 2 pts with ASM evolved to acute myeloid leukemia (AML, 2.8% of all series, 28% of ASM), 1 ISM pts developed organ damage and shifted to ASM; 4 pts died (4/70, 5.7%). Skin involvement was found in 80.6% of pts, 31.3% had history of anaphylaxis and 27.8% osteoporosis. Median serum tryptase levels were 28.7 ng/mL (range 3-192) for ISM, 190 (60-591) for SSM, 64 (23-300) for ASM and 2000 in MCL. All but 3 pts (2 ISM, 1 ASM) were cKITD816V mutated by RT-PCR in bone marrow aspirate (BM) or peripheral blood (PB) (in pts with punctio sicca). 54/67 (80%) had a cKITD816V mutation detectable in PB, underlying that even with high sensitivity methods PB cannot replace BM analysis for diagnosis. Median VAF was 0.34% (range 0.03-38.4) in ISM, 17.2% (0.3-45) in SSM, 26.7% (0.9-81.7) in ASM and 71.7% in MCL. Pts with at least 1 additional mutation were 6.9% (4/54) ISM, 50% (2/4) SSM and 57% (4/7) ASM(p In univariate analysis the following parameters were associated with shorter survival: one or more additional mutations (HR 19.2, CI 2-185, p2% (68.8% versus 8.3%, P None of the analyzed molecular features correlated with anaphylaxis, osteoporosis or skin involvement. Conclusions: With the limitations due the small number of events recorded in this series, these data suggest that knowledge of molecular asset in pts with SM might provide prognostically relevant information. Disclosures Vannucchi: Novartis: Consultancy, Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Shire: Speakers Bureau.

The revised 2016 World Health Organization (WHO) classification of myeloid neoplasms dictated distinct criteria for prefibrotic (prePMF) and overt primary myelofibrosis (PMF), based on bone marrow (BM) morphology, including fibrosis grade (G) AIM: to describe the characteristics and outcome of patients (pts) with a diagnosis of prePMF versus PMF according to the 2016 WHO criteria. METHODS. We used a database of ca 800 pts collected in 5 Italian tertiary centers of the AGIMM project. Pts annotated with diagnosis of pre/early PMF and PMF were identified, and BM biopsies were re-classified according to current criteria. A total of 639 pts with full information were retrieved; all were annotated for both driver mutations (JAK2V617F, MPLW515x, CALR) and High Molecular Risk mutations (HMR; Vannucchi et al, Leukemia 2013;1861), including ASXL1, EZH2, SRSF2, IDH1/2. RESULTS. Of the 639 pts, 274 (42.8%) were re-classified as prePMF and 365 (57.2%) as PMF. After a median follow-up (FU) of 3.6y, 212 pts (33.2%) had died, 23% prePMF vs 40.8% PMF (P65y old individuals (36.1% vs 46.8%, P= .004), had higher Hb (12.7 vs 10.7g/dL; P1% were found in 12.0% vs 26.3% (P25x109/L were similar. Abnormal karyotype in 18.5% prePMF vs 38.6% PMF (n=317; P10cm in 10.5% vs 24.1% (P The proportion of patients with JAK2V617F (and their median allele burden), MPLW515x and CALR (type I and type II) mutations was similar in prePMF and PMF; triple-negative (TN) pts were slightly more frequent in PMF (14%) than prePMF (9.9%; P=.041). Conversely, 24.8% of prePMF pts vs 41.1% PMF were HMR (P2 HMR mutations, that are prognostically negative (Guglielmelli et al, Leukemia 2014; 28), were found in 11.8% of PMF pts vs 4.7% prePMF (P Median survival (OS) was 17.6y in prePMF vs 7.2y in PMF (P2 HMR mutations (HR 9.3, 4.5-19.0 and 3.4,2.1-5.3, respectively; P CONCLUSIONS. This analysis of pts with contemporary diagnosis of prePMF and PMF disclosed important clinical, hematologic and molecular differences between the two, and indirectly suggested that they might represent a phenotypic continuum where increased grade of fibrosis associates with worsening of disease manifestations and outcome. Disclosures Vannucchi: Novartis: Consultancy, Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Shire: Speakers Bureau.

Primary Myelofibrosis (PMF) is a Philadelphia-negative myeloproliferative neoplasm characterized by the presence of hyperplastic/dysplastic megakaryocytes and the development of myelofibrosis and osteosclerosis. This disruption of the bone marrow (BM) structure results from a deregulated release of growth factors by malignant hematopoietic cells, particularly monocytes and megakaryocytes, that activate BM stromal cells (Desterke-C, Mediators Inflamm, 2015). Therefore, it remains of primary importance to identify novel therapeutic strategies to restore BM microenvironment homeostasis in PMF patients. In order to better characterize the molecular mechanisms underlying PMF pathogenesis, we have recently compared the gene expression profile (GEP) of CD34+ hematopoietic progenitor cells (HPCs) from PMF patients and healthy donors, and we observed the upregulation of the transcription factor MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) (Norfo-R, Blood, 2014). With the aim to investigate the role of MAF in PMF pathogenesis and to reproduce the condition observed in PMF CD34+ cells, we studied the effects of MAF overexpression on HPCs differentiation. Human cord blood-derived CD34+ cells were transduced with a retroviral vector carrying the coding sequences for the two MAF transcript variants (LMAFVAR1IDN and LMAFVAR2IDN). The megakaryocyte lineage-committed CD41+ population was increased in LMAFVAR1IDN (38.2±2.3%) and LMAFVAR2IDN (39.4±2.3%) samples compared with the empty vector LXIDN (16.1±2.7%) at day 3 after NGFR+ cells purification. Values are reported as mean ± SEM from 3 independent experiments, p Next, we analyzed the GEP of MAF-overexpressing CD34+ cells and observed the upregulation of genes involved in megakaryopoiesis, such as MERTK and RCAN1, and in monocyte/macrophage differentiation (e.g. EGR2 and KLF4). In particular, GEP revealed the upregulation of genes related to processes deregulated in PMF, such as fibrosis (i.e. ENPP2, ADAM9) and inflammation (i.e. TNF, CCL2 and CCL3). Noteworthy, several upregulated transcripts encoded for secreted proteins. Based on their role in inflammation and fibrosis, we selected a set of secreted molecules (IL8, MMP9, CCL2, SPP1, LGALS3 and PLAUR) and assessed them by quantitative enzyme-linked immunoassay (ELISA) in the culture supernatants from LMAFVAR1IDN-, LMAFVAR2IDN- and LXIDN-transduced cells. The levels in culture supernatants of all these molecules were increased in MAF-overexpressing samples compared to control. In order to correlate the expression of these molecules with MAF upregulation in PMF, we assessed them in plasma samples of PMF patients (n=30) and healthy donors (n=10). All the selected molecules displayed higher plasma levels in PMF patients than in healthy controls (Figure 1). Since the increase of SPP1 and LGALS3 has never been described before in PMF, we investigated which cell type could be responsible for SPP1 and LGALS3 production. Real Time qRTPCR performed on healthy donors peripheral blood-derived cells showed that the highest SPP1 expression levels were observed in CD34+ cells-derived megakaryocytes and granulocytes, whereas LGALS3 was expressed at higher levels in monocytes compared to the other cell types. Because the increased proliferation of fibroblasts is involved in BM fibrosis and is a PMF feature, we investigated the effects of LGALS3 and SPP1 on normal human primary dermal fibroblasts. The treatment with recombinant human LGALS3 (1000 ng/ml) or SPP1 (500 ng/ml) caused an increase in fibroblasts cell count of 20.9±2.7% (p Finally, we observed that increased SPP1 plasma levels in PMF patients correlate with a more severe fibrosis and lower overall survival (p In conclusion, our data unveil the involvement of MAF in PMF pathogenesis through the induction of a pro-fibrotic and pro-inflammatory phenotype in the malignant myeloid differentiated progeny. Disclosures Vannucchi: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Abstract 2854 Molecular mechanisms underlying Philadephia-negative myeloproliferative neoplasm (MPN) pathogenesis were partially unraveled in 2005–2006 with the identification of somatic gain-of-function of JAK2 and MPL, after which many other mutated genes were found. Recently, several new molecular pathogenetic mechanisms were identified. Among them, aberrant microRNA (miRNA) expression especially seems to add to the molecular complexity of MPNs, as specific miRNA signatures capable of discriminating MPN cells from those of normal donors were previously reported (P. Guglielmelli et al., Exp Hematol, 2007). In order to have a comprehensive picture of miRNA deregulation and its relationship with differential gene expression in primary myelofibrosis (PMF) cells, we obtained coding gene- (GEP) and miRNA expression profiles (miEP) in the same CD34+ sample from 31 healthy donors and 42 PMF patients by means of Affymetrix technology (HG-U219 and miRNA 2.0 arrays). 726 genes were found as differentially expressed (DEG) (fold change contrast ≥2, false discovery rate ≤0.05) (FIG. 1) and further analysis pointed out that several DEG are related to processes involved in PMF progression as megakaryocyte (MK) differentiation, fibrosis and migration. Of interest, we found the upregulation of some putative cancer markers, such as WT1 (K. Inoue et al., Blood, 1994) and ANGPT1 (C.L. Cheng, Br J Cancer, 2011) whose expression has already been associated with poor prognosis in hematological neoplasms and in other malignancies. Figure 1 Figure 1. Among the deregulated transcription factors, we detected several genes involved in CD34+ commitment, and potentially in their transformation, such as NFE-2 (C. LAbbaye et al., J Clin Invest, 1995) and KLF3 (A.P. Funnell, Mol Cell Biol, 2012). As regards miEP, we achieved a list of 74 human miRNAs modulated in PMF (DEM) (fold change contrast ≥1.5, false discovery rate ≤0.05), some of which associated with hematological malignancies or known as oncomirs are upregulated, i.e. hsa-miR-155-5p (S. Jiang, Cancer Res, 2010), miRNAs belonging to the miR-17–92 cluster (L. Venturini et al., Blood, 2007), whereas other aberrantly expressed miRNAs have never been described in any hematological context. Next, we performed an in silico integrative analysis (IA) with Ingenuity Pathway analysis software, which combines the computational predicted targets with the gene expression data, in order to construct regulatory networks of the functional human miRNA-target interactions. IA between DEG and DEM disclosed a high number of predicted targets with anti-correlated expression to the trend of their targeting miRNAs. This approach allowed the identification of different networks potentially involved in PMF onset and progression, such as MK differentiation and chromatin remodeling, highlighting the potential contribution of miRNAs to PMF pathogenesis. In particular, the integrative analysis has identified an interaction network involving the oncomirs miR-155-5p and miR-29a-3p (R. M. O'Connel et al, J Exp Med, 2008, Y.C. Han et al, j Exp Med, 2010) and their targets (FIG. 2). Figure 2 Figure 2. In this network the upregulation of miR-155-5p and mir-29a-3p could explain the negative regulation of two tumor suppressor genes, HBP1 and TP53INP1, and of SPTB1, CDC42 and KLF3, whose downregulation is involved in malignant hematopoiesis (L.Yang et al, Blood 2007). This network also shows the upregulation of some miRNAs whose function is unknown in the hematopoietic context as miR-335-5p, with the negative regulation of its predicted targets, NR4A3 and PRDM2, which are described as implicated in myeloproliferation (AM Ramirez-Herrick et al, Blood 2001). The present findings lay the groundwork for functional in vitro validation of selected networks in normal and PMF CD34+ cells by means of DEG/DEM overexpression and silencing experiments; furthermore, expression data will be helpful in order to find a clinical correlation between mRNA/miRNA expression levels and diagnostic/prognostic relevance. In conclusion, GEP and miEP pointed out genes and miRNAs candidate for elucidating some of the pathogenetic features of PMF CD34+ cells, whereas IA uncovered potential regulatory networks in which aberrantly expressed miRNAs and genes interact contributing to the malignant phenotype. Disclosures: Vannucchi: Novartis: Membership on an entity's Board of Directors or advisory committees.

Intrauterine growth restriction is a condition fetus does not reach its growth potential and associated with perinatal mobility and mortality. Intrauterine growth restriction is caused by placental insufficiency, which determines cardiovascular abnormalities in the fetus. This condition, moreover, should prompt intensive antenatal surveillance of the fetus as well as follow-up of infants that had intrauterine growth restriction as short and long-term sequele should be considered.

To measure aortic intima media thickness and diameter by ultrasonography in fetuses with intrauterine growth restriction (IUGR) and in appropriate for gestational age (AGA) fetuses and in the same children after a mean follow-up of 18 months.This was a prospective study performed between January 2006 and August 2008. Fetuses were classified as having IUGR if the estimated fetal weight was below the 10th percentile and umbilical artery pulsatility index was greater than 2 standard deviations; they were classified as AGA if the estimated fetal weight was between the 10th and 90th percentiles. Abdominal aortic intima media thickness and diameter were measured in each fetus with IUGR and in each AGA fetus at a mean gestational age of 32 weeks. The same measurements were taken in the children after a mean follow-up of 18 months.Thirty-eight fetuses with IUGR and 32 AGA fetuses were enrolled in the study. Aortic intima media thickness median values were significantly higher in IUGR than in AGA both in utero (1.9 mm compared with 1.15 mm; P.001) and after birth (2.4 mm compared with 1.03 mm; P.001) and were significantly correlated (P=.018, r=0.48). At 32 weeks of gestation, aortic intima media thickness in fetuses with IUGR was inversely correlated with estimated fetal weight (P.003; r=-0.58). Median diameter of the abdominal aorta and blood-flow velocity at 32 weeks of gestation were significantly higher in fetuses with IUGR compared with AGA fetuses (median diameter 4.5 mm compared with 3.6 mm, P.001, blood-flow velocity 42.5 cm/s compared with 23.3 cm/s, P.001). At follow-up, in 25 children who had had IUGR and 25 children who had been AGA, there was no significant difference in median diameter of the abdominal aorta (6.8 mm compared with 7.5 mm, P=.21).Aortic wall thickening in fetuses and children with IUGR shows differences with respect to those who were AGA. This may reflect a correlation between impaired growth in utero, Doppler abnormalities, low birth weight, and early signs of vascular dysfunction.II.

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 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.

BACKGROUND. Calreticulin (CALR) mutations (mut) are associated with a IPSS/DIPSSplus and mutated ASXL1-independent favorable outcome in patients (pts) with primary myelofibrosis (PMF) compared to those harboring JAK2 V617F and MPL W515 mut and those lacking driver mutations ("triple negative", TN). Spliceosome mut were reported to be less represented in CALR - mut pts compared with other genotypes (Leukemia 2014;28:1472), but no other molecular or cytogenetic correlate is known. It is also debated whether there are prognostic differences between type 1/type 1 like (Ty1; a 52 bp deletion; p.L367fs*46) and type 2/type 2 like (Ty2; a 5 bp TTGTC insertion; p.K385fs*47) CALR mut. Tefferi et al (Blood 2014;124:2465) reported that only Ty1 was prognostically favorable, while either the opposite or no effect was found in other studies (Leukemia 2015; 29:249). AIMS and METHODS. To evaluate the prognostic relevance of different types of CALR mut in a series of 396 PMF pts (2008 WHO criteria) seen in our center. CALR mut were analyzed by high-resolution capillary electrophoresis and confirmed by Sanger sequencing. Deep sequencing based on Ion Torrent PGM platform was used to analyze mutations in 17 genes previously shown to recur at discrete frequencies in chronic phase and leukemic transformation (LT) of PMF. Comparisons of quantitative variables between groups were carried out by the nonparametric Wilcoxon rank-sum test. RESULTS. 251 pts (63.4%) harbored JAK2 V617F mut, 21 (5.3%) MPL W515 mut, 74 (18.7%) CALR mut, of which 53 (71.6%) Ty1 and 21 (28.4%) Ty2; 50 pts (12.6%) were TN. There was no statistically significant difference between Ty1 and Ty2 for common hematologic and clinical variables. Conversely, both Ty1 and Ty2 differed from JAK2 V617F mut counterpart for younger age, lower leukocyte and higher platelet counts; males were more represented among Ty1 pts than other genotypes. Among CALR Ty1, more pts were in lower (low+Intermediate-1) IPSS risk categories (80%) compared to Ty2 (62%; P=0.04). With a median follow-up of 6.4 and 5.4 yrs for CALR mut Ty1 and Ty2 pts, respectively, there were less pts who died among Ty1 (17%) compared to Ty2 (47.6%) (P=0.007). Median survival of CALR Ty1 was 26.4 yr (range, 15.5-37.3) vs 7.4 yr (4.6-10.2) for CALR Ty2, 7.2 yr (5.7-8.6) for JAK2 V617F and 2.0 (1.6-2.4) for TN (P Since some subclonal mutations are prognostically relevant in PMF (Leukemia 2013; 27:1861), we compared the mutational profile of 17 such genes, functionally clustered as epigenetic (DNMT3A, EZH2 TET2, IDH2, IDH1, ASXL1), of thespliceosome (SF3B1, SH2B3 U2AF1, SRSF2) orassociated with LT (cKIT, RUNX1, NRAS, KRAS, IKZF1, TP53 CBL) in 44 and 20 CALR mut Ty1 and Ty2 pts, respectively. There was a greater proportion of pts harboring at least one mutated gene among CALR Ty2 than Ty1 (70% vs 56.8%; P=0.02). Also, the proportion of pts with >2 mut was significantly greater in CALR mut Ty2 compared to Ty1 (40% vs 18%; P=0.01); in particular, 15% of CALR Ty2 mut pts had >3 mut compared to 2.2% in Ty1 (P=0.04). Frequency of individual mutations in Ty2 vs Ty1 was: epigenetic, 50% vs 50%; spliceasome 30% vs 4.5% (P=0.009); LT 30% vs 13.6% (P=0.06). Of note, SF3B1 mutations were 10-fold more represented in CALR mut Ty2 (25%) compared to Ty1 (2.2%; P CONCLUSIONS. These findings support previous report that the prognostic advantage of CALR mutation in PMF regards only pts harboring type 1/type 1-like abnormalities. A greater mutational complexity involving subclonal mutations, particularly in genes of the spliceosome with an unusually high incidence of SF3B1 mutations, as well as increased number of mutated genes, may contribute to the dismal outcome of CALR mut Ty2 pts. Disclosures Vannucchi: Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees; Shire: Speakers Bureau.

Background. In primary myelofibrosis (PMF), mutations of JAK2, CALR and MPL driver genes can be detected in about 60%, 20% and 5% of the patients (pts), respectively. Therefore, about 10% of the pts lack any of the 3 driver mutations and are operationally called "triple negative" (TN). TN pts present a higher risk of developing anemia and thrombocytopenia, suffer from reduced overall survival compared to other genotypes, particularly to CALR type1/type1-like mutations, and may be at higher risk of leukemic transformation (Blood 2014; 124:1062; Leukemia 2014; 28:1472; Blood 2014;124:2465). Aims The aim of the study was to analyse the molecular landscape of TN PMF pts by genotyping a set of myeloproliferative-neoplasms associated genes whose mutated status was shown to be prognostically relevant in previous studies (Leukemia 2013;27:1861; Blood 2014;123:2220; Leukemia 2014;28:1804;Leukemia 2014;28:1494). Methods. Diagnosis ofPMF was made according to WHO2008 criteria. All pts provided informed written consent. Previously published methods were used to screen mutations involving JAK2, MPL and CALR. A deep sequencing custom panel was designed to genotype the following genes: EZH2, ASXL1, IDH1/2, SRSF2, TP53, TET2, RUNX1, CBL, NRAS, KRAS, DNMT3A, SF3B1, IKZF1, NFE2, SH2B3. Analysis was performed using the Ion torrent PGM platform. Comparisons of quantitative variables between groups were carried out by the nonparametric Wilcoxon rank-sum test. The prognostic value of the molecular variables with regard to OS was estimated by the Kaplan-Meier method and Cox regression. Results. We analysed 28 TN PMF collected at the time of diagnosis. Median age was 66.7y, 57% were male. Median follow up was 2.1y (0.3-14.5). Overall, 8 patients (28.6%) progressed to AML. Death occurred in 20 pts (71.4%) after a median follow up of 2.3y (1.4-3.3y); progression to leukemia was the cause of death in 5 pts (25%). Overall, 22 pts (78.6%) presented at least one mutation in any of the 15 genes of the panel; 14 pts (50%) presented at least 2 mutations in different genes. The frequency of mutated genes was: SRSF2 39.3%, ASXL1 28.6%, EZH2 21.4%, NRAS 21.4%, TET2 10.7%, CBL 10.7%, IDH 3.6%, DNMT3A 3.6%, SH2B3 3.6%, U2AF1 3.6%. Twenty pts (71.4%) were classified as high molecular risk (HMR: ie, any mutated gene of ASXL1, EZH2, SRSF2, IDH1/2), a proportion significantly higher (P Among the mutated genes, SRSF2 was associated with shorter survival [1.9y (1.6-2.2)] compared to pts with un-mutated SRSF2 [3.2y (0.9-5.4y)] (HR 2.3, 95%CI 0.9-6.4). SRSF2 mutations were also associated with shorter leukemia free survival (LFS): LFS not reached in un-mutated pts compared to 2.2y (1.8-2.7y) for mutated pts, with a HR=4.5 (95%CI 10.3-19.9). We also found that in pts with grade 1 bone marrow fibrosis the splicing and the leukemic pathway were more frequently mutated compared to grade 2-3 fibrosis (57% vs 28.5% and 50% vs 28.5% respectively). Conclusions. "Triple-negative" pts with PMF present high rate of mutations of MPN-associated genes, most of them are classified as "high molecular risk" and harbor >2 mutations. Mutated SRSF2 was particularly associated with shorter LFS. Such complex molecular landscape might help to explain the negative outcome of TN PMF pts. Disclosures Vannucchi: Baxalta: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Speakers Bureau.

Background. The prognostic significance of bone marrow (BM) fibrosis grade in pts with primary myelofibrosis (PMF) is debated. A fibrosis grade greater than 1 was associated with a 2-fold higher risk of death compared with pts with early/prefibrotic MF (grade 0) [Thiele J, Ann Hematol 2006]. Recent data suggest that more accurate prediction of survival is achieved when fibrosis grade is added to IPSS [Verner C, Blood 2008; Giannelli U, Mod Pathol 2012]. Aim. To analyze the prognostic impact of fibrosis in diagnostic BM samples of 540 WHO-2008 diagnosed PMF pts with extensive clinical and molecular information collected in 6 Italian centers belonging to AGIMM (AIRC-Gruppo Italiano Malattie Mieloproliferative). Methods. The clinical variables assessed were those previously identified as prognostically relevant in the IPSS score. Published methods were used to screen mutations of JAK2, MPL, CALR, EZH2, ASXL1, IDH1/2 and SRSF2. European consensus scoring system was used to grade fibrosis (on a scale of MF-0 to MF-3). The prognostic value of fibrosis with regard to overall survival (OS) was estimated by Kaplan-Meier method and Cox regression. Results. Pts' median age was 61y; median follow-up 3.7y; median OS 10.5y; 184 pts (34.1%) died. IPSS risk category: low 33.7%, Int-1 27.7%, Int-2 19.1%, High-risk 19.5%. Mutational rate: JAK2 V617F 62.6%, CALR 20.7% (type-1/1-like 77.7%, type2/2-like-2 21.4%), MPL W515 5.9%; 62 (11.5%) were triple negative (TN). 171 pts (31.7%) were High-Molecular Risk (HMR) category (Vannucchi AM, Leukemia 2013); mutation rate: EZH2 7.2%, ASXL1 22.2%, IDH1-2 2.4%, SRSF2 8.3%. According to fibrosis grading, 50 pts were MF-0 (9.3%), 180 MF-1 (33.3%), 196 MF-2 (36.3%), 114 MF-3 (21.1%). Compared with both MF-0 and MF-1, MF-2 and MF-3 pts presented more frequently constitutional symptoms (P There was no correlation between fibrosis grade and phenotypic driver mutations; in particular, TN pts were equally distributed among MF fibrosis grades (10%, 10.6%, 14.3% and 8.8% from MF-0 to -3, respectively). Conversely, the frequency of HMR pts increased progressively according to fibrosis grade: 8 pts MF-0 (16%), 46 MF-1 (25.6%), 66 MF-2 (33.7%) and 51 MF-3 (44.7%) (P Median OS was significantly shorter in pts with MF-2 (OS 6.7y, HR 7.3, IC95% 2.7-20.0; P Conclusions. Overall, these results indicate that higher grades (MF-2 and MF-3) of fibrosis correlate with defined clinical and molecular variables and independently negatively impact on OS in PMF, suggesting the opportunity to explore its value in the setting of clinical and molecular prognostic scores for PMF. Table. Multivariate Analysis Variables HR 95% CI P value HMR status 2.4 1.5-3.7 Figure 1. Figure 1. Disclosures Passamonti: Novartis: Consultancy, Honoraria, Speakers Bureau. Barbui:Novartis: Speakers Bureau. Vannucchi:Shire: Speakers Bureau; Novartis: Other: Research Funding paid to institution (University of Florence), Research Funding; 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.

Background: Philadelphia-negative Myeloproliferative Neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis, both Primary (PMF) and secondary to PV or ET (PPV-MF and PET-MF). A MPN is frequently the underlying cause of splanchnic vein thrombosis (SVT). Ruxolitinib, a JAK1/2 inhibitor, efficiently reduced spleen volume and improved symptoms in patients (pts) with MF and PV in the COMFORT-I/II and RESPONSE phase III trials, and in ET pts in a phase II study. We reported (Blood 2014 124:3192) that ruxolitinib was safe in pts with MPN associated to SVT and effective in reducing spleen size at the planned primary endpoint analysis at 24 weeks (w) in a phase II clinical trial. Herein we present follow up data with cut off at 1 year after core period (a total of 72 w of treatment). Methods: Main enrolment criteria included diagnosis of PV, ET, PMF or PPV-/PET-MF associated with SVT, splenomegaly >5 cm below costal margin (bcm), active anticoagulant or antiaggregant thrombosis prophylaxis, platelet count (plt) >100 x109/L, neutrophils count >1x109/L, normal hepatic and renal function, absence of esophageal varices >grade 2. Pts who completed the 24 w of study treatment and tolerated well the drug and had evidence of clinically-significant improvement were allowed to enter an extension phase aimed at collecting and reviewing safety and efficacy data. The drug was provided free of charge by Novartis, that had no role in trial design nor in data analysis. Results: Diagnosis of MPN were: PMF 8 (38.1%), PV 5 (23.8%), ET 4 (19.1%), PPV-MF 3 (14.3%), PET-MF 1 (4.8%). Nineteen pts had spleno-porto-mesenteric thrombosis and 3 Budd-Chiari syndrome (BCS); one pt had both sites involved. Initial dose of ruxolitinib was 10 mg BID for PV, 25 mg BID for ET, 15 mg BID for MF pts with baseline (bl) platelet count of 100 to 200x109/L and 20 mg BID for platelet count >200x109/L. Currently 17/21 pts are on active treatment, 14 completed w72; final data for all 17 pts will be available at meeting. One pt with MF discontinued from the study being shifted to commercial ruxolitinib at w60, one ET and one MF pt discontinued for inefficacy at w24 and one MF pt for an unrelated adverse event after w72. Efficacy: 13/21 (61.9%) pts obtained a ≥50% spleen length (sl) reduction by palpation at w24, that was maintained at w72 in 8/14 pts (57.1%). Median sl reduction at w72 was 63% (range 0-100). No significant differences in resistive or pulsatility index of splanchnic artery were noted, nor in esophageal varices status evaluated at w72. 10/11 evaluated pts with echocardiography at w72 showed a median reduction of the cardiac output of 20.1% (range 2.3-42.2) mainly due to a reduction of heart rate and of cardiac index (-21.9%, range 8.8-44.3) due to increase in body surface area. The first effect could be attributed to decrease of proinflammatory cytokines, the second to weight gain associated with ruxolitinib. Symptomatology was evaluated by MPN-SAF up to w24, showing a median total symptom score reduction from 65 to 42. Safety: regardless of drug relationship, the most common adverse events (AE) (% any grade, % grade ≥3) were thrombocythopenia (57.1%; 14.3%) and anemia (33.3%, 19%) that were the main reasons for dose adjustments. Other AE included AST or ALT increase (42.9%, 0%), diarrhea (28.6%, 0%), abdominal pain (23.8%, 0%), ascites (19%, 0%), fever (23.8%, 0%), neutropenia, (9.5%, 9.5%), upper airways infection (19%, 0%), weight gain (14.3%, 4.8%), muscle cramps (14.3%, 0%). Three serious AE occurred: one case of hepatocarcinoma in a pts with BCS, one grade 2 pneumonia and one grade 2 haematemesis not related to esophageal varices. Median ruxolitinib total daily dose at w72, after dose adjustments, was 19.1 mg for MF, 16 mg for PV and 28.3 mg for ET. Median hemoglobin reduced from 12.9 gr/dL (range 9.4-16.7) at bl to 10.7 (8.4-14.4) at w16 and recovered at w72 (12.1, range 10.8-14.7). No pts received transfusions. Median platelet count was 212 x109/L (100-389) at bl, reached to the lowest level at w4 (139, range 48-252) and improved to 160 (69-285) at w72. Median leukocyte count decreased from 7.3 x109/L (1.8-16.4) at bl to 4.08 (1.2-21.7) at w 24, and remained substantially stable through w 72 (4.96; range 2.45-17.3). Median reduction of JAK2 allele burden at w72 was 9% (range 0-38). Conclusions: At w 72 follow up, ruxolitinib continues to be safe in pts with MPN associated to SVT and maintains efficacy against splenomegaly in 57% of the pts. Disclosures De Stefano: Roche: Research Funding; GlaxoSmithKline: Speakers Bureau; Bruno Farmaceutici: Research Funding; Novartis: Research Funding, Speakers Bureau; Janssen Cilag: Research Funding; Celgene: Speakers Bureau; Shire: Speakers Bureau; Amgen: Speakers Bureau. Barbui:Novartis: Speakers Bureau. 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.

Introduction The Myeloproliferative Neoplasms (MPNs) are characterized by a recurrent point mutation of JAK2 gene (JAK2 V617F). This mutation, which usually affects only one of the JAK2 gene alleles in Essential Thrombocythemia (ET), frequently becomes homozygous in Polycythemia Vera (PV) and Myelofibrosis (MF) due to homologous mitotic recombination. A JAK2 V617F-mutated disease is strongly associated with a specific constitutional JAK2 haplotype, designated 46/1(GGCC), (Nat Genet 2009; 41:446) with complete linkage disequilibrium although the risk of developing MPNs is independent of the 46/1 haplotype. Furthermore, JAK2 V617F specifically arises on the 46/1 allele in most cases, thus predisposes to the development of MPN (Leukemia 2010; 24:1533; Nat Genet 2009; 41:446). These observations suggest that it is possible to define the JAK 2V617F clonal architecture starting from 46/1 SNP allele burden and the homologous recombination frequency. Aim and Methods The aim of the study was to investigate changes in the JAK2 V617F clonal structure in patients affected by PV, ET or MF and treated with ruxolitinib, a JAK1/JAK2 inhibitor recently approved for MF and PV and under investigation in ET pts intolerant of or resistant to hydroxyurea. We used a recently described methodology (Leukemia 2014;28:460) combining allele burden evaluations of both 46/1 and JAK2 and the frequency of mitotic recombination to derive the percentages of JAK2 V617F clones in MPN patients in 46/1 heterozygous patients for rs12343867 polymorphism (C/T). The JAK2 allele burden values were confirmed independently by two RTQ-PCR methods, according to Lippert (sensitivity, 0.8%) and to Larsen (sensitivity, 0.08%) method. Results We analysed 40 patients (pts) affected by MPNs (12 PV, 1 ET and 27 MF) treated with ruxolitinib over 1 to 5 years (yrs) of follow up (FU); 13 patients (32.5%) presented a reduction of at least 10% of JAK2 V617F allele burden values at latest FU: 7/12 PV (58.3%), 1/1 ET (100%), 6/27 PMF (22.2%). Among these, 9 pts with at least two years of FU were eligible for the study. A quantification of JAK2 homozygous (V617F/V617F), heterozygous (JAK2 V617F/wt) and wild type (wt/wt) clones was obtained using the methodology described above. We found a median reduction of JAK2 V617F/V617F and JAK2 V617F/wt clones of 32.31 % and 8.82 %, respectively, in PV (n.4); 12.22% and 17.86%, respectively, in MF (n.4); 35.99% and 100%, respectively, in ET (n.1). Furthermore, an almost complete molecular remission (CMR) was seen in two PV patients with 4 and 5 yrs of FU respectively and in one ET patient after 5 years of treatment. In these patients we observed reduction of homozygous clones of 99.60%, 86.91% and 100%, respectively, and the residual JAK2 allele burden was due to the persistence of JAK2 V617F/wt clones (Figure). Conversely, in one patients with an increase in JAK2 allele burden from 39% to 45% after 1,5 yrs under ruxolitinib, we observed an increase in JAK2V617F/wt but not in JAK2 V617F/V617F clones. Conclusion. Taken together, these results showed that ruxolitinib may preferentially target the homozygous clones inducing in some cases an almost complete molecular remission with prolonged treatment. Future analysis will be performed to study the clonal architecture in an increased number of patients treated with JAK inhibitors as single agent or in combination with other drugs. Figure 1. Figure 1. Disclosures Vannucchi: Shire: Speakers Bureau; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees.

The discovery of mutations in calreticulin (CALR) in patients with primary myelofibrosis (PMF)1, 2 prompted a reappraisal of the clinical correlates and prognostic impact of the so-called driver mutations that include JAK2V617F, MPLW515L/K/A and CALR in ~60%, 5–10% and 20–25% of patients, respectively. As compared with their JAK2V617F counterpart, PMF patients harboring CALR mutations showed younger age, higher platelet and lower hemoglobin and leukocyte counts. The cumulative incidence of anemia, leukocytosis and thrombocytopenia was significantly lower in CALR-mutated patients who were also less likely to be red cell transfusion-dependent;3, 4 in addition, they had significantly longer large splenomegaly-free survival compared with the other genotypes as well as patients lacking the three driver mutations (triple-negative (TN) patients).3, 4 Interestingly, spliceosome mutations were significantly less represented in CALR-mutated patients; however, no additional molecular or cytogenetic correlate was highlighted.3 These data suggested a milder disease in patients harboring the CALR mutation, and conceivably the presence of CALR mutation was associated with better overall survival (OS) when compared with JAK2V617F- and MPLW515-mutated patients, and particularly TN patients.3, 4 In multivariable analysis, CALR mutations had a favorable impact on survival that was independent of International Prognostic Scoring System (IPSS), Dinamic IPSS (DIPSS)4 or DIPSS-plus risk stratification,3 and also of ASXL1 mutation, known for its dismal impact on survival in PMF.5 At this regard, we found that DIPSS-plus-independent OS was significantly longer in CALR-mutated/ASXL1-unmutated compared with CALR-unmutated/ASXL1-mutated patients.6

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms (MPN) characterized by the presence of JAK2V617F mutation in >95% and 60% of patients (pts), respectively. This mutation usually affects one allele in ET while most PV pts are homozygous due to mitotic recombination. Acquisition of the JAK2V617F mutation is strongly associated with the germline 46/1 predisposition haplotype. Ruxolitinib is a JAK1/JAK2 inhibitor recently approved for myelofibrosis (MF) and under investigation in PV and ET pts intolerant or resistant to hydroxyurea. We enrolled 24 pts, 11 with PV and 13 with ET, in the phase II INCB18424-256 trial that overall included 34 PV and 39 ET pts. 21/24 pts were still on treatment at 5 years (yr), of which 19 JAK2V617F mutated. Results of the PV cohort have been reported recently (Verstovsek et al. Cancer, 2014): with a median follow up of 35 months (mo), the JAK2V617F allele burden decreased by a mean of 8%, 14%, and 22%, respectively, after 12, 24 and 36 mo. The proportion of pts who achieved a reduction ≥50 % at any time during the 1st yr, 2nd yr, and 3rd yr were 5.9%, 14.7%, and 23.5%, respectively, but no patients achieved a complete remission. In our series of pts we evaluated the JAK2V617F allele burden by two RTQ-PCR methods, according to Lippert (sensitivity, 0.8%) and to Larsen (sensitivity, 0.08%) method. We also analysed by next generation sequencing (NGS; Ion Torrent platform) a series of MPN-associated mutations including TET2, ASXL1, IDH1/2, LNK, CBL, SRSF2, EZH2 and MPL at baseline and at 5 yr of treatment in ruxolitinib treated pts who achieved a >25% JAK2V617F allele burden reduction at 5 yr (n=13/19). JAK2V617F allele burden decreased by a mean of 7%, 11%, and 19% at 12, 24 and 36 mo, and decreased further by a mean of 28% after 60 mo. Three (1 PV, 2 ET) of 19 pts (16%) achieved a 50% or greater allele burden reduction after 2 yr; no additional pts achieved this degree of allele burden reduction even in prolonged follow up. These 3 pts further improved their molecular response to a complete molecular response (CMR) after 5 yr of treatment. Their mean JAK2V617F allele burden was 46.6% at baseline, 28.3%, 16.3%, 8.7% and 0% after 1 yr, 2 yr, 3 yr and 5 yr, respectively. The JAK2 CMR was confirmed in at least one independent sample at 3 mo after first discovery. At this last timepoint, the PV pt was in complete haematological remission according to ELN criteria, the 2 ET pts were in partial remission due to platelet count still >400x109/L: 422x109/L and 812x109/L, respectively. BM histopathology in the 2 ET pts at 5 yr, while they were in CMR, showed still evidence of megakaryocyte hyperplasia. In the PV pt, histopathology at 5 yr is pending; evaluation at 3 yr, a time when she was in complete hematologic remission and JAK allele burden had decreased from 69 to 8%, showed normalization of cellularity, megakaryocyte and myeloid lineage compared to baseline but still slight erythroid hyperplasia. All 3 pts had normal karyotype at baseline that remained unchanged thereafter. CMR for JAK2V617F was confirmed by NGS. The 2 ET pts achieving CMR did not show any additional mutations, while the PV pts presented a TET2 Y867H mutation with an allele burden of 48.9% and 52%, respectively at baseline and 5 yr. No recurrent mutations in genes other than JAK2 were found in all other examined cases at baseline or at 5 yr. In 3 informative pts, we also analysed the proportion of JAK2V617F homozygous, heterozygous and wild type clones by the method of Hasan et al (Leukemia 2013) based on allelic discrimination of 46/1 haplotype and JAK2. We found that JAK2V617F/V617F clones were reduced by a mean of 95.5%, JAK2V617F/WT showed an uneven trend with a mean reduction of 45.54% while JAK2WT/WT conversely increased (mean 61.43%) at 5 yr, suggesting that in a subset a patients who present significant reduction of VF allele burden ruxolitinib may preferentially target the homozygous clones. Until now, complete molecular remission in PV pts has been described only in patients treated with interferon. Our data suggest that a subset of pts who present a rapid and sustained reduction of the JAK2V617F allele burden under ruxolitinib may eventually reach a condition of CMR with prolonged treatment. However, similar to findings with interferon, mutations establishing clonality, such as in TET2, may still persist in patients who eventually show the disappearance of JAK2V617F mutated subclones. Disclosures Verstovsek: Incyte: Research Funding. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Ph-negative myeloproliferative neoplasms (MPNs) are characterized by many somatic mutations which have already been shown useful in the prognostic assessment of MPN patients [A.M. Vannucchi et al., Leukemia, 2013]. Moreover, aberrant microRNA (miRNA) expression seems to add to the molecular complexity of MPNs, as specific miRNA signatures capable of discriminating MPN cells from those of normal donors were previously reported [P. Guglielmelli et al., Exp Hematol, 2007]. In order to have a comprehensive picture of miRNA deregulation and its relationship with differential gene expression in primary myelofibrosis (PMF) cells, we obtained gene- (GEP) and miRNA expression profiles (miEP) of CD34+ cells from 31 healthy donors and 42 PMF patients using Affymetrix technology (HG-U219 and miRNA 2.0 arrays). Among 726 differentially expressed genes (DEG) we found that several putative cancer markers (WT1, ANGPT1) and several genes related to PMF progression, i.e. involved in megakaryocyte (MK) differentiation (NFE2, CD9), and fibrosis development (DLK1, LEPR1), were significantly more expressed in PMF samples than in the normal counterpart. Similarly, as regards the miEP, among 74 human differentially expressed miRNAs (DEM) in PMF compared to controls we found the upregulation of several miRNAs associated with hematological malignancies or known as oncomiRs (i.e. hsa-miR-155-5p [S. Jiang et al., Cancer Res, 2010], miRNAs belonging to the miR-17-92 cluster [L. Venturini et al., Blood, 2007]), and other aberrantly expressed miRNAs never described in hematopoiesis (i.e. hsa-miR-335-5p). Then, in order to construct regulatory networks of the functional human miRNA-target interactions, we performed an integrative analysis (IA) with Ingenuity Pathway analysis software, which combines the miRNA expression profile with computational predicted targets and with the gene expression data. IA between DEG and DEM disclosed a high number of predicted targets with anti-correlated expression to the trend of their targeting miRNAs. Of note, IA identified an interaction network (see Figure) in which the upregulated oncomirs miR-155-5p [R.M. O'Connel et al., J Exp Med, 2008], miR29a-3p [Y.C. Han et al., J Exp Med, 2010] and miR-19b-3p [K.J. Mavrakis et al., Nat Cell Biol, 2010] could explain the downregulation of targets whose lower expression was already described as involved in myeloproliferative phenotypes, such as NR4A3, CDC42, HMGB3. Additionally, IA disclosed the chromatin remodeler JARID2, which is frequently deleted in leukemic transformation of chronic myeloid malignancies, as a shared target of several upregulated miRNAs in PMF samples (i.e. miR-155-5p, miR-152-3p). Noteworthy, these miRNA-mRNA interactions were functionally confirmed by 3' UTR luciferase reporter assays. Next, in order to characterize the role of JARID2 in PMF pathogenesis, we performed RNAi-mediated gene silencing experiments on CD34+ cells of healthy donor. Interestingly, inhibition of JARID2 expression produces in silenced cells a significant increase of CD41 expression when compared with control (28.6±3.1% vs 15.3±1.8% at day 8, 52.6±7.6% vs 35.4±4.9% at day 12 of serum free liquid culture) and a remarkable increase in CFU-MK colonies (59.6±6.5% vs 39.8±5.9%). The values are reported as mean ± 2S.E.M from five independent experiments. Moreover, morphological analysis after May-Grunwald-Giemsa staining showed that JARID2 silencing induces in normal CD34+ cells a considerable enrichment in MK precursors at different stages of maturation. This study allowed the identification of different networks possibly involved in PMF onset, highlighting the potential contribution of miRNAs to PMF pathogenesis. Furthermore, for the first time, we demonstrated that the JARID2 downregulation in CD34+ cells might contribute to the abnormal megakaryopoiesis typical of PMF. Disclosures: Rambaldi: Novartis: Honoraria; Sanofi: Honoraria; Italfarmaco: Honoraria.

Philadelphia-negative Myeloproliferative Neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis, both Primary (PMF) and secondary to PV or ET (PPV-MF and PET-MF). A MPN is frequently the underlying cause of splanchnic vein thrombosis (SVT), accounting for 31.5% of portal vein thrombosis (PVT) and 40.9% of Budd Chiari syndrome (BCS). In patients (pts) with MPN and SVT, splenomegaly can arise as the consequence of the hematological disease and/or blood flow abnormalities consequent to the thrombosis itself. Splenomegaly and the compensatory enlarged splanchnic vessels are responsible for several complications including esophageal and gastric varices. Splenomegaly may cause abdominal discomfort; furthermore pts may present symptomatic burden due to the MPN. Current treatment strategies for MPN pts with SVT include anticoagulants and cytoreductive therapy (ie hydroxyurea, interferon) that have little influence in the control of splenomegaly and symptoms and do not improve flow abnormalities. Ruxolitinib, a JAK1/2 inhibitor, was highly effective in reducing spleen volume and improving symptoms in patients with MF and PV in phase II and III studies. We hypothesized that the decrease of the enlarged spleen determined by Ruxolitinib could result in a reduction of the local pressure in splanchnic vessels, producing both symptomatic improvement of splenomegaly-related symptoms and of splanchnic circulation. We designed an investigator-initiated multicentre phase 2 study of Ruxolitinib in pts with splenomegaly due to an underlying MPN associated with SVT. The drug was provided free of charge by Novartis, that had no role in trial design nor in data analysis. The primary study objective was to evaluate the proportion of subjects achieving ≥ 50% reduction in spleen length from left costal margin (LCM) measured by palpation at any time from baseline to week 24 (w24) and at w24, or a ≥ 35% reduction in spleen volume by MRI or CT at week 24. The secondary objectives included: evaluation of safety of Ruxolitinib in MPN-associated SVT; assessment of splanchnic circulation through Doppler analysis, measurement of hyperdynamic arterial circulation by echocardiography and stiffness of hepatic/splenic parenchyma by fibroscan; status of esophageal varices at w24 compared to baseline. Quality of Life assessment was performed using MPN-SAF questionnaire. Exploratory objectives include evaluations of changes in JAK2V617F or MPLW515 allelic burden, association of baseline mutations with response to treatment, changes in cytokine and microRNAs profiles, quantification of circulating endothelial cells. At the time of abstract submission 7 out of 21 pts have been enrolled, of which 5 completed the 24 weeks of treatment; two additional pts are in screening phase. Three pts had PMF, two ET, one PV and one PPV-MF, associated to spleno-porto-mesenteric thrombosis (5 pts) and Budd Chiari syndrome (2 pts). All pts were under oral anticoagulation therapy. Initial dose of Ruxolitinib was 10 mg BID for PV, 25 mg BID for ET, 15 mg BID for MF pts with baseline platelet count of 100 to 200x109/L and 20 mg BID for those with baseline platelet count >200x109/L. A palpable splenomegaly greater than 5 cm below LCM was a criterion for enrollment; the 5 patients who completed the 24 weeks of treatment had a median splenomegaly of 8 cm below LCM at baseline, and obtained a median reduction of 69% measured by palpation at week 24, associated with a significant reduction in abdominal discomfort as measured by MPN-SAF questionnaire (median score at screening 5 vs 1.5 at week 24). The total symptom score calculated by using BFI and MPN-SAF was reduced from 50 at screening to 35 at week 24. Instrumental evaluations of splanchnic and systemic circulation showed that 3 pts obtained a reduction of the spleen stiffness from a median value of 66 to 49.6 kilopascals (KPa), 2 pts had a reduction of the liver stiffness from a median value of 23.85 to 18.2 KPa and 1 pt a reduction of the cardiac output from 5.871 to 4.6 L/min. Evaluation of esophageal varices at week 24 showed stabilization with neither worsening nor need of banding. Ruxolitinib was well tolerated, with no SAE reported; one pt developed anemia G2 and one G3 leading to dose reduction. Other adverse events include G1 asthenia and G≤2 AST/ALT increase in 3 pts, one case of Herpes Zoster and one case of abdominal pain both G1. Updated results will be presented at the meeting. Disclosures: Marchioli: Novartis: Research Funding; Ospedali Riuniti di Bergamo: Research Funding; AIFA (Italian Regulatory Agency): Research Funding; AMGEN S.p.A.: Research Funding; Genzyme Olanda: Research Funding; Gruppo Italiano Trapianti di Midollo (GITMO): Research Funding; Pierre Fabre Italia S.p.A.: Research Funding; Università Cattolica del Sacro Cuore, Roma: Research Funding; Sigma-Tau: Research Funding; Myeloproliferative disorder Research Consortium: Research Funding; Celgene: Research Funding; Associazione Italiana Linfomi (AIL): Research Funding; Fondazione Italiana Linfomi (FIL): Research Funding; LaRoche: Research Funding; Università degli Studi di Firenze: Research Funding. Vannucchi:NOVARTIS: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

The discovery of the JAK2V617F mutation in 2005 [Kralovics R, N Engl J Med 2005] represented a major breakthrough in the understanding of the molecular pathogenesis of Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPN). Nevertheless several observations suggest that the JAK2V617F mutation may not be the disease funding mutation, at least in most instances. Therefore, a great deal of effort is ongoing with the aim to identifying novel genetic lesions contributing to the disease pathogenesis. The two major theoretical and technical drawbacks to the identification of new somatic mutations are represented, respectively, by the huge number of genes potentially involved in tumorigenesis of MPN and by the availability of a “pure” germline control DNA. Buccal swabs and saliva have been generally considered as readily available sources of DNA of non-hematopoietic origin, but detection of the JAK2V617F mutation in at least some of these samples indeed suggested the presence of myeloid cell contamination [Levine RL, Cancer Cell 2005]. So, in order to discover novel mutations in MPN using upfront technologies based on next-generation sequencing (NGS) we designed a “cancer exome” capture panel of 2000 unique genes and microRNAs. This panel was used to capture libraries generated from genomic DNA extracted from granulocytes and in vitro expanded CD3+ T-lymphocytes as germline control, in a cohort of 20 MPN patients. These captured libraries were than massively sequenced using the Roche 454 FLX platform. DNA samples had been collected at the diagnosis of PV in 9 subjects and PMF in 6 subjects, while the remaining 5 DNA samples were from 5 of the 9 PV patients at the time they evolved to post-PV myelofibrosis. After extensive bioinformatics analysis and multiple control adjustments, we finally produced a list of 171 novel “true” somatic mutations occurring in genes and microRNAs coding regions of those MPN samples; some of these mutations have been already described in MPN, whereas novel variants represent the vast majority. Despite patients harbored different numbers of somatic mutations, spanning from four to twenty-one variants, only 22 genes appear recurrently mutated. It is worth of notice the acquisition of additional mutations and/or the occurrence of loss of some mutations at the time of disease evolution from PV to a post-PV Myelofibrosis in the five patients for whom samples were available at both disease phases. Some of them, either acquired (NTRK1, PRDM2, BRCA2 and BARD1) or lost (APC, CARS, MLL3 and FAT2) had been found also in another PV or PMF sample. To test the recurrence of these mutations, we screened a different cohort of 189 patients composed by PMF (91 samples), PV (50 patients) and post-PV Myelofibrosis (48 samples) by Ion AmpliSeq technology on an Ion Torrent PGM platform. Deep amplicon sequencing of granulocytes DNA achieved a sample median of 1000-fold coverage. Excluding JAK2, MPL, IDH2, ASXL1 known variants, for 7 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, NRAS) we demonstrated in MPN a global mutation frequency greater than the 3%. Whereas some new variants need functional validation to prove causal mechanisms, some other mutations have a well-known pathogenic role in solid cancers but here are described for the first time in MPN. Disclosures: No relevant conflicts of interest to declare.