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Myeloproliferative neoplasms (MPNs) are uncommon in children/young adults. Here, we present data on unselected patients diagnosed before 25 years of age included from 38 centers in 15 countries. Sequential patients were included. We identified 444 patients, with median follow-up 9.7 years (0-47.8). Forty-nine (11.1%) had a history of thrombosis at diagnosis, 49 new thrombotic events were recorded (1.16% patient per year [pt/y]), perihepatic vein thromboses were most frequent (47.6% venous events), and logistic regression identified JAK2V617F mutation (P = .016) and hyperviscosity symptoms (visual disturbances, dizziness, vertigo, headache) as risk factors (P = .040). New hemorrhagic events occurred in 44 patients (9.9%, 1.04% pt/y). Disease transformation occurred in 48 patients (10.9%, 1.13% pt/y), usually to myelofibrosis (7.5%) with splenomegaly as a novel risk factor for transformation in essential thrombocythemia (ET) (P= .000) in logistical regression. Eight deaths (1.8%) were recorded, 3 after allogeneic stem cell transplantation. Concerning conventional risk scores: International Prognostic Score for Essential Thrombocythemia-Thrombosis and new International Prognostic Score for Essential Thrombocythemia-Thrombosis differentiated ET patients in terms of thrombotic risk. Both scores identified high-risk patients with the same median thrombosis-free survival of 28.5 years. No contemporary scores were able to predict survival for young ET or polycythemia vera patients. Our data represents the largest real-world study of MPN patients age < 25 years at diagnosis. Rates of thrombotic events and transformation were higher than expected compared with the previous literature. Our study provides new and reliable information as a basis for prospective studies, trials, and development of harmonized international guidelines for the specific management of young patients with MPN. ispartof: BLOOD ADVANCES vol:6 issue:17 pages:5171-5183 ispartof: location:United States status: published

Essential thrombocythemia (ET) is a Philadelphia-negative myeloproliferative neoplasm with high platelet counts and an increased risk of thrombosis. Acquired somatic mutations delineate four ET subtypes: JAK2-V617F , CALR -mutated, MPL -mutated, and “Triple-Negative” (TN).[1][1] CALR mutations

We have previously described the safety and efficacy of pegylated interferon-α2a therapy in a cohort of 62 patients with myeloproliferative neoplasm-associated myelofibrosis followed in centers affiliated to the French Intergroup of Myeloproliferative neoplasms. In this study, we report their long-term outcomes and correlations with mutational patterns of driver and non-driver mutations analyzed by targeted next generation sequencing. The median age at diagnosis was 66 years old, the median follow-up since starting pegylated interferon was 58 months. At the time of analysis, 30 (48.4%) patients were alive including 16 still being treated with pegylated interferon. The median survival of patients with intermediate and high-risk prognostic Lille and dynamic International Prognostic Scoring System scores treated with pegylated interferon was increased in comparison to that of historical cohorts. In addition, overall survival was significantly correlated with the duration of pegylated interferon therapy (70 versus 30 months after 2 years of treatment, P

Myeloproliferative neoplasms (MPN) are most common in old people (>60 years) and are rarely identified in children and young adults where information about complication rates and long-term data are lacking. To improve our knowledge, we retrospectively collected cases of young patients diagnosed with MPN before 25 years of age and analysed data of their disease to date, including vascular events and disease evolution. Data were collected in 29 hospital centres from 12 countries. Between 1971 and 2018, 335 young patients were diagnosed for an MPN before the age of 25. They were mostly females (n=246; 73.4%) with a median age of 20.3 years at diagnosis (2.5 months-25 years). Essential thrombocythemia was diagnosed in 234 patients (69.8%), polycythemia vera in 60 (17.9%) and myelofibrosis or unclassified MPN in 41 (12.3%). Most of the diagnoses were made following a coincidental blood count analysis (n=75; 51%) some based on symptoms (n=57; 38.8%) or thrombotic events (n=15; 10.2%). In terms of complications before or at diagnosis, 31 (9.3%) patients experienced thrombosis, mostly venous (75%) and 13 (3.9%) had hemorrhage. At diagnosis, the median leukocyte count was 9x109/l (range: 3-22.8), median hemoglobin count 140 g/l (65-220) and median platelet count 900x109/l (99-3290). To assess the diagnosis, 158 patients (47.2%) had had bone marrow aspirates and 214 (63.9%) a bone marrow biopsy. Mutational status was available in 319 (90%) cases: 194 (60.8%) were JAK2V617F positive, 48 (15%) had a calreticulin mutation, 76 (23.8%) were triple-negative and 1 patient had MPL mutation. The median follow-up of the cohort was 7.7 years (0-46.8) with 134 patients (40%) having follow-up for more than 10 years. 81 female patients (32.9%) experienced pregnancies. During this period, 295 patients (88%) received at least one drug for their MPN: 254 patients (77.2%) received antithrombotic drug and 222 patients (66.5%) a cytoreductive drug. As first line of treatment, hydroxycarbamide was given to 111 patients (50%) whereas anagrelide was given to 56 patients (25.2%) and interferon to 50 (22.5%). During the follow-up, 97 patients (29%) experienced at least one complication. In terms of cardiovascular events, 38 (11.3%) patients experienced thromboses with 50 events in total (recurrences in 12 cases), including 33 venous events (66%) of which 15 were localized in the splanchnic territory (45.5%). Hemorrhagic events were recorded in 34 cases (10.1%). During the follow-up, 39 patients (11.6%) have evolved: 11 from ET to PV (28.2%), 26 into MF (66.7%) and 2 into AML (5.1%). All evolutions were exclusive: one event per patient. At the time of the analysis, 66 patients (19.7%) were declared as lost of follow up and 4 were dead (1.2%). This is the largest cohort of patients aged below 25 years at the time of diagnosis demonstrates that despite their youth most of them (88%) received drug(s) for the management of their MPN. There was a high incidence of complications (29%), with vascular events and disease evolution occurring at equal frequency although death was uncommon (1.2%). Rates of events were disease evolution: 1.51/100pts/y; thromboses: 1.47 and hemorrhages: 1.32. No specific national or international guidance exists for MPN patients of this age; but our data suggest that these are not benign conditions and patients need to be carefully followed and treated. Table. Table. Disclosures Kiladjian: Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding. Giraudier:Novartis: Research Funding. Griesshammer:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Harrison:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria; Gilead: Honoraria, Speakers Bureau; CTI BioPharma: Consultancy, Honoraria.

Background: Myeloproliferative neoplasms (MPN) including polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF) are clonal hemopathies characterized by burdensome symptom profiles and impaired quality of life. Treatments include pharmacologic approaches, phlebotomy, and bone marrow transplant. Outcome studies have historically focused on hematologic improvement and survival benefit. Few prospective studies have evaluated patient-reported symptoms and quality of life outcomes after standard therapy. The Myeloproliferative Neoplasm Quality of Life (MPN-QOL) Study Group aims to objectively quantify symptomatic response to standard treatments. Here we provide updated results for the prospective MPN Experimental Assessment of Symptoms by Utilizing Repetitive Evaluation (MEASURE) trial. Methods: The MEASURE trial is a prospective international cohort study evaluating responsiveness of the Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score (MPN-SAF TSS or MPN-10) in an anticipated 480 ET, PV, and MF patients receiving non-experimental medical therapy and/or phlebotomy. Patients complete the MPN-SAF TSS (JCO 2012) for seven consecutive days at enrollment then again for seven days between 90 days and six months later. Patients also complete the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core-30 (EORTC QLQ-C30) and M.D. Anderson Symptom Inventory (MDASI) instruments at enrollment and on the first day of the second assessment. Physicians report demographic, laboratory, and clinical data. Results: Demographics To date, 340 patients have enrolled and 270 have completed both study visits. Participants include ET (60%), PV (29%), and MF (12%; including 68% primary MF, 12% post-ET, 20% post-PV) patients. Enrolled patients are 51% male. The most common therapies received prior to enrollment were aspirin (34%), phlebotomy (15%), hydroxyurea (14%), and warfarin/clopidogrel/anticoagulation (6%). The most common current MPN therapies were hydroxyurea (67%), aspirin (24%), phlebotomy (9%), and ruxolitinib (8%). Information on mutational analysis was also collected; 77% of patients have a known JAK2 V617F mutation, 3% have an MPL W515 mutation. Symptom Measures On the MPN-SAF TSS, the majority of individual symptoms assessed did not change significantly between the two assessment time points while on standard therapy. Notable exceptions were a significant decrease in weight loss (mean 2.0 vs. 1.7 on a 0=absent to 10=worst imaginable scale, p=0.005) and a significant worsening in quality of life (mean 3.6 vs. 3.8 on a 0=as good as it can be to 10=as bad as it can be scale, p=0.04). No changes were seen in other symptoms including early satiety, abdominal discomfort, night sweats, fatigue, inactivity, poor concentration, bone pain, itching, or fever. Similarly, the cumulative MPN-SAF TSS score remained stable at a mean of 24.4 over the treatment period. These findings were congruent with those observed using the EORTC QLQ-C30 instrument, with no significant changes seen in emotional, physical, role, cognitive, or social functioning. Finally, the MDASI also revealed a lack of change in either symptom severity (mean 2.7) or symptom distress (mean 3.0) scores, which remained stable over the study period. Discussion: Myeloproliferative neoplasms are associated with burdensome symptoms that significantly compromise quality of life. To date, no studies have quantified symptomatic response to standard-of-care treatments. Overall, results from the MEASURE study suggest that standard treatments have limited systematic impact on patient symptomatology over time, with no improvements in overall quality of life. However, we note the relatively low proportion of patients in this cohort treated with ruxolitinib, which has been shown previously to improve symptom burden, and this may have impacted the symptom responses seen in the group as whole; detailed responses by treatment and MPN subtype will be presented. Figure. Figure. Disclosures Dueck: Pfizer: Honoraria; Bayer: Employment; Phytogine: Employment. Ross:BMS: Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Research Funding. Tam:Janssen: Honoraria, Research Funding; Gilead: Honoraria; AbbVie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Travel funding; AbbVie: Honoraria, Research Funding; Roche: Honoraria; Beigene: Honoraria, Other: Travel funding; Pharmacyclics: Honoraria; Gilead: Honoraria; Roche: Honoraria; Beigene: Honoraria, Other: Travel funding. Radia:Novartis: Speakers Bureau; Blueprint: Consultancy. Mesa:UT Health San Antonio - Mays Cancer Center: Employment; Celgene: Research Funding; NS Pharma: Research Funding; Pfizer: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Promedior: Research Funding; CTI Biopharma: Research Funding; Incyte Corporation: Research Funding; Novartis: Consultancy.

International audience; Myeloproliferative neoplasm-related myelofibrosis is associated with cytopenic or proliferative phases, splenomegaly and constitutional symptoms. Few effective treatments are available and small series suggested that interferon could be an option for myelofibrosis therapy. We performed a retrospective study of pegylated-interferon α-2a (Peg-IFNα-2a) therapy in myelofibrosis. Sixty-two patients treated with Peg-IFNα-2a at 17 French and Belgian centres were included. Responses were determined based on the criteria established by the International Working Group for Myelofibrosis Research and Treatment. Mean follow-up was 26 months. Sixteen of 25 anaemic patients (64%) (eight concomitantly receiving recombinant erythropoietin) achieved a complete response and transfusion-independence was obtained in 5/13 patients (38*5%). Constitutional symptoms resolved in 82% of patients. All five leucopenic patients normalized their leucocyte counts, whereas a normal platelet count was obtained in 5/8 thrombocytopenic patients. Splenomegaly was reduced in 46*5% of patients, and complete resolution of thrombocytosis and leucocytosis were observed in 82*8% and 68*8% of patients, respectively. Side effects (mostly haematological) were mainly of grade 1-2. The only factor independently associated with treatment failure was a spleen enlargement of more than 6 cm below the costal margin. In conclusion, Peg-IFNα-2a induced high response rates with acceptable toxicity in a large proportion of patients with primary and secondary myelofibrosis, especially in early phases.

hma.2013.0802 Auteur(s) : Jean-Loup Demory1 Demory.JeanLoup@ghid.net, Brigitte Dupriez2 1 Service d’oncohematologie, laboratoire d’hematologie, groupe hospitalier de l’Universite catholique de Lille 2 Service d’hematologie clinique, centre hospitalier de Lens Tires a part : J. Demory La myelofibrose primitive est le plus grave des syndromes myeloproliferatifs Phi-negatifs (Phi-). Dans une etude europeenne recente portant sur plus d’un millier de patients, la mediane de survie [...]

Purpose Despite the lack of major improvements in the treatment of primary myelofibrosis (PMF), there are recent indications that the survival of patients might have increased over the years. This study was aimed at ascertaining whether survival prolongation has actually occurred in PMF. Patients and Methods A total of 802 patients diagnosed with PMF in four European countries were compared for the presentation of features and survival according to the diagnostic periods 1980 to 1995 (n = 434) and 1996 to 2007 (n = 368); relative survival was estimated for the two groups. Results Patients diagnosed between 1996 and 2007 more often had constitutional symptoms (31% v 23%) but a lower incidence of marked anemia (31% v 39%), leukocytosis greater than 25 × 109/L (9% v 13%), and blood blasts (27% v 33%); risk distribution was comparable between the two groups. Median survival was 4.6 years (95% CI, 4.0 to 5.1) for patients from 1980 to 1995 and 6.5 years (95% CI, 5.5 to 7.4) for patients from 1996 to 2007 (P < .001). The latter group of patients showed improved relative survival, especially for women, patients younger than age 65 years, and patients with low or intermediate-1–risk disease. Rates of PMF-attributable mortality at 5 and 10 years were significantly lower in the second period; this reduction in disease-specific mortality occurred across all patient subgroups, except in intermediate-2–risk or high-risk patients. Conclusion Survival of PMF is steadily improving, except in patients in poor-risk categories. This observation must be taken into account at the time of evaluating the survival impact of newer therapies for PMF, which are currently being tested in these patient subpopulations.

The median survival of patients with primary myelofibrosis ranges from 3.5 to 5.5 years, and most patients die from cause related to the disease, including blast phase (BP, in 5%-30% of cases). Because identification of high-risk patients might use information collected during the clinical course, we assessed the prognostic value of time-dependent covariates for 2 competing risks (death and BP) in a series of 172 patients. Significant (P < .01) adverse time-dependent prognostic factors for the risk of death were the time to onset of anemia (hemoglobin < 100 g/L [10 g/dL]), leukocytosis (leukocyte count > 30 × 109/L), thrombocytopenia (platelet count < 150 × 109/L), presence of circulating blasts, intermediate-high or high International Working Group for Myelofibrosis Research and Treatment score, and time to splenectomy. The first 3 dependent covariates and the time to chemotherapy initiation (P = .05) were prognostic factors for the risk of BP. The prognostic effect of onset of leukocytosis was significantly more pronounced for BP than for death. Thus, occurrence during the follow-up of characteristics associated with an adverse prognostic value at diagnosis also influenced the risks of death and BP. Patients with leukocytosis should be closely monitored. These data might efficiently help to evaluate the severity of the disease before treatment decision during the clinical course.

In a case with secondary myelofibrosis occurring after essential thrombocythemia, cytogenetic analysis revealed an isolated translocation t(X;17)(q27;q22) in all cells. We found that a bacterial artificial chromosome (BAC) encompassing the breakpoint on chromosome 17 long arm contained only one gene, NOG. We therefore investigated the occurrence of this rare breakpoint in myeloproliferative disorders (MPDs). We identified three more patients with a 17q abnormality in MPDs: myelofibrosis with myeloid metaplasia (MMM); chronic myeloid leukemia positive for t(9;22)(q34;q11) with additional t(4;17)(p15;q22) at diagnosis; and myelofibrosis complicating polycythemia vera. All three cases exhibited a split of BACs containing NOG. The protein encoded by NOG, noggin, acts as an antagonist to bone morphogenetic secreted protein 2 and 4 (BMP2 and BMP4). A comparative analysis of gene expression on Agilent 22K oligonucleotide microarrays in purified CD34+ cells from the blood of MMM patients showed significant downregulation of BMPR2, BMPR1B, BMP2, and BMP8; upregulation of BMP3 and BMP10; and a trend to lower expression of NOG. Thus, given that expression and release of BMPs are important in the induction of osteosclerosis and angiogenic activity, the observed BMP deregulations could be triggered by potential NOG genetic alterations in the four cases here described, and may contribute to the myelofibrotic process characterized by bone marrow stromal reaction including collagen fibrosis, osteosclerosis, and angiogenesis.

Myelofibrosis with myeloid metaplasia (MMM) is a rare myeloproliferative disorder which associates clonal proliferation of multi-potent hematologic progenitors and reactive fibrosis [1]. The karyot...

Among cytogenetic studies of patients affected with myelofibrosis with myeloid metaplasia (MMM), a rare chronic myeloproliferative disorder, we found several reports of structural abnormalities of the long arm of chromosome 12. Two MMM patients had a balanced translocation involving 12q: t(4;12)(q32;q15) and t(5;12)(p14;q15), respectively. FISH (fluorescence in situ hybridization) analysis showed that BAC (bacterial artificial chromosome) RP11-366L20 overlaps the breakpoint in both cases. A gene, HMGA2, most of which is included in that BAC, thus was identified as a potential candidate. Using reserves transcriptase–polymerase chain reaction (RT-PCR), we looked for expression of HMGA2 in blood mononuclear cells from these 2 patients and demonstrated a transcript in both. Moreover, we found the gene expressed in the hematopoietic cells of 10 of 10 additional patients bearing no 12q anomalies. HMGA2, not expressed in normal subjects, is implicated in benign solid tumors such as lipomas, leiomyomas, and other rare tumors of mesenchymal origin. We postulate that its dysregulation and overexpression in myeloid progenitors contribute also to the pathogenesis of MMM. © 2003 Wiley-Liss, Inc.

Primary myelofibrosis is characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in the bone marrow and spleen. The expression of CD9, a tetraspanin known to participate in megakaryopoiesis, platelet formation, cell migration and interaction with stroma, is deregulated in patients with primary myelofibrosis and is correlated with stage of myelofibrosis. We investigated whether CD9 participates in the dysmegakaryopoiesis observed in patients and whether it is involved in the altered interplay between megakaryocytes and stromal cells. We found that CD9 expression was modulated during megakaryocyte differentiation in primary myelofibrosis and that cell surface CD9 engagement by antibody ligation improved the dysmegakaryopoiesis by restoring the balance of MAPK and PI3K signaling. When co-cultured on bone marrow mesenchymal stromal cells from patients, megakaryocytes from patients with primary myelofibrosis displayed modified behaviors in terms of adhesion, cell survival and proliferation as compared to megakaryocytes from healthy donors. These modifications were reversed after antibody ligation of cell surface CD9, suggesting the participation of CD9 in the abnormal interplay between primary myelofibrosis megakaryocytes and stroma. Furthermore, silencing of CD9 reduced CXCL12 and CXCR4 expression in primary myelofibrosis megakaryocytes as well as their CXCL12-dependent migration. Collectively, our results indicate that CD9 plays a role in the dysmegakaryopoiesis that occurs in primary myelofibrosis and affects interactions between megakaryocytes and bone marrow stromal cells. These results strengthen the “bad seed in bad soil” hypothesis that we have previously proposed, in which alterations of reciprocal interactions between hematopoietic and stromal cells participate in the pathogenesis of primary myelofibrosis.

Background: Myeloproliferative neoplasms (MPN) including polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF) are clonal hemopathies characterized by burdensome symptom profiles and impaired quality of life. Few studies have evaluated patient-reported outcomes during treatment with non-experimental pharmacological regimens. Aims: The Myeloproliferative Neoplasm Quality of Life (MPN-QOL) Study Group aims to objectively quantify MPN symptom severity, frequency and quality of life at baseline and throughout treatment with non-experimental therapies utilizing the Myeloproliferative Neoplasm Symptom Assessment Form - Total Symptom Score (MPN-SAF TSS; JCO 2012). In this abstract, we provide updated results for the prospective international cohort trial currently in active enrollment: the MPN Experimental Assessment of Symptoms by Utilizing Repetitive Evaluation (MEASURE) trial. Methods: This study aims to recruit 180 international ET, PV, and MF (including primary MF and post-ET or post-PV MF) patients receiving non-experimental medical therapy and/or phlebotomy. Patients complete the MPN-SAF for seven consecutive days at enrollment and repeat the survey for an additional seven consecutive days between 90 days and six months. Patients also complete the European Organisation for Research and Treatment of Cancer (EORTC) and M.D. Anderson Symptom Inventory (MDASI) instruments at enrollment and on the first day of the second assessment. At visits, physicians acquired demographic, laboratory, physical examination, and radiographic data. Descriptive statistics were used to summarize data. Results: Clinical Data The MEASURE trial opened for enrollment in 2012 and remains in recruitment phase with 15 participating international sites. To date, 39 patients have been enrolled and 25 have completed both study visits. Participants include ET (28%), PV (24%), and MF (48%; 50% primary MF, 8% post-ET, 42% post-PV) patients. The majority of patients are male (64%) and of expected age (mean 69.3, range 39-89) for the disorders. Seventeen percent had prior thrombosis, 9% required red blood cell transfusion, and none reported prior splenectomy or hemorrhage. Mean hematologic measures included hemoglobin 13.2 g/dL, WBC count 11.4 x109/L, ANC 8.5 x109/L, and platelets 514 x109/L. Therapies received prior to enrollment included aspirin (n=16), hydroxyurea (n=11), phlebotomy (n=8), warfarin/clopidogrel/anticoagulation (n=8), erythropoietin (n=2), and interferon (n=1). The most common current MPN therapies were hydroxyurea (n=9), aspirin (n=9), interferon (n=4), and phlebotomy (n=2). Symptom Assessment In comparing MPN-SAF TSS mean symptom scores, all symptoms except bony pain improved between the first and second visits, including fatigue, early satiety, abdominal discomfort, inactivity, concentration, night sweats, itching, fever, weight loss, and overall quality of life (Figure1). Total MPN-SAF TSS scores improved from a mean of 32.3 to 25.9. On the EORTC, mean scores for physical, role, emotional, and social functioning improved from the first to the second visit (Figure 2). Cognitive functioning showed a slight decline. Global health status measure improved from 60.2 to 72.9. On the MDASI, symptom severity scores decreased from 3.6 to 2.8 from the first to second visit (Figure 3). Symptom distress measure decreased from 4.1 to 3.0. Discussion: Interim results from the MEASURE trial demonstrate that standard, non-experimental treatment regimens offer improvement in quality of life-related symptoms on multiple patient-reported survey instruments including the MPN-SAF TSS, EORTC QLQ-C30, and MDASI. Updated data including symptom correlations and mutational analysis to be presented at the 2016 ASH conference. Disclosures Ross: Novartis Pharmaceuticals: Honoraria, Research Funding; BMS: Honoraria. Radia:Novartis: Honoraria; Pfizer: Honoraria. McMullin:Novartis: Honoraria, Speakers Bureau. Cargo:Novartis: Honoraria; Celgene: Honoraria, Research Funding. Sekhar:Novartis: Research Funding. Mesa:Gilead: Research Funding; CTI Biopharma: Research Funding; Galena: Consultancy; Ariad: Consultancy; Incyte: Research Funding; Novartis: Consultancy; Celgene: Research Funding; Promedior: Research Funding.

Background: Myelofibrosis (MF) is the less frequent Philadelphia-negative myeloproliferative neoplasms (MPN). We have included in a nationwide database primary (P), post-polycythemia (PPV) and post-essential thrombocythemia (PET) MF diagnosed in France since 2005. Methods: Inclusion criteria were: diagnostic of MF after 2005; according strictly to WHO (bone marrow biopsy mandatory); informed consent. The registry was launched in Oct. 2013, and 26 hematology centers included patients (pts). Summary statistics were reported, namely median [Interquartile range, IQR] or percentage. Baseline characteristics were compared across IPSS groups using chi-square test or Mann and Whitney test. Kaplan-Meier survival curves were plotted and compared by the log-rank test. Results: At time of analysis (June 2016) a total of 527 pts were included in the registry, complete baseline data were available in 499 (95%), and follow-up (FU) data in 433 (87%). Median [IQR] age and M/F sex ratio were 71 [63-78] years and 315/184 (1.7), respectively (resp). 301 (60%) pts had PMF, 182 (36%) had secondary MF (including 64 PPVMF and 118 PETMF) and 16 had pre-fibrotic MF. Five percent of pts had a familial history of hematologic malignancy, and 22% a history of thrombosis or hemorrhage. Splenomegaly was present in 386 (77%) with a median [IQR] spleen size of 4 cm [1-8] below costal margin, and was symptomatic in 11% of pts. Constitutional symptoms were present in 107 (21%) (weight loss in 56, night sweats in 61, fever in 14), and ECOG score was 0, 1, 2, and 3 in 53%, 36%, 11% and 0.3% of pts, resp. Median [IQR] Hemoglobin, WBC and platelet counts were 109 g/L [94-122], 9.3 G/L [5.7-16.0] and 257 G/L [138-430], resp. Circulating blast cells were present in 41%, LDH was above normal value in 95% of pts, median EPO level was 54 [11 - 57] U/L. Grade of fibrosis (WHO) was 1, 2, and 3 in 2%, 66% and 32% of pts, resp. Karyotype was done in 321 pts, normal in 173 (54%), abnormal with favorable prognostic value in 89 (30%) and unfavorable in 30 (10%) (29 failures). A total of 461 (92%) pts had molecular testing: 60% were JAK2V617F positive, 4% had MPL and 7% had CALR mutations, and 99 (28%) over the 352 pts with triple testing were triple negative. IPSS risk categories were low, int-1, int-2 and high in 68 (14%), 168 (34%), 158 (32%), and 105 (21%) pts, resp. In addition to constitutional symptoms, there was a significant increase in the prevalence of clinical signs across IPSS categories (from low to high risk): symptomatic spleen (p=0.016) -though no difference in spleen size was found (p=0.18)-, early satiety (p=0.013), ECOG score (p= 0.0001), bone pain (p=0.002). Moreover, among biological parameters, there was an increase across IPSS groups in WBC (p=0.029), LDH (p=0.0007), ferritin (p=0.003), circulating CD34+ cells (p=0.020), EPO level (p=0.035). In contrast, a decrease was seen for hemoglobin and platelets (p=0.0001 for both). Lastly, frequency of grade 3 fibrosis increased with IPSS (p=0.043), while no evidence of difference was found regarding abnormal karyotype and mutational pattern. Median FU was 33 months [9-63 months]. Among those 433 pts with FU data, median FU was 38 months [19-68], and 124 (29 %) pts had died at the time of analysis, including 13%, 17%, 31%, and 40% of pts from the Low, Int-1, Int-2, and High risk groups, resp (p= 0.0001, figure 1). In the 450 pts with treatment data, treatments received during FU included cytoreductive drug (41%), Jak-inhibitors (35%), Interferon alpha (18%), IMIDs (4%). Splenectomy was performed in only 14 (3%) pts. Forty percent of pts received packed red blood cells, and 12% platelets transfusions. 49 (11%) patients participated in a clinical trial, and 27 (6%) were allografted. Conclusion: This is the first analysis of the French MF observatory after inclusion of more than 500 pts diagnosed and treated during the past 10 years. Complete baseline data and follow-up information available for the majority of pts should allow for new studies of outcomes and influence of clinical and biological parameters, as well as reassessment of prognostic models in the era of new targeted therapies. Figure 1 Comparison of overall survival according to IPPS Figure 1. Comparison of overall survival according to IPPS Figure 2 Figure 2. Disclosures Etienne: Pfizer: Speakers Bureau; BMS: Speakers Bureau; ARIAD: Speakers Bureau; novartis: Consultancy, Speakers Bureau. Tavitian:Novartis: Membership on an entity's Board of Directors or advisory committees. Ugo:Novartis: Membership on an entity's Board of Directors or advisory committees. Kiladjian:Novartis: Honoraria, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Auteur(s) : Jean-Loup Demory1, Jean-Francois Abgrall2 1Service d’hematologie, Hopital Saint-Vincent de Paul, Lille 2Laboratoire d’hematologie, Hopital de la Cavale Blanche, Brest J.-L. Demory Les classifications recentes font effectivement mention d’une « forme prefibrotique » de myelofibrose primitive (MP). Cette notion est issue d’une serie impressionnante de publications redondantes de l’equipe de Thiele (Cologne) depuis les annees 1990 : chez certains patients [...]

Two main types of therapy-related acute myeloid leukemias (tAML) and myelodysplastic syndromes (tMDS) have been described. The first classical type typically occurs late after use of alkylating agents and presents as MDS with -7/del 7q and/or -5/del5q. The second form occurs early after the use of agents targeted at topoisomerase II, and presents as AML with 11q23 or other rearrangements of de novo AML. Recently, we and others reported, in AML and MDS, a strong correlation between cytogenetic rearrangements leading to 17p deletion, a specific type of dysgranulopoiesis and p53 mutation; several of those cases of 17p- syndrome were therapy-related. Over the last 15 years, we observed 25 cases of tAML and tMDS with 17p deletion, which represented 36% of the AML and MDS with 17p deletion diagnosed during that period. Median age was 59 years. Twenty-one patients had tMDS and four tAML. Typical dysgranulopoiesis and p53 mutation and/or overexpression were seen in 22 of 24 and 16 of 19 evaluable patients, respectively. 17p deletion resulted from unbalanced translocations involving 17p (18 cases), monosomy 17 (five cases), i(17q) (one case) or del 17p (one case). Twenty-one patients also had -5/del 5q, and/or -7/del 7q. Median interval from treatment of the first tumor of tAML and tMDS was 94 months (range 19-252). Median survival was only 7 months. Based on primary tumor and antineoplastic agents used, patients could be relatively well divided into two groups: a first group of 11 cases, occurring mainly after a lymphoid neoplasm (eight cases) treated by chemotherapy with an alkylating agent (10 cases), and a second group of 14 cases occurring after essential thrombocythemia (ET) or polycythemia vera (PV) treated mainly by hydroxyurea (10 cases), pipobroman (eight cases), 32P (six cases) but rarely by alkylating agents (two cases). -7/del 7q was found in 10 of the 11 patients in the first group, as compared to three of the 14 patients of the second group (P = 0.0001). Therefore, therapy-related cases represent a high proportion of AML and MDS with the 17p- syndrome. They have many features in common with classical tMDS and tAML, including long interval from the first tumor, a usual preleukemic phase, and frequent occurrence of -5/del 5q. About one half of them, in addition, occur after alkylating agents and generally carry -7/del 7q. The other half, however, occur mainly after ET or PV treated by hydroxyurea or other non-alkylating agents, and usually have no -7/del 7q. These findings bring further support to a possible relationship between prior drugs used and cytogenetic rearrangements in tAML and tMDS.

Myelofibrosis with myeloid metaplasia (MMM) is an uncommon disorder in young individuals, for whom haemopoietic stem cell transplantation offers the only possibility of cure. However, although the latter procedure is associated with significant morbidity and mortality, the clinical course of MMM is variable, with some patients surviving for less than a year and others showing an indolent course. Selection of young MMM patients for transplantation or other newer therapies is currently difficult since no prognostic data exists for this subgroup. In the present collaborative study a number of initial clinical and laboratory parameters have been evaluated for prognosis in 121 MMM patients aged 55 years or less. Median survival of the series was 128 months (95% CI 90-172). In the Cox proportional hazard regression model three initial variables were independently associated with shorter survival: Hb /=1% (P=0.003). Based on the above three criteria, of the 116 patients with complete data, two groups were identified: a 'low-risk' group, characterized by 88 patients with up to one adverse prognostic factor, in whom MMM had an indolent course (median survival 176 months, 95% CI 130-188), and a 'high-risk' group, including 28 patients with two or three factors, who had a more aggressive disease (median survival 33 months, 95% CI 20-42). The above prognostic scoring system showed a high positive predictive value, sensitivity and specificity to predict survival in the series, and could be of help in making treatment decisions in young patients with MMM.

Idiopathic myelofibrosis, or agnogenic myeloid metaplasia, is a chronic myeloproliferative disorder characterized by clonal expansion and marrow fibrosis. Although marrow fibrosis appears to be a reactive process, it substantially contributes to impaired haemopoiesis. During the last few years the implication of megakaryocyte-derived growth factors in its pathogenesis has been documented. We previously reported increased expression of TGF-beta in patients with idiopathic myelofibrosis. In the present study we show that circulating megakaryocytic cells from such patients expressed high levels of basic fibroblast growth factor (bFGF). An increased expression of bFGF was also detected in patients' platelets. Under culture conditions, bFGF present in megakaryocytic cells was not exported into the medium. consistent with the fact that bFGF is devoid of a secretion peptide signal. Interestingly, this lack of bFGF secretion was observed in all patients but one, who was in an accelerated phase of the disease and presented an important percentage of circulating megakaryoblasts.

We studied the survival of 195 patients with agnogenic myeloid metaplasia (AMM) diagnosed between 1962 and 1992 in an attempt to stratify patients into risk groups. Median survival was 42 months. Adverse prognostic factors for survival were age > 60 years, hepatomegaly, weight loss, low hemoglobin level (Hb), low or very high leukocyte count (WBC), high percentage of circulating blasts, male sex, and low platelet count. A new scoring system based on two adverse prognostic factors, namely Hb < 10 g/dL and WBC < 4 or > 30 x 10(3)/L, was able to separate patients in three groups with low (0 factor), intermediate (1 factor), and high (2 factors) risks, associated with a median survival of 93, 26, and 13 months, respectively. An abnormal karyotype (32 cases of 94 tested patients) was associated with a short survival, especially in the low-risk group (median survival of 50 v 112 months in patients with normal karyotype). The prognostic factors for acute conversion were WBC > 30 x 10(3)/L and abnormal karyotype. Thus, hemoglobin level and leukocyte count provide a simple prognostic model for survival in AMM, and the adverse prognostic value of abnormal karyotype may be related to a higher rate of acute conversion.

Survival of patients with primary myelofibrosis (PMF) is highly variable, ranging from months to even decades. Although treatment of PMF remains mostly palliative, the increasing use of allogeneic hemopoietic stem cell transplantation and introduction of newer therapies has raised the need for a better prognostic stratification of these patients. The initial features of 1,054 patients (638M/416F; median age 64 years, range 10–90) consecutively diagnosed with PMF in seven institutions from Europe and USA between 1980 and 2007 were evaluated for prognostic significance. Patients with the so-called “pre-fibrotic” form of PMF were not considered. The variables assessed were those previously shown to be of prognostic value in PMF, those clinically meaningful, and possible confounders (namely, diagnostic period and series of origin). The effect of the potential prognostic factors on survival was evaluated by the Cox proportional hazards regression and the proportional hazards assumption checked in every model by graphical methods and the Grambsch-Therneau test. A prognostic model was identified through a stepwise selection process, based on a z-test of the regression coefficients, and evaluated by calculating its discriminating power (measured by Harrell’s C concordance index), as compared to that of current PMF prognostic systems, and its positive predictive accuracy for actual survival longer (or shorter) than definite periods of time from diagnosis. The replicability of both the prognostic variables and the new prognostic score was tested by bootstrap re-sampling, in which 1,000 samples of the same size than the original series were built by random extraction with reposition. Median survival of the overall series was 69 months (95% CI: 61–76), with 49% of patients having died at the time of study. At multivariate analysis, five initial variables were associated with shorter survival: age > 65 years, constitutional symptoms (weight loss, fever, sweats), Hb < 10 g/dL, leukocyte count > 25×109/L, and blood blasts 1%. The hazard ratios of these variables did not differ substantially, ranging from 2.89 (95% CI: 2.46–3.61) for Hb < 10 g/dL to 1.80 (95% CI: 1.50–2.17) for blood blasts 1% and, therefore, one point was assigned to each one. Based on this, among the 1,001 patients of the series with all the necessary data available, four risk groups were identified with no overlapping in the 95% CI of their survival curves (figure): low risk (no factor, 22% of patients; median survival 135 months); intermediate risk-1 (one factor, 29% of patients; median survival 95 months); intermediate risk-2 (two factors; 28% of patients; median survival 48 months); and high risk (three factors, 21% of patients; median survival 27 months). The new prognostic score showed high predictive accuracy and replicability and had higher discriminating power than current risk-based PMF classifications. In the 409 patients with assessable metaphases, presence of cytogenetic abnormalities (observed in 30% of cases) was associated with shorter survival and contributed to prognosis, but only in patients in the intermediate-risk groups. No prognostic influence for either JAK2 V617F mutational status (n= 345) or blood CD34+ cell count (n= 150) was noted. This new prognostic scoring system substantially improves existing risk-based classifications of PMF and may help in treatment-decision making and in the design and analysis of newer therapies for the disease. Figure Figure

Therapeutic decision-making in primary myelofibrosis (PMF) is becoming more challenging because of the increasing use of allogeneic stem cell transplantation and new investigational drugs. To enhance this process by developing a highly discriminative prognostic system, 1054 patients consecutively diagnosed with PMF at 7 centers were studied. Overall median survival was 69 months (95% confidence interval [CI]: 61-76). Multivariate analysis of parameters obtained at disease diagnosis identified age greater than 65 years, presence of constitutional symptoms, hemoglobin level less than 10 g/dL, leukocyte count greater than 25 × 109/L, and circulating blast cells 1% or greater as predictors of shortened survival. Based on the presence of 0 (low risk), 1 (intermediate risk-1), 2 (intermediate risk-2) or greater than or equal to 3 (high risk) of these variables, 4 risk groups with no overlapping in their survival curves were delineated; respective median survivals were 135, 95, 48, and 27 months (P < .001). Compared with prior prognostic models, the new risk stratification system displayed higher predictive accuracy, replicability, and discriminating power. In 409 patients with assessable metaphases, cytogenetic abnormalities were associated with shorter survival, but their independent contribution to prognosis was restricted to patients in the intermediate-risk groups. JAK2V617F did not cluster with a specific risk group or affect survival.

Myelofibrosis with myeloid metaplasia is the rarest myeloproliferative syndrom. Diagnosis is often easy in classical form, characterised by splenomegaly, leukoerythroblastic blood reaction and tear-drop erythrocytes on blood count and myelofibrosis on bone marrow biopsy. Evolution is highly variable with a median overal survival of 40 to 60 months and numerous prognostic factors especially anemia. No treatment has been demonstrated to improve survival (apart from allogenic bone marrow transplant). The rarity and the complexity of the disease are the most important difficulties for the definition of standardized diagnostic, prognostic and therapeutic criteria.

A minority of patients with newly diagnosed polycythemia vera (PV) have an abnormal karyotype in their myeloid cells but no invariant chromosomal aberration has been found. The most frequent visible alteration is a 20q deletion, also characterized in other myeloproliferative diseases (MPD) and myeloid malignancies; among other chromosomal changes, trisomy 9 appears more common in PV than in other MPDs. When a myelofibrosis complicates the course of the disease, cytogenetic anomalies become quite common with a striking frequency of partial duplication 1q; an evolution towards myelodysplasia or acute leukemia is almost always associated with nonspecific chromosomal aberrations. Modern cytogenetic methods have disclosed cryptic anomalies and pointed out the high frequency of 9p alterations affecting a restricted region, thus stimulating an active search for candidate genes or specific mutations.

Rearrangements of 6p are frequent in both myeloid and lymphoid malignant hematological disorders. High-mobility group AT-hook 2 (HMGA2) rearrangements have been described in myelofibrosis with myeloid metaplasia (MMM) and also in myelodysplasia. High-mobility group A proteins are nonhistone nuclear proteins that bind DNA and regulate the transcriptional activity of many genes. We used FISH, with bacterial artificial chromosome RP11-513I15 probe, to study 16 cases of myeloid malignancies with chromosome 6 short arm rearrangements, most of them following myeloproliferative disorders. Among these we found two 6p21.3 duplications and one 6p21.3 triplication involving HMGA1 in four cases of myelodysplasia with and without myelofibrosis. In these four cases, duplications and triplication were partially masked at the cytogenetic level by a derivative chromosome 6 resulting from translocation with another chromosome. HMGA1 proteins have been recently found overexpressed in human leukemias, but to our knowledge this is the first reported duplication of HMGA1.

Among cytogenetic studies of patients affected with myelofibrosis with myeloid metaplasia (MMM), a rare chronic myeloproliferative disorder, we found several reports of structural abnormalities of the long arm of chromosome 12. Two MMM patients had a balanced translocation involving 12q: t(4;12)(q32;q15) and t(5;12)(p14;q15), respectively. FISH (fluorescence in situ hybridization) analysis showed that BAC (bacterial artificial chromosome) RP11-366L20 overlaps the breakpoint in both cases. A gene, HMGA2, most of which is included in that BAC, thus was identified as a potential candidate. Using reserves transcriptase-polymerase chain reaction (RT-PCR), we looked for expression of HMGA2 in blood mononuclear cells from these 2 patients and demonstrated a transcript in both. Moreover, we found the gene expressed in the hematopoietic cells of 10 of 10 additional patients bearing no 12q anomalies. HMGA2, not expressed in normal subjects, is implicated in benign solid tumors such as lipomas, leiomyomas, and other rare tumors of mesenchymal origin. We postulate that its dysregulation and overexpression in myeloid progenitors contribute also to the pathogenesis of MMM.

Polycythemia vera (PV) is a chronic myeloproliferative disease characterized by an increase of total red cell volume; in 10% to 15% of cases, bone marrow fibrosis complicates the course of the disease after several years, resulting in a hematologic picture mimicking myelofibrosis with myelocytic metaplasia (MMM). This condition is known as post polycythemic myelofibrosis (PPMF). Among 30 patients with PPMF followed in Northern France, 27 (90%) expressed one or two abnormal clones in myelocytic cell cultures. Of these, 19 (70%) had partial or complete trisomy 1q. This common anomaly either resulted from unbalanced translocations with acrocentric chromosomes, that is, 13, 14, and 15, or other chromosomes, that is, 1, 6, 7, 9, 16, 19, and Y, or from partial or total duplication of long arm of chromosome 1. A single patient had an isochromosome 1q leading to tetrasomy 1q. In all cases, a common trisomic region spanning 1q21 to 1q32 has been identified. Given that most patients had previously received chemotherapy or radio-phosphorus to control the polycythemic phase of their disease, this study illustrates the increased frequency of cytogenetic abnormalities after such treatments: 90% versus 50% in de novo MMM. Moreover, karyotype can be used to distinguish PPMF-where trisomy 1q is the main anomaly-from primary MMM where trisomy 1q is rare and deletions 13q or 20q are far more common. Whether trisomy 1q is or is not a secondary event remains a matter of debate, as well as the role of cytotoxic treatments.

Among cytogenetic studies of 205 patients diagnosed as myelofibrosis with myeloid metaplasia, we found seven cases with structural abnormalities of the long arm of chromosome 12. The karyotype showed six balanced translocations, that is, t(4;12)(q33;q21), t(5;12)(p14;q21), t(1;12)(q22;q24), t(12;17)(q24;q11), t(7;12) (p11;q24), and t(1;12)(p12;q24), as well as other cytogenetic abnormalities such as del(12)(q21;q24) and inv(12) (p12q24). Some isolated cases involving the 12q21 region have also been described in the literature. Importance of rearrangement of chromosome 12 in 12q21 or 12q24 is underlined by the authors suggesting a proto-oncogene accountable mechanism of leukemogenesis.

Aspirin is recommended and widely prescribed to MPN patients without any evaluation of its efficacy. We report on 46 patients followed in our institution for a Myeloproliferative Neoplasm and treated by low-dose aspirin (75 to 160 mg p. day) alone or aside a cytoreductive therapy, mainly hydroxy-urea ; on the occasion of a routine visit we measured the response of their platelets to arachidonic acid in order to assess their sensibility to aspirin. Most patients (25) had ET, 15 had PV, 4 had CML and 2 Myelofibrosis. Response to arachidonic acid was measured on a citrated whole blood sample using an impedance-based semi-automatic aggregometer (Multiplate®) ; TRAP (Thrombin Receptor Activating Peptide) aggregation was performed systematically as a positive control. In 20 patients out of 46 (43,5%) there was persistence of a significant aggregability to arachidonic acid defining the resistance to aspirin according to our reference values : 10/25 ET (38%), 9/15 PV (62%)and 1/4 LMC. These proportions are much higher than those we note in heart or vascular diseases as well as those reported in published series. The observance of aspirin treatment could be considered doubtful only in a minority of patients (5/46) ; as the resistance appeared more frequent in PV we could assume a biais linked to the utilization of whole blood, but most PV patients were well controlled and did not have an elevated hematocrit. In untreated or refractory ET patients, the persistence of an increased platelet count suggests that aspirin dosage was not adequate although there was no statistical significance between daily dose and resistance. We observed an ischemic complication (transient stroke) in a single patient whose platelet count was not controlled despite hydroxy-urea but remained sensible to aspirin. We conclude that aspirin resistance is frequent in MPN treated with low-dose aspirin and that a monitoring is worth to increase the dose or change for another inhibitor. Disclosures: No relevant conflicts of interest to declare.

Introduction Myelofibrosis (MF) and Post PV/ET MF represents a group of debilitating hematological disorders in which quality of life (QOL) is severely compromised in most patients as a result of persistent constitutional symptoms, progressive cytopenias, and splenomegaly. Conventional therapeutic modalities have largely focused on symptoms palliation rather than cure. Allogeneic stem cell transplantion (ASCT) remains the only potential curative option for intermediate and high risk disease. Till now, outcome data for MF have historically focused on survival benefit. Until now, few studies have evaluated the QOL, financial burden, and symptom response in MF patients undergoing treatment including ASCT. The development of the Myeloproliferative Symptom Assessment Form Total Symptom (MPN-SAF TSS) score in 2012 allows us to objectively quantify with a standardized tool these crucial aspects of patients care. The Myeloproliferative Neoplasm Quality of Life (MPN-QOL) Study Group aims to objectively quantify symptomatic response to standard available treatments by utilizing the MPN-SAF TSS. We introduce two MPN-QOL prospective trials currently in active enrollment: The MPN Experimental Assessment of Symptoms by Utilizing Repetitive Evaluation (MEASURE) Trial and the Symptoms Yielded in Myelofibrosis Patients after Transplant as Objectified by MPN-SAF TSS (SYMPTOMS) Trial. Our intention with these studies is to quantify the QOL and symptom burden of PMF and post PV/ET MF patients undergoing treatment including ASCT. Methods The MEASURES trial is a prospective questionnaire based study evaluating the responsiveness of the MPN-SAF TSS in detecting symptomatic changes in target symptoms for an anticipated 180 ET, PV and MF (including primary MF, post-ET and post PV MF) patients receiving non-experimental medical therapy (aspirin, hydroxyurea, anagrelide, interferon, busulfan, melphalan, cladribine, thalidomide, lenalidomide, prednisone, danazol, ruxolitinib) and/or phlebotomy. Patients complete the MPN-SAF for seven consecutive days at the time of enrollment and repeat the survey for an additional seven consecutive days between 90 days and six months. Patients also complete the MDASI, EORTC and Global Impression of Change Items on the first day of the second assessment. Physicians acquire demographic, laboratory, physical examination and radiographic data, along with serial response assessments. In parallel, the SYMPTOMS trial is prospective questionnaire based study evaluating the QOL of patients undergoing ASCT utilizing MPN-SAF TSS, the FACT-BMT, Global Impression of Change, and a financial questionnaire. Patients will be evaluated at various time points pre-transplant, day 30, day 100 and 1 year post-transplant. Participants (N=110) will be prospectively enrolled from Mayo Clinic Arizona (MCA) with other centers to soon join. To date we have enrolled 5 patients from MCA on the SYMPTOMS trial and 32 patients on the MEASURES trial. Results Both trials began open enrollment in the summer of 2012 and remain in recruitment phase. Our updated preliminary data will be presented at the ASH 2013 meeting Conclusion Myeloproliferative Neoplasms have been associated with debilitating symptom profile that can significantly impair the QOL. We recognize the burden of this disease and treatment and have therefore initiated two ongoing parallel prospective trials with goals to quantify the QOL and symptom burden of MPN patients. By quantifying degree of burden and impairment in QOL in a standardized format in MPN patients undergoing a range of treatments, we will in the future be better able to inform our patients of the likely benefits and toxicities of the available treatment options. Disclosures: Birgegard: Vifor Pharma: Honoraria.

According to strict clinical, hematological and morphological criteria, the Philadelphia (Ph) chromosome negative chronic myeloproliferative disorders essential thrombocythemia (ET), polycythemia vera (PV), and agnogenic myeloid (megakaryocytic/granulocytic) metaplasia (AMM) or idiopathic myelofibrosis (IMF) are three distinct disease entities with regard to clinical manifestations, natural history and outcome in terms of life expectancy. As clonality studies have clearly demonstrated that fibroblast proliferation in AMM, as well as in many other conditions such as advanced stages of Ph(+)-essential thrombocythemia, Ph(+)-granulocytic leukemia, and Ph(-)-polycythemia vera, is polyclonal indicating that myelofibrosis is secondary to the megakaryocytic granulocytic metaplasia in these various conditions, AMM is illogically labeled as IMF. As abnormal megakaryocytic granulocytic metaplasia is the essential feature preceding the early prefibrotic stage of AMM, the term essential megakaryocytic granulocytic metaplasia (EMGM) can readily be used to characterize this condition more appropriately at the biological level. Clinical, hematological and morphological characteristics, in particular megakaryocytopoiesis and bone marrow cellularity, reveal diagnostic features, which enable a clear-cut distinction between ET, PV and EMGM or classical IMF. The characteristic increase and clustering of enlarged megakaryocytes with mature cytoplasm and multilobulated nuclei and their tendency to cluster in a normal or only slightly increased cellular bone marrow represent the hallmark of ET. The characteristic increase and clustering of enlarged mature and pleiomorphic megakaryocytes with multilobulated nuclei and proliferation of erythropoiesis in a moderate to marked hypercellular bone marrow with hyperplasia of dilated sinuses are the specific diagnostic features of untreated PV. EMGM, including the early prefibrotic stages as well as the various myelofibrotic stages of classical IMF appear to be a distinct neoplastic dual proliferation of abnormal megakaryopoiesis and granulopoiesis. The histopathology of the bone marrow in prefibrotic EMGM and in classical IMF is dominated by atypical, enlarged and immature megakaryocytes with cloud-like immature nuclei, which are not seen in ET and PV at diagnosis and during follow-up. Myelofibrosis in ET, PV and EMGM is graded into: no reticulin fibrosis (MF0), early reticulin fibrosis (MF1), advanced reticulin sclerosis with minor or moderate collagen fibrosis (MF2) and advanced collagen fibrosis with osteosclerosis (MF3). Myelofibrosis is not a feature of ET at diagnosis and during long-term follow-up. Myelofibrosis may be present in a minority of PV-patients at diagnosis and usually becomes apparent during long-term follow-up in the majority of PV-patients. Myelofibrosis secondary to the abnormal megakaryocytic and granulocytic myeloproliferation constitutes a prominent feature in the majority of EMGM/IMF at time of diagnosis and usually progresses more or less rapidly during the natural history of the disease. Life expectancy is normal in ET, normal during the 1st ten years and compromised during the 2nd ten years follow-up in PV, but significantly shortened in the prefibrotic stage of EMGM as well as in the various myelosclerotic stages of classical IMF. First line treatment options in prospective randomized clinical trials of newly diagnosed MPD-patients are control of platelet function with low-dose aspirin versus reduction of platelet count with anagrelide, interferon or hydroxyurea in ET; control of platelet and erythrocyte counts by interferon alone versus bloodletting plus hydroxyurea on indication in PV; interferon versus no treatment in the early stages of EMGM; a wait and see strategy in the fibrotic stages of EMGM or classical IMF with favorable prognostic factors, and bone marrow transplantation in classical IMF with poor prognostic factors at presentation or during short-term follow-up.

Treatment with alkylating agents or radiophosphorous (32P) has been shown to carry a certain leukemogenic risk in myeloproliferative disorders (MPDs), including essential thrombocytemia (ET). The leukemogenic risk associated to treatment with hydroxyurea in ET, on the other hand, is generally considered to be relatively low. Between 1970 and 1991, we diagnosed ET in 357 patients, who were monitored until 1996. One or several therapeutic agents had been admistered to 326 patients, including hydroxyurea (HU) in 251 (as only treatment in 201), pipobroman in 43, busulfan in 41, and32P in 40. With a median follow-up duration of 98 months, 17 patients (4.5%) had progressed to acute myeloid leukemia (AML; six cases) or myelodysplastic syndrome (MDS; 11 cases). Fourteen of these patients had received HU, as sole treatment in seven cases, and preceded or followed by other treatment in seven cases, mainly pipobroman (five cases). The remaining three leukemic progressions occurred in patients treated with 32P (two cases) and busulfan (one case). The incidence of AML and MDS after treatment, using 32P alone and 32P with other agents, busulfan alone and with other agents, HU alone and with others agents, and pipobroman alone and with other agents was 7% and 9%, 3% and 17%, 3.5% and 14%, and 0% and 16%, respectively. Thirteen of 17 patients who progressed to AML or MDS had successful cytogenetic analysis. Seven of them had rearrangements of chromosome 17 (unbalanced translocation, partial or complete deletion, isochromosome 17q) that resulted in 17p deletion. They also had a typical form of dysgranulopoiesis combining pseudo Pelger Hüet hypolobulation and vacuoles in neutrophils, and p53 mutation, as previously described in AML and MDS with 17p deletion. Those seven patients had all received HU, as the only therapeutic agent in three, and followed by pipobroman in three. The three patients who had received no HU and progressed to AML or MDS had no 17p deletion. A review of the literature found cytogenetic analysis in 35 cases of AML and MDS occurring after ET, 11 of whom had been treated with HU alone. Five of 35 patients had rearrangements that resulted in 17p deletion. Four of them had been treated with HU alone. These results show that treatment with HU alone is associated with a leukemic risk of approximately 3.5%. A high proportion of AML and MDS occurring in ET treated with HU (alone or possibly followed by pipobroman) have morphologic, cytogenetic, and molecular characteristics of the 17p− syndrome. These findings suggest that widespread and prolonged use of HU in ET may have to be reconsidered in some situations, such as asymptomatic ET.

Abstract 1762 Introduction The primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by myeloproliferation, splenomegaly with hematopoietic metaplasia and dysmegakaryopoieisis. We have previously described an increase in Flt3 ligand (FL) in plasma and spleen of patients with PMF and its role in the dysmegakaryopoiesis (Desterke and al, Cancer Res, 2011). Account the importance of FL in development of splenic myeloid dendritic cells (mDC), we studied the differentiation of mDC in patients and its potential impact on dysmegakaryopoiesis. Patients and Methods The mDCs were obtained from cell culture of blood and spleen mononuclear cells in presence of fetal calf serum (FCS) and bacterial lipolysaccharides (LPS). The megakaryocytes were obtained by culturing CD34+ cells from blood or spleen in specific medium serum free in presence of IL-3, IL-6, IL-11 and Tpo. Gene expression was quantified by microarray and RT-QPCR, protein analysis by immunofluorescence and flow cytometry, and migration experiments were performed in Boyden chamber. Results Transcriptome of circulating CD34+ cells from PMF patients showed an increase in expression of genes encoding for integrin CD11c and also TLR4 and a decrease in the expression of gene encoding TLR9: suggesting the presence of progenitor mDCs in the blood. These results have been confirmed 1/ in cytometry by an increase in the number of CD34+ CD11c+ HLA-Dr+ cells in the blood; 2/ in cell culture by the presence of adherent cell colonies positives for TLR4+ CD11c+ HLA-Dr+ in the blood. The in vitro differentiation of mDC cells and the proportion of mature mDCs HLA-Dr+ CD11c+ cells are decreased in blood of PMF patients. Myeloid nature of circulating DCs was confirmed by the absence expression of the plasmacytoid membrane marker CD123 and by an increased of TLR4 and myeloid PU-1 (myeloid transcription factor) expression in opposite to a decreased of plasmacytoid markers: IL-23, HMGB1 and TLR9. Moreover in PMF patients, circulating adherent mDCs overexpressed CXCL12 chemokine and also FL which have abnormal chemottractant ability with respect to PMF megakaryocytic precursors still expressing flt3 receptor. Finally, in PMF patients, coculture of MK with mDC promotes their survival, differentiation and maturation (MK ploidy and transcriptional program). Primary results confirmed the presence of these mDCs precursors (CD34+ CD11c+ HLA-Dr+) in the spleen of PMF patients which harbored also an extramedullary megakaryopoiesis. These mDCs precursors are absent of the spleen from healthy subjects. Conclusion Our results show an increased presence of mDC progenitor population CD34+ HLA-Dr+ CD11c+ in the blood and the spleen of PMF patients. They also suggest that these cells are involved in migration, survival and differentiation/maturation of megakaryocytes, particulary in the spleen of patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 2841 Introduction: Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) characterized by bone marrow fibrosis, splenomegaly, cytopenias and constitutional symptoms. Ruxolitinib was recently approved by the FDA for the treatment of MF in the USA; its approval in Europe is still pending. However, EU patients may access to ruxolitinib through compassionate programs. In France, health authorities opened a compassionate patient-named program (Authorization for Temporary Utilization [ATU] program. Methods: 241 French patients (pts) with MF, including primary (PMF), post-polycythemia vera (PPV) and post-essential thrombocythemia (PET) MF were granted ruxolitinib therapy through ATU program, independently of their JAK2 mutational status, between April 15, 2011 and May 31, 2012. Physicians were asked to provide information on disease characteristics, treatment history, constitutional symptoms, spleen size, platelet and neutrophils count, as well as the ruxolitinib dose prescribed and adverse events (AE). Request forms had to be submitted at the time of initial application and every 3 months upon drug resupply or in case of treatment discontinuation. This analysis has been performed based on data available at baseline (n= 241), after 3 months (n= 101), 6 months (n= 57), 9 months (n= 21) and 12 months (n= 4). Results: In the entire cohort, 138 pts were men and 103 women. Median age was 68.3 years. 51.5% of pts had PMF, 22.8% PPV-MF and 23.8% PET-MF. 99.2% of pts had received ≥1 lines of therapy for MF prior to ruxolitinib (hydroxyurea: 56%; pipobroman: 15.4%; iMIDs: 13.7%; interferons: 13.7%; erythropoietins: 6.6%; spleen irradiation: 6.6%; anagrelide: 5.8%; corticosteroids: 4.9%). Despite these therapies, 93.7% had constitutional symptoms and 94.2% of patients presented a palpable splenomegaly (median 15 cm below costal margin) at inclusion. Efficacy: According to the baseline platelet count, ruxolitinib therapy was initiated at 15 mg BID in 132 pts (54.8%) or 20 mg BID in 103 pts (42.7%), or other doses in a minority of pts (n=6). Among the pts who were evaluable after 3 and 6 months of therapy, 96.5% and 90% presented a mean reduction in the spleen size (by palpation) by 47.2% and 46% from baseline, respectively. Constitutional symptoms resolved in 65.3% and 70.2% of pts at the aforementioned time points, respectively. In pts who completed 9 months follow-up (n=21), benefits in spleen size reduction and symptoms resolution were durable (95% and 71.4%). Safety: Since the beginning of the ruxolitinib ATU program, 83 pts presented at least one AE, including 27 pts with serious AE (SAE), with or without causal relationship to ruxolitinib therapy. AE, all grades, were essentially hematologic abnormalities (51.6%), gastro-intestinal 6.3%, cardiac 3.1%, musculoskeletal 3.1%, hepatic 2.3%, infection 2.3%. Dose adjustments were reported in 60 pts, mainly due to thrombocytopenia (n=36) and anemia (n=13). However, no patient discontinued ruxolitinib therapy because of cytopenia. Treatment was discontinued in 11 patients after a median duration of 2.6 months (range 0.3–7.9 months). Reasons for discontinuation were death (n=6), AE (n=2), inefficacy (n=2), and patient decision (n= 1). Conclusion: In this large, unselected population of heavily pretreated MF pts, ruxolitinib therapy appeared to be effective in treating both constitutional symptoms and splenomegaly, in line with previously reported efficacy in clinical trials. The safety profile seems also comparable. Comprehensive data and updated follow-up of the cohort will be presented. Disclosures: Off Label Use: compassionate use of ruxolitinib for myélofibrosis in France (indication not yet approved by EMEA). Rey:Novartis: Consultancy. Nicolini:novartis, Bristol myers Squibb, Pfizer, Ariad and Teva: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Recher:Celgène, Genzyme, Sunesis, Jansen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel to ASH Other. Ranta:Novartis: Membership on an entity's Board of Directors or advisory committees. Legros:Novartis, Bristol Myers-Squibb: Research Funding, Speakers Bureau, Travel to meeting Other. Viallard:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dupriez:novartis: Membership on an entity's Board of Directors or advisory committees. Coiteux:Novartis, Bristol Myers-Squibb: Speakers Bureau. Demory:Novartis: Honoraria. Giraudier:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ugo:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel to ASH Other. Kiladjian:Incyte: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding. Roy:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel to ASH Other.

Abstract 1745 Rationale: The diagnosis of Polycythemia Vera (PV) and Essential Thrombocytemia (ET) relies on a set of criteria gathered in the 2008 WHO classification; however, it may occasionnaly be difficult, especially in the absence of molecular markers such as Jak2V617→F or Mpl mutations. Having previously reported a significant increase in circulating platelets microparticles (PMP) in patients bearing Myeloproliferative neoplasms versus healthy donors (V.Tintillier-Colin, ASH Annual Meeting 2009, Abstr. 1906), our purpose has been to assess the usefulness of blood PMP count as an additional criteria in dubious cases of ET and PV. Patients and methods: We performed PMP counts in 100 newly diagnosed and still untreated patients (55 ET and 45 PV in accordance to WHO criteria), 40 secondary or reactive states (erythrocytosis and thrombocytosis, 20 cases each) and 75 controls matched for age and sex. The PMP were characterized by their size and co-expression of Annexin V and CD41; their concentration was measured on plasma extracts using the FC500 flow-cytometer (Beckman-Coulter™). Pre-analytical and testing procedures complied with the recommendations of the ISTH Standardization Sub-committee with each test performed in duplicate. Results: The 3 groups were homogeneous regarding age and sex-ratio. Mean platelet counts were 703.109/l in the TE group, 652.109/l in the cohort with secondary thrombocytosis and 247.109/l in the control group. Mean Hb level was 18.0g/dl in the PV group, 18.1g/dl in the secondary erythrocytosis group and 15g/dl in controls. The median value of PMP concentration is much higher (p < 0.05, Krusall-Wallis test) in the TE group (5900/μl) than in the reactive thombocytosis group (1283/μl), this latter being no different from the control group (996/μl). In the ET group, PMP count is similarly increased whether patients beared Jak2V617→F mutation or not. Using the ROC statistical method, we defined a threshold value of 3400 PMP/μl able to discriminate ET from reactive thrombocytosis with 93% specificity and 67% sensitivity. Similarly, the median value of PMP count was neatly higher in the PV group (3258/μl) than in secondary erythocytosis (671/μl) and healthy controls (996/μl) (p < 10−5, Krusall-Wallis test). The ROC method fixed a 1300/μl threshold separating PV from secondary erythrocytosis with 89% sensibility and 63% specificity. Conclusion: We report an evaluation of the blood PMP count in a quite large series of newly diagnosed untreated ET and PV patients compared to patients with secondary thrombocytosis or erythrocytosis and healthy volunteers; this study emphasizes the interest of the circulating PMP count in order to differentiate MPN neoplasms from normal subjects as well as patients with reactive conditions. This test, quick, reproducible and cheap, especially shows its value as an additional diagnostic criteria in ET or PV when clonal markers are lacking. Disclosures: No relevant conflicts of interest to declare.

Abstract 3834 Introduction: CD9, a four transmembrane glycoprotein belonging to the tetraspanin family, is suggested to regulate cell motility and adhesion and to play a role in megakaryopoiesis. It has been reported to be a molecular marker of primary myelofibrosis (PMF) being characterized by myeloproliferation, dysmegakaryopoiesis, alterated bone marrow/spleen stroma and extramedullary haematopoiesis. CD9 mRNA has been shown to be overexpressed in CD34+ PMF HPs and its membrane expression level was correlated with platelet counts. Our recent data evidencing an alteration of CD9 expression in PMF megakaryocytes (MK) have encouraged us to investigate whether CD9 participates in the dysmegakaryopoiesis and whether it is involved in the dialogue between MK and stromal cells in PMF patients. Patients and Methods: CD34+ cells were MACS selected from the peripheral blood of PMF patients (n=67) and of unmobilized healthy donors (n=61). Functional studies were performed on MK precursor-derived from CD34+ cells cultured in MK medium with ou without monoclonal antibodies (Syb mAb) or siRNAs targeting CD9. CXCL12-induced MK migration was performed in Boyden chambers. Bone marrow mesenchymal stromal cells (MSC) from healthy donors and PMF patients were cultured in DMEM+10%FCS. Results: Our results showed that CD9 membrane expression was altered on CD34+ cells and on MK precursor-derived from PMF CD34+ cells. Binding of CD9 with Syb mAb restored the in vitro megakaryocyte differentiation process that was altered in patients as shown by an increase in: i) megakaryocytic colony formation in semisolid medium, ii) CD41 and CD62p MK differentiation marker and GATA-1 expression, iii) MK cytoplasmic maturation, iv) apoptotic MK number (reduced AKT phosphorylation and Bcl-XL expression and increased percentage of Annexin+ cells). Activation of CD9 was also associated with regulation of MAPK and AKT-GSK3β pathways whose balance is involved in MK differentiation. Treatment of PMF MK precursors by Syb modulated activation of the MAPK pathway as shown by an increased of p38, JNK and GSK3β phosphorylation and of AP-1 mRNA expression. Taking into account the structure of the tetraspanin molecular network, binding with Syb mAb might also impact the effects associated to the multimolecular complex in which CD9 is involved. This prompted us to study the effects of a molecular silencing of CD9 on the PMF MK differentiation. We showed that, in contrast to the Syb mAb, addition of CD9 siRNA to PMF megakaryocytes reduced their transcriptional program including c-Myb, a transcription factor that is involved in CD9 regulation during megakaryopoiesis. Given the role of CD9 in cell migration, we further investigated whether it could be involved in the megakaryocytic precursor migration observed in patients. We showed that silencing CD9 reduced the CXCL12-dependent megakaryocytic precursor migration as well as the CXCR4 and CXCL12 transcription and that this migration involved actin polymerization. c-Myb siRNA restored CXCR4 and CXCL12 expression and reduced actin polymerization suggesting that CD9 was involved, via c-Myb, in the CXCL12-dependent megakaryocytic precursor migration. Effect of CD9 on cell migration is often interpreted as related to modulation of integrins participating in the integrin/tetraspanin network and of their interaction with mesenchymal stromal cells (MSC). We showed that several genes involving the CD9 partner interactome were over-expressed in MSC from PMF bone marrow as compared to MSC from healthy donors. Preliminary results showing that PMF MK precursors display different behaviour in terms of cell survival and adhesion when co-cultured on bone marrow MSC from PMF patients as compared to healthy donors suggest that interactions between MKs and bone marrow MSC is involved in PMF dysmegakaryopoiesis. Addition of Syb reverses these alterations suggesting the participation of CD9 in the abnormal dialogue between MK and MSC. Conclusion: Our results show a deregulation of CD9 expression in megakaryocytes from PMF patients. They also suggest that CD9 i) participates in PMF dysmegakaryopoieis in terms of MK differentiation and survival and ii) is involved in the increased MK precursor migration through alterations of the CXCL12/CXCR4 axis. Our data further support the role of bone marrow stroma in PMF dysmegakaryopoeisis through CD9 interactions. Disclosures: No relevant conflicts of interest to declare.

Abstract 5166 Rationale: In Ph1 myeloproliferative neoplasms (MPN), the blood stream carries a variety of microparticles and exosomes released by cells from myeloid lineages and endothelium; we had already demonstrated an excess of platelet microparticles (PMP) in ET and PV patients (ASH Annual Meeting 2009, Abstr. 1906). A further step has been to characterize five categories of MP depending on their origin. Then we used this method to compare the sub-populations found in Polycythemia vera (PV) and secondary erythrocytosis (SE). Patients and methods: We analyzed plasma samples from 20 PV diagnosed in accordance to WHO classification and 20 SE patients. SE was defined by elevated hematocrit, normal or increased serum EPO, Jak2V617F negative and a presumable cause, mainly respiratory. The 2 groups were comparable for age and sex-ratio. Mean Hb level was 18.0g/dl in the PV group and 18.1g/dl in the SE group. The concentration of MP was measured by flow cytometry using a FC500 cytometer (BeckmanCoulter™). All the MP subtypes express Annexin V thus defining the total MP. Additional co-expression of either CD41, CD144, CD14, CD11b or GPA identify MP populations originating respectively from platelets (PMP), endothelial cells (EMP), monocytes (MoMP), granulocytes (GrMP) and red cells (EryMP). Pre-analytical and testing procedures complied with the recommendations of the ISTH Standardization Sub-committee. Results: MP from endothelial cells (EMP) and leukocytes (GrMP and MoMP) are minor populations whose proportions are similar in both groups of patients (7%, 4% and 2% respectively). Conversely, EryMP and PMP are significantly different (p So, in PV most of the MP are PMP while EryMP widely predominate in SE; in an attempt to explain such unexpected results we postulate that the cells generating MP are more or less mature depending on the physiopathology: clonal hematopoietic progenitors in MPN and well-differentiated cells in reactive conditions. However, even in SE, the EryMP concentration does not correlate with RBC count, haemoglobin or hematocrit. Conclusion: This study demonstrates a different phenotypic profile of circulating MP in PV and SE, possibly helpful to precise the origin of an elevated hematocrit, especially when molecular markers are negative. The significance of the MP subtype pattern observed in PV and SE remains unclear, calling for further studies. Disclosures: No relevant conflicts of interest to declare.

Abstract 440 Background: The JAK2V617F mutation is found in a small proportion of MDS, especially in RARS-T and occasionally in other MDS subtypes, but the overall impact of JAK2V617F on MDS characteristics and outcome remains unclear. Method: Diagnostic and follow up data on MDS patients (pts) with known JAK2V617F mutation status were collected from 19 centers of the Groupe Francophone des Myélodysplasies (GFM) and the French Intergroup of MPN (FIM). MDS post MPN and CMML were excluded. Patient characteristics and outcome according to JAK2V617F status were analyzed by univariate analysis. Survival analysis with Cox model matched on age, IPSS score and sex according to JAK2 status was also made. Analysis was performed using STATA 10.0 software. Result: 161 cases were collected, including 65 JAK2V617F mutated (JAK2 pos) and 96 unmutated (JAK2 neg) cases. Median age was 75 years and M/F ratio 1.2 in JAK2 pos vs 71 years (p=NS) and 1 (p=NS) in JAK2 neg pts, respectively (resp). WHO 2008 distribution was RA (8%), RARS (12%), RARS-T (41%), CRDM (15%), RAEB-1 (11%), RAEB-2 (5%), 5q- (3%), unclassified (5%) in JAK2 pos pts and RA (25%), RARS (9%), RARS-T (1%), CRDM (14%), RAEB-1 (28%), RAEB-2 (19%), 5q- (1%), unclassified (3%) in JAK2 neg pts, resp (p Conclusion: We found JAK2V617F mutation in MDS to be associated with higher WBC, ANC and platelet counts, lower % marrow blasts, less progression to AML and better survival than JAK2V617F neg MDS. This positive prognostic impact persisted after exclusion of RARS-T. However, our results will require confirmation in a prospective study. Disclosures: Fenaux: CELGENE, JANSSEN CILAG, AMGEN, ROCHE, GSK, NOVARTIS, MERCK, CEPHALON: Honoraria, Research Funding.

Abstract 4103 Background: The moderate effect of most palliative treatments in primary and secondary myelofibrosis (MF), in addition to the limited possibilities of allogeneic stem cell transplantation, has incited physicians to look for alternative treatments. Since 1987, several studies have suggested that interferon may be beneficial in the treatment of MF. However, important hematological and general limiting toxicities frequently occur in MF patients (pts), leading to rapid treatment discontinuation in more than 50% of pts. Better results were recently reported in a small series of 13 MF pts treated with Peg-Interferon-α2a (Ianotto et al., Br J Haematol, 2009). The present study aimed to collect data of pts with primary and secondary MF treated with Peg-Interferon-α2a in French centers members of the FIM (French Intergroup of Myeloproliferative disorders) and GEM (Groupe d'Etudes des Myelofibroses) groups, to better assess tolerance and efficacy of this form of interferon in MF. Patients and Methods: Between Dec 2006 and Feb 2010, 39 MF pts treated with Peg-Interferon-α2a were registered from 10 different French centers affiliated to FIM and GEM groups. Age of pts ranged from 41 to 81 years, 21 were men and 18 women. Sixteen pts had primary MF, 13 had post-PV and 10 post-ET MF, respectively. Twenty-five patients (64%) were JAK2V617F positive. Twenty-eight patients had previously received other cytoreductive treatment. Clinical and biological parameters were collected at diagnosis and every 3 months. Responses were assessed according to the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) criteria. Analyses were performed in July 2010, after a median follow-up of 18 months (range: 3 – 42 months). Results: Among the 28 patients with splenomegaly, we observed 10 responses (36%) including 7 complete and 3 partial responses. Fourteen patients had constitutional symptoms which resolved in 8 of them (57%). Seven of 15 patients (47%) with an initial hemoglobin level below 100 g/L achieved complete response (CR). Three of 8 (37%) transfused pts became transfusion-independent. Twenty-two patients had abnormal WBC count which normalized in 13 of them (59%). Platelet count was abnormal in 27 patients, and 14 (52%) achieved CR with Peg-Interferon-α2a treatment. The evolution of the JAK2V617F allele burden is currently under investigation and will be presented at the meeting. The initial median dose of Peg-Interferon-α2a effectively received was 103 μ g/wk, further decreased to a median of 85 μ g/wk after one year. At time of analysis, treatment was stopped in 11/39 (28%) pts due to side effects, inefficacy or hematologic evolution. Conclusion: In this observational study, we found higher efficacy and better tolerance of interferon than previously reported in patients with primary or secondary MF. Such results were possibly due to a better tolerance of the pegylated form used, and to the low-dose schedule applied by the physicians. Our results suggest at least that Peg-Interferon-α2a should be considered as a possible therapeutic option in selected MF patients. Future clinical trials in MF will hopefully involve combinations of low dose Peg-Interferon-α2a with JAK2-inhibitors or immunomodulatory agents in order to improve tolerability and increase efficacy. Disclosures: Off Label Use: This is an observational study of the off-label use of peg-Interferon-alfa2a in myelofibrosis.

Abstract 1906 Poster Board I-929 Microparticles (MP) are plasma membrane vesicles bearing potent procoagulant proteins and released into the circulation by various blood and endothelial cells during cellular activation and apoptosis. Their concentration has been shown markedly increased in vascular and thromboembolic diseases and in several types of cancer. Essential Thrombocythemia (ET), Polycythemia Vera (PV) and Primary Myelofibrosis (PM) are Bcr-Abl-negative chronic myeloproliferative disorders (MPD) with common biological and clinical features, especially thrombocytosis and more or less effective megakaryocytic proliferation. ET, PV and PMF are also characterized by an increased risk of thrombotic complications. Given the involvement of megacaryocytopoiesis in this group of diseases, we chose to focus on circulating platelet-derived microparticles (PMP) whose count can be established by flow cytometry. Our goals were to look for an increased release of PMP in MPD and if so, try to establish a correlation between thrombotic events recorded retrospectively and PMP level. Patients and methods: Plasma samples were collected from 85 patients with MPD (40 ET, 28 PV, 17 PMF) at any time of the disease course except blastic phase and 31 healthy age-paired controls ; the quantity of PMP (positive for the platelet marker CD41 and the microparticle marker AnnexinV) was measured by flow cytometric analysis using the FC500 cytometer from Beckmann-Coulter™. Pre-analytic and testing procedures complied with the recommendations of the ISTH Standardization Subcommittee and besides, our laboratory participates in a multicenter program to standardize the enumeration of cellular microparticles. Results: The number of PMP is significantly higher in ET, PV and MFP patients (median: 4862 MPP/mL, 3289 MPP/mL and 6114 MPP/mL respectively) than in controls (median 1310 MPP/mL) [p=0.000018, p=0.0013, p=0.0009]. Neither in MPD patients nor in control group PMP correlates with platelet count and appears thus as an independent parameter. ROC method allows to fix a cut-off at 3121 MPP/mL to discriminate MPD from controls. This value has a sensibility of 75 % and a specificity of 94%. Interestingly, the total number of PMP is not different between untreated patients (mean=7246 MPP/mL) and those who received myelosuppressive therapy (mean=6320 MPP/mL). Regarding thrombotic events, we failed to demonstrate any significant difference between patients with previous thrombosis and those without; however, the small size of the series and the retrospective assessment are possible bias. Conclusion: Patients with Phi negative MPDs have a high number of circulating PMP, irrelevant to platelet count or treatment by Hydroxy-urea or aspirin. A role for microparticles in the development of these diseases and in the occurrence of thrombotic complications can be hypothesized but requires further studies -;preferably prospective- on larger cohorts of patients. Disclosures: Charpentier: Schering-Plough: Research Funding.

Background: The prognosis of liver iron overload is highly dependent on liver iron content (LIC) and the extent of liver fibrosis. Liver biopsy is the invasive reference method for fibrosis evaluation but with several limitations. Non-invasive biological methods (FibroTest, APRI) are not applicable in blood diseases. Transient elastography (TE) is a new non-invasive and rapid bedside method used to measure liver stiffness.This technique has been extensively studied with success in evaluation of liver fibrosis in HCV infection (Ziol, Hepatology 2005; Castera, Gastroenterology 2005). Until now, no data are available on evaluation of liver fibrosis by TE in highly transfused patients with iron overload. Methods: We assessed liver stiffness (kPa) by elastography measurement (Fibroscan; Echosens, Paris, France). Ishak’s and Metavir’s scales were used to stage fibrosis and hepatitis activity; LIC was measured by atomic absorption spectometry in a central laboratory (Rennes, France) and by MRI procedure according to Gandon (Lancet 2004). Cardiac MRI T2* (indirect estimation of iron heart content) was also measured by MRI according to Anderson (Eur Heart J 2001). Serum ferritin level and HCV serology were assayed in all cases. Correlation statistical analysis used Spearman’s Rho and Pearson tests and mean comparisons were done by a non-parametric Mann-Whitney test. Results: 15 liver samples (weight >1mg except 1) from monthly transfused patients (13 major beta Thalassemia, 1 sickle cell disease, 1 myelodysplastic syndrome) were analyzed. Median age was 37 years (7–75). Histological results were: 3 cirrhosis (METAVIR F=4 (F4), or Ishak’s Staging =6 (IS6)) among 5 patients with severe fibrosis (F3,F4) or (IS4-6) and 10 patients with no or mild fibrosis (F0-2) or (IS0-3). Mean ferritin was 2579 ng/ml CI 95% [1372–3786]. 2 patients were HCV positive. Correlation between LIC and ferritin was 0.82 (p Conclusion: A significant correlation between Metavir Fibrosis scale and elastography values was found. This new non-invasive method would be helpful to evaluate liver fibrosis and to determine patients who could avoid invasive procedures in particular in regularly transfused low risk myelodysplastic patients at risk of bleeding due to abnormal platelet function. These preliminary results will have to be confirmed in a larger population. Figure Figure

MMM is a myeloproliferative disorder characterized by extramedullary hematopoiesis and reactive myelofibrosis. Recently, HMGA2 dysregulation has been demonstrated in 2 MMM patients showing 12q15 rearrangement and confirmed in 25 consecutive MMM patients without cytogenetic abnormalities (Andrieux, 2004). HMGA2 proteins belong to the high mobility group A (HMGA) family of architectural transcription factors regulating the expression of several genes. As MMM is a clonal disorder of CD34+ hematopoietic progenitors, we analyzed HMGA2 expression in peripheral blood sub-populations of 5 MMM patients and 7 healthy donors to determine in which sub-population HMGA2 was dysregulated. RNA was extracted from peripheral blood mononuclear cells (PBMC) and CD15+ granulocytic cells (PBCD15+) separated through Ficoll centrifugation or from immunomagnetically selected circulating CD34+ cells (PBCD34+). Real-time quantitative PCR (RQ-PCR) using Taqman technology was performed on cDNA. As different isoforms were described in malignancies, we used two primer sets : the first one allowing the amplification of all HMGA2 isoforms (exon 1 to 3) (HMGA2 1–3), the second one allowing the amplification of the full length HMGA2 isoform (exon 1 to 5)(HMGA2 1–5). In healthy donors and in MMM, PBMC HMGA2 expression levels were heterogeneous, depending of the cellular sub-population purity. HMGA2 1–3 or HMGA2 1–5 were both expressed in MMM and normal PBCD34+ cells, but with a higher expression level for HMGA2 1–3 as compared to HMGA2 1–5. Furthermore, both HMGA2 1–3 and HMGA2 1–5 expression levels were significantly increased in PBCD34+ MMM patients (p98%) nor in PB neutrophils from other myeloproliferative disorders (Polycythemia Vera and Essential Thrombocythemia). To determine if HMGA2 level was modified during hematopoietic differentiation, we quantified HMGA2 1–3 and 1–5 isoform expression on purified healthy donor PB CD34+ and MMM CD34+ before and after culture with specific lineage growth factors (12 day-culture). Primary results showed that both HMGA2 isoform expression levels were higher during granulocytic differentiation. Our results demonstrate that HMGA2 1–5 isoform is discriminately overexpressed in MMM PBCD15+. The persistence of this HMGA2 full length expression in MMM myeloid lineage could be considered as a marker of the disease.