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Introduction The JAK2V617F allele burden at progenitor levels increases over time in polycythemia vera (PV), with the dominance of the JAK2V617F-positive clone at the CD34+ compartment being an important modifier of the disease phenotype. The relationship between clonal dominance and the evolutionary pattern of JAK2V617F granulocytic load is presently unknown. Objective To analyze the relationship between clonal dominance of CD34+ cells with the evolution of the granulocytic JAK2V617F mutant load in PV patients. Methods A total of 37 patients with PV were included in the study. At time of the study, 30 patients corresponded to the chronic phase of the disease, whereas 7 patients had evolved to myelofibrosis (post-PV MF). Seventeen patients were in early chronic phase (less than 5 years from diagnosis) and 13 patients in late chronic phase (more than 5 years from diagnosis). Granulocytes were isolated from peripheral blood by density gradient, whereas CD34+ cells were purified by immunomagnetic positive selection. Stem cells (CD34+, CD38-) and progenitors (CD34+, CD38+) populations were further separated by fluorescence-activated cell sorting. JAK2V617F allele burden was measured by quantitative PCR in all three cell fractions. Clonal dominance was defined as an absolute difference ≤ 10% in JAK2V617F between granulocytes and CD34+ populations. Granulocytic JAK2V617F mutant load was measured every year with the evolutionary pattern of the JAK2V617F allele burden being stratified as follows: stable < 50%, stable ≥ 50%, progressive increase and unexplained decrease. The study was approved by the local Ethics Committee and informed consent was obtained according to the Declaration of Helsinki. Results JAK2V617F allele burden in CD34+/CD38- cells was 8%, 24.9% and 71.4% in early chronic phase, late chronic phase and post-PV MF, respectively (p < 0.001). Similar results were observed in CD34+/CD38+ cells (JAK2V617F allele burden: 19.1%, 36.1% and 71.8% in early chronic phase, late chronic phase and post-PV MF, respectively, p < 0.001). No significant differences were observed among the three groups in granulocyte JAK2V617F allele burden. Six patients (16.2%) presented clonal dominance. According to disease status, clonal dominance was observed in 5 out of 7 (71.4%) patients with post-PV MF and in 1 out of 13 (7.7%) patients in late chronic phase, whereas no patient in early chronic phase showed clonal dominance. JAK2V617F monitoring was performed in 29 patients. According to their granulocytic JAK2V617F evolutionary patterns, 9 patients remained with a stable JAK2V617F below 50%, 13 patients showed a stable JAK2V617F ≥ 50%, 6 patients experienced a progressive increase of the JAK2V617F mutant load and 1 case presented an unexplained decrease of JAK2V617F. Clonal dominance was observed in 5 out of 20 (25%) patients with a stable JAK2V617F >50% or a changing profile in comparison to 0 out of 9 patients with stable JAK2V617F < 50% (p=0.05). Conclusion Clonal dominance correlates with JAK2V617F allele burden evolutionary pattern.This may be a factor to be taken into account in the therapeutic strategy of PV patients and supports the need to monitor JAK2V617F burden during clinical follow-up. Funding this study was supported by grants from the Ministry of Education and Science of Spain and Instituto de Salud Carlos III FEDER (EC10-136, FIS PI10/018087, RD09/0076/00036 and RD12/0036/0010), 2009SGR929 and by grant from Asociación Española Contra el Cáncer Catalunya. Disclosures: No relevant conflicts of interest to declare.

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

Background MicroRNAs (miRNAs) act as negative regulators of expression of genes that participate in cellular proliferation, apoptosis and/or carcinogenesis. MiRNAs have been shown to play a role in both solid and hematological tumors. Interestingly, several miRNA profiles have been differentially related to the mutational status of multiple tumors. A mutation in the JAK2 kinase (V617F) has been associated with essential thrombocythemia (ET) in around 40–50% of cases. However, the biological basis of ET in patients lacking the JAK2V617F mutation is still unknown. miRNAs may be crucial to the development of ET in these patients, yet their role in JAK2V617F-negative ET remains to be elucidated. Aim To characterize the expression pattern of miRNAs involved in JAK2V617F-negative ET and to identify the targets of these miRNAs. Methods Total RNA was extracted from isolated platelets from 19 ET patients (10 JAK2V617F-positive and 9 JAK2V617F-negative) and 10 healthy controls. The expression of 670 mature miRNAs was determined by TaqMan Human MicroRNA Arrays v2.0 (Applied Biosystems) in an ABI 7900 HT real time PCR system. miRNA expression data was analyzed by the 2-ΔΔCt method, using RNU48 as endogenous control. Statistical analyses were performed with TiGR MultiExperiment Viewer and R software v 2.13.To identify molecular pathways potentially altered by the expression of multiple miRNAs, we used Diana-mirPath, which performs an enrichment analysis of multiple miRNA target genes, comparing each set of miRNA targets to all known KEGG pathways. The mRNA expression of identified putative targets was analyzed by TaqMan gene expression assays (Applied Biosystems) and correlated with miRNA expression in order to select candidate genes for further target validation by Renilla-Luciferase assay and Western Blot. For Renilla-Luciferase assay, 100nM of the pre-miRNAs of interest or pre-miR negative control were transfected in K562 together with 1μM of modified psicheck2 vector containing the 3’UTR region to be validated. Renilla luciferase levels were measured at 24h post-tranfection. The miRNAs identified in the Renilla-Luciferase assay were further confirmed by Western Blot. 100nM of pre-miR/pre-miR negative control were transfected in the HL60 cell line and protein levels were measured at 24h post-transfection. Anti-SOCS1 (ab119954) and Anti-SOCS3 (ab62584) primary antibody (abcam) were used. Results Hierarchical cluster analysis showed two well-separated clusters between patients and controls, indicating that ET platelets had a characteristic miRNA signature (p Conclusion A 40-miRNA signature is differentially expressed in JAK2V617F-negative and JAK2V617F-positive ET patients. Two of these miRNAs, miR-221 and miR-203, target SOCS1 and SOCS3, which are involved in the JAK-STAT pathway. Further investigation is warranted to shed light on the role of these miRNAs in the development of JAK2V617F-negative ET. Acknowledgments This study was supported in part by grants EC10-136, FISPI10/01807, RD09/0076/00036, RD12/0036/0010, 2009SGR929 and AECC Catalunya 2011 Disclosures: No relevant conflicts of interest to declare.

Background We have previously reported on the high prevalence and severity of insomnia amongst a large international cohort of MPN patients (Emanuel JCO 2012). We sought to further analyze the relationships between insomnia and the MPN disease features (in particular splenomegaly, status of blood counts), individual MPN symptom prevalence and severity, language, and overall quality of life. Methods Data was collected among an international cohort of patients with MPNs. Subjects completed the BFI, MPN-SAF, and EORTC QLQ-C30 instruments. Surveyed symptoms on the MPN-SAF included the patient’s perceptions of common MPN-related symptoms and overall quality of life (QOL) on a 0 (absent) to 10 (worst imaginable) scale. Specifically, the MPN-SAF insomnia item asked about “difficulty sleeping”. Total symptom score (TSS) was computed based on 10 symptom items using the published scoring algorithm on a 0 (all reported symptoms absent) to 100 (all reported symptoms worst imaginable) scale. Pairwise associations between the MPN-SAF insomnia item and continuous and categorical covariates were investigated using Pearson correlations and analysis of variance/t-tests, respectively. Multivariate regression models were used to investigate impact of groups of covariates on the insomnia item with the final multivariate model selected using forward regression. Results Demographics A total of 1992 MPN patients (essential thrombocythemia=834, polycythemia vera=703, myelofibrosis=449, unknown=6) completed the insomnia item. Participants were of typical age (median=61, range-15-94) and gender (female=54%). Overall, 1307 subjects endorsed the MPN insomnia item (score >0) with an overall mean symptom score of 3.0 (median=2.0, SD=3.1. range=0-10); 603/1992 (30%) of patients had severe insomnia related complaints (score >4). Univariate Analysis Among QLQ-C30 functioning scales, emotional functioning most highly correlated with the MPN-SAF insomnia item (r=-.47, p Multivariate Analysis In a multivariate model of MPN-SAF insomnia containing all QLQ-C30 functioning scales, physical, emotional, and cognitive functioning along with global health status/QOL were all significant (all p Conclusions Insomnia is highly prevalent and severe in MPN patients and closely correlates with most other MPN-related symptoms and functional domains bearing a multi-faceted impact on overall quality of life. Correlations between insomnia and emotional, cognitive, and physical complaints including depression/sad mood, concentration problems, night sweats, and numbness/tingling in the extremities suggest that the cause of MPN-related sleep complaints is likely complex. Future studies should evaluate the impact of interventions on MPN associated insomnia as well as its biological underpinnings. Disclosures: Etienne: novartis: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Pfizer: Membership on an entity’s Board of Directors or advisory committees; Ariad: Membership on an entity’s Board of Directors or advisory committees. Roy:Novartis, BMS: Honoraria. Harrison:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; YM Bioscience: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Honoraria; Shire: Speakers Bureau; S Bio: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Birgegard:Vifor Pharma: Honoraria.

PURPOSE To evaluate prospectively the feasibility and results of bone marrow transplantation (BMT) after induction and intensification chemotherapy (CT) in patients with de novo acute myeloid leukemia (AML). PATIENTS AND METHODS A total of 159 patients less than 51 years of age were treated. Induction CT consisted of daunorubicin 60 mg/m2 for 3 days, cytarabine (ARA-C) 100mg/m2 for 7 days, and etoposide 100 mg/m2 for 3 days. The first intensification therapy included mitoxantrone 10 mg/m2 for 3 days and ARA-C 1.2 g/m2 every 12 hours for 4 days. Amsacrine (100 or 150 mg/m2 for 3 days) and ARA-C (1.2 g/m2 every 12 hours for 2 or 4 days) were given as the second intensification therapy. Depending on the availability of a human leukocyte antigen (HLA)-identical sibling, the intention of treatment after CT was allogeneic BMT (allo-BMT) or autologous BMT (ABMT). RESULTS Complete remission (CR) was obtained in 120 patients (75%) and partial remission (PR) in 11 (7%), while 15 patients (10%) were refractory and 13 (8%) died during induction. There was a trend for better leukemia-free survival (LFS) at 4 years for patients assigned to the ABMT group (50% +/- 6%) compared with the allo-BMT group (31% +/- 7%) (P = .08). This difference in LFS reached statistical significance when considering only transplanted patients (63% +/- 3% at 4 years after ABMT and 38% +/- 11% after allo-BMT, P = .02). The favorable results in patients who received ABMT (no toxic deaths and 37% +/- 7% probability of relapse at 4 years) contrast with the poor outcome of allografted patients (11 patients with transplant-related mortality). CONCLUSION Our study reflects the difficulties in the completion of a therapeutic strategy that include BMT and suggests that intensification before BMT may be useful in the setting of ABMT, but this approach was associated with a high mortality rate in allo-BMT patients.

Cytogenetic analysis was successfully performed in 45 consecutive multiple myeloma (MM) patients. Cytogenetic abnormalities were observed in 13 of 45 patients (29%). Eleven patients showed numerical changes and 9 showed structural abnormalities in chromosomes 5, 9, 11, 14, 15, and 19 were most frequently gained. Structural abnormalities preferentially involved chromosomes 6, 13, and 14.

Abstract 2842 Background: As some patients (pts) with essential thrombocythemia (ET) may discontinue their cytoreductive treatment because of drug intolerance/inefficacy, combinations of drugs with different activity/tolerability patterns are being utilized in clinical practice. Objectives: To evaluate the clinical relevance and patterns of cytoreductive combination treatment in ET in Evaluation of Xagrid™ Efficacy and Long-term Safety (EXELS), an observational study in 3643 European pts with high-risk ET. Patients and Methods: Data on pts receiving cytoreductive treatments in combination for ≥30 days (COMB) were collected from a planned data cut in September 2011. This timepoint was 2.5 years after enrollment completed. Results: COMB was recorded in 347 pts (9.5%), the vast majority (87.6%) of whom received anagrelide + hydroxycarbamide (ANA+HC). Other combinations were anagrelide + interferon-α (ANA+IFN), anagrelide + pipobroman (ANA+PIPO; Table). Six patients also received anagrelide + other drugs (ANA+OTH). In 333 pts, the initial drug was HC (n=167; 50.2%), ANA (n=138; 41.4%), IFN (n=14; 4.2%), PIPO (n=9; 2.7%), or OTH (n=5; 1.5%), while the added drug was ANA (n=195; 58.6%), HC (n=123; 36.9%), IFN (n=12; 3.6%), PIPO (n=2; 0.6%), or OTH (n=1; 0.3%). Fourteen pts started ANA+HC concomitantly. The median duration of monotherapy treatment before COMB start was 139 months for HC, 53 months for ANA, 71 months for IFN, and 237 months for PIPO. In pts receiving ANA+HC, the median weekly dose of the initial drug, both before and during COMB, was 10.5 mg for ANA and 7.0 g for HC, while the median weekly dose of the added drug during COMB was 7.0 mg for ANA and 3.5 g for HC. In these pts, the median platelet count ≤6 months before COMB start, during COMB (first test), and during COMB (last test) was 581, 411, and 434 ×109/L, respectively (with counts ≤600 ×109/L in 52.9%, 79.2%, and 77% of cases, respectively); the median white blood cell count was 6.9, 6.8, and 6.8 ×109/L, respectively; the median hematocrit was 38.0%, 36.3%, and 39.0%, respectively; and median hemoglobin was 11.9, 11.6, and 12.0 g/dL respectively. Similar laboratory results were obtained in pts receiving ANA+IFN or ANA+PIPO. Before, during and after COMB, anti-aggregatory treatment, usually low-dose aspirin, was given in 66.3%, 71.8%, and 33.1% of pts, respectively. In the same time periods, thrombotic (3, 11, and 11 cases, respectively) and hemorrhagic events (1, 2, and 5 cases, respectively) occurred. The COMB discontinuation rate was 54.5% globally (186/341 pts). COMB was discontinued by stopping ANA in 67 cases (36.0%), HC in 76 (40.9%), IFN in 8 (4.3%), PIPO in 4 (2.2%), both ANA and HC in 19 (10.2%), both ANA and IFN in 3 (1.6%), both ANA and PIPO in 3 (1.6%), or by adding a third drug in 6 cases (3.2%). In the 158 pts who discontinued ANA+HC, reasons were: intolerance/side effects (n=79, 50.0%), lack of efficacy (n=35, 22.2%), investigator decision (n=34, 21.5%, mainly with switch to ANA monotherapy), patient preference (n=10, 6.3%), missing, other (including economic) or unknown (n=17, 10.2%). Similarly, intolerance/side effects was the most frequent reason for discontinuing ANA+IFN (12/20; 60.0%). Overall, there were 63 patients with suspected severe adverse reactions (SSARs) and 45 with predefined events (PDEs) during COMB. The most frequent SSARs were myelofibrosis, palpitations, myocardial infarction and tachycardia. The most frequent PDEs included cardiovascular symptoms, myocardial infarction, stroke and transformation. Pts on COMB, the majority of whom received ANA+HC, accounted for 3.8% of all pts at registration and 5.0–5.8% of all pts in the following 5 years. Conclusion: COMB was performed in almost 10% of the 3643 pts with ET in the EXELS study, mainly with use of ANA+HC (87.6%). COMB appears to be a useful approach to treating patients for whom monotherapy was not well tolerated or ineffective since platelet levels were reduced to Disclosures: Gugliotta: Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Besses:Shire: Honoraria; Novartis: Honoraria. Griesshammer:Shire: Honoraria. Harrison:Novartis: Consultancy, Honoraria, Research Funding; YM Biosciences: Consultancy; S*Bio: Consultancy; Shire: Honoraria, Research Funding; Sanofi Avensis: Honoraria. Kiladjian:Shire: Membership on an entity's Board of Directors or advisory committees; Incyte: 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. Coll:Shire Pharmaceuticals: Employment. Smith:Shire Pharmaceuticals: Employment. Abhyankar:Shire Pharmaceuticals: Employment. Birgegård:Shire Pharmaceuticals: Honoraria; Pharmacosmos: Research Funding; Shire Pharmaceuticals Sweden: Consultancy.

Abstract 2828 Antiplatelet therapy with low-dose aspirin is commonly used in combination with cytoredution for the prevention of thrombosis in patients with high-risk essential thrombocythemia (ET), as determined by age > 60 years and/or previous history of thrombosis. However, it is uncertain whether low-dose aspirin adds any benefit to cytoreductive therapy in patients without a history of thrombosis. In this study, the probability of thrombosis and bleeding was retrospectively analyzed in 248 patients with ET (median age: 66 years; 83 males, 165 females) treated with cytoreduction plus low-dose aspirin (n=170) or with cytoreduction only (n=78). Patients with a history of thrombosis or bleeding, as well as those receiving anticoagulant therapy, were excluded from the study. The indication of cytoreduction was age > 60 years (n=198), extreme thrombocytosis (n=37), microvascular disturbances (n=6), and others (n=7) First-line cytoreductive therapy consisted of hydroxyurea (n=216), anagrelide (n=27), interferon-α (n=4), and busulfan (n=1). During a median follow-up of 7.4 years (range: 0.1–26), a total of 28 thromboses (arterial, n=22; venous, n=6) were registered. At 5 years, the probability of thrombosis in patients receiving cytoreduction plus low-dose aspirin or cytoreduction only was 8% and 17%, respectively (p=0.006). No significant differences were observed depending on gender, presence of cardiovascular risk factors, JAK2 V617F mutational status, WBC count at diagnosis or type of cytoreductive therapy. At multivariate analysis, patients not receiving antiplatelet therapy were found to have a higher risk of thrombosis (HR: 3.3, 95%CI: 1.3–7.9, p=0.009). During the study period, a total of 13 major hemorrhagic events were registered (digestive, n=7, intracranial, n=3, others, n=3). At 5 years, the probability of major bleeding in patients receiving cytoreduction plus low-dose aspirin or cytoreduction as monotherapy was 6% and 4%, respectively (p=0.1). In conclusion, in patients with ET receiving cytoreductive therapy as primary prophylaxis of thrombosis, the addition of low-dose aspirin reduces the risk of thrombosis without increasing the risk of bleeding. Disclosures: No relevant conflicts of interest to declare.

Abstract 2852 Background: The 18-item Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF, Scherber et al Blood 2011) given in conjunction with the 9-item Brief Fatigue Inventory (BFI, Mendoza et al Cancer 1999) is a patient-completed questionnaire for assessing symptoms in persons with MPNs. The MPN-SAF has been translated and validated in 9 languages to date. The Total Symptom Score (TSS) is computed from 10 of the most pertinent MPN-SAF items to assess symptom burden in MPN patients and to evaluate response to therapy. Psychometric properties of the TSS have been previously reported (Emanuel et al Blood 2012). The purpose of this analysis is to compare MPN-SAF symptoms and psychometric properties of the TSS across 9 languages in an international sample. Methods: Data were collected in an international cohort of subjects with MPNs. Surveyed symptoms included fatigue, early satiety, abdominal pain and discomfort, inactivity, headaches, concentration, dizziness, extremity tingling, insomnia, sexual problems, mood changes, cough, night sweats, pruritus, bone pain and fever on a 0 (absent) to 10 (worst imaginable) scale. TSS was computed using the published scoring algorithm on a 0 (all symptoms absent) to 100 (all symptoms worst imaginable) scale. Demographic and disease-related data including disease type, gender, and age had to be present to be included in analysis. Demographics were compared across languages groups using ANOVA and chi-squared tests. Symptoms and TSS were compared across language groups using a general linear model adjusting for disease type, age, and gender with post-hoc Tukey pairwise comparisons. Internal consistency and factor structure of the TSS were investigated overall and within language groups using Cronbach's alpha and principal-axis factoring analysis. Results: Subject Demographics and Disease Type: 1,851 subjects with polycythemia vera (PV N=655), essential thrombocythemia (ET N=769) and myelofibrosis (MF N=427; 286 primary MF, 61 PV-MF, 80 ET-MF) were prospectively enrolled and administered the MPN-SAF and BFI in 1 of 9 languages: English [UK] 55, English [US] 102, Italian 186, Swedish 114, German 112, French 457, Spanish 192, Dutch 236, and Chinese 397. Age (median 61, range, 15–94) and gender (55% F) were typical. Disease type and age varied across language groups (both p MPN-SAF Symptoms and TSS: Symptom frequencies ranged from 19% (fever) to 88% (fatigue) overall with mean severities ranging from 0.4 (SD=1.3, fever) to 4.3 (SD=2.3, fatigue). Fatigue had the highest mean severity among all symptoms within each language group. Overall, mean TSS was 21.5 (SD=16.7) with the Swedish (mean=18.1, SD=15.2) and Dutch (mean=27.6, SD=17.1) cohorts reporting the lowest and highest unadjusted TSS means, respectively. When comparing symptom items across languages (adjusting for disease type, age, and gender), concentration and sexual problems had the most statistically significant pairwise differences (11 and 10, respectively, out of a possible 36) followed by dizziness and overall quality of life (9 each, out of a possible 36). No statistically significant pairwise differences were observed for abdominal discomfort, headache, extremity tingling, or insomnia. For the TSS, the Dutch cohort appeared to statistically significantly differ (all p TSS Internal Consistency and Factor Structure: The TSS had excellent internal consistency overall (Cronbach's alpha 0.83) as well as within language groups (Cronbach's alpha 0.81–0.86). Overall factor analysis identified a single underlying construct among the 10 TSS items. Factor loadings ranged from 0.41 for fever to 0.73 for inactivity. A single factor solution was appropriate for each language group with factor loadings ranging from 0.18 to 0.85. Conclusion: This analysis suggests that the available translations of the MPN-SAF are generally acceptable for use in a broad context. The TSS demonstrated acceptable internal consistency and similar factor structure across all language groups. Most symptom and TSS comparisons between languages were not statistically significant, but for the few which differed, further studies are needed to evaluate whether these variances are due to disease-related factors or due to linguistic or cultural influences present in the cohorts. Disclosures: Kiladjian: Novartis: Honoraria, Research Funding; Celgene: Research Funding; Shire: Honoraria. Griesshammer:Shire: Honoraria. Roy:Novartis, BMS: Speakers Bureau. Harrison:Novartis: Honoraria, Research Funding, Speakers Bureau; YM Bioscience: Consultancy, Honoraria; Sanofi Aventis: Honoraria; Shire: Honoraria, Research Funding. Passamonti:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Mesa:Incyte: Research Funding; Lilly: Research Funding; Sanofi: Research Funding; NS Pharma: Research Funding; YM Bioscience: Research Funding.

We report a case of multiple myeloma with a t(8;22)(q24;q11) found during the progression of the disease. The relation between the association of a Burkitt's type translocation with cytological characteristic features is presented. To our knowledge, there is no report of a multiple myeloma with t(8;22)(q24;11).

We investigated the expression of the lymphocyte function associated (LFA)-1 adhesion molecule, recognized by the CD1 la and CD 18 monoclonal antibodies, in 53 patients with B-cell chronic lymphocytic leukemia (B-CLL) and in 37 controls. The mean percentage of control lymphocytes expressing CD1 la and CD 18 positivity was 92 ± 5.5 and 90 ± 8.7, respectively and, in patients with B-CLL, 14.2 ± 10.3 and 14.8 ± 10.3 respectively (p < 0.001). No statistical difference was found between CDlla and CD 18 expression and the Rai clinical stages. In our experience a decrease of LFA-1 is a constant finding in B-CLL, in the early stages of the disease. It may, therefore, be useful to differentiate between slight increases of neoplastic cells and reactive blood lymphocytosis.

9033 Background: The rapid microbiologic diagnosis in the course of fever of unknown origin plays an important role for the appropriate treatment in neutropenic patients. Blood culture helps identi...

Abstract 4631 BACKGROUND The precursor of nucleotide biosynthesis acadesine or 5-aminoimidazole-4-carboxamide (AICA) riboside induces apoptosis in CLL cells, and other lymphoproliferative diseases such as splenic marginal zone lymphoma and mantle cell lymphoma. This effect is selective for B-cells, at least ex vivo, however there is no evidence of whether this cytotoxic effect depends on well known prognostic variables such as ZAP-70 expression or IgVH mutational status. AIM To analyse ex vivo the cytotoxic effect of acadesine on peripheral CLL cells and correlate it with prognostic variables. METHODS Cryopreserved cells from 62 CLL patients were incubated ex vivo with acadesine at 0.2, 0.5, and 1 mM for 24 hours. Viability was determined by flow cytometry using Annexin V-FITC and DAPI staining combined with CD19-PE and CD3-PerCP to differentiate cell viability of B- and T-cells. Cells were considered sensitive to the drug when the percentage of acadesine-induced apoptosis was equal to or higher than 15% with respect to the viability of control cells. The mutational status of IgVH genes was determined by RT-PCR amplification using a set of six VH family-specific primers (VH1 through VH6) along with primers complementary to the constant region (IgM and IgG). Products were directly sequenced from both strands using the Big Dye Terminator Cycle Sequencing Ready Reaction (version 3.1, Applied Biosystems). Sequencing analysis and alignments were performed with use of V-QUEST software and the online international immunogenetics information (IMGT) data library. Samples in which fewer than 2 percent of base pairs differed from those of the consensus sequence have been considered unmutated. ZAP-70 expression was quantified by flow cytometry (cut-off:20%) and cytogenetic alterations associated with CLL (trisomy 12, del13q, del 17p and del11q) were determined by FISH. RESULTS After 24h of ex vivo incubation, 0.2 mM acadesine induced a significant cytotoxic effect (> 15%) in 31 out of 62 patients (50%). Higher concentrations, 0.5 mM and 1 mM, induced a significant effect in 91.4% (57 of 62 patients) and 98% (61 of 62 patients), respectively. The viabilities (mean ±SD) of the different culture conditions are shown in the table. The cytotoxic effect induced by acadesine was analyzed with respect to the IgVH mutational status and ZAP-70 expression. No significant differences were observed between cases with unmutated IgVH (n=20) and mutated IgVH(n=37), or between ZAP-70 positive (n=24) and ZAP-70 negative cases (n=34). Interestingly, 5 cases showing deletion of 17p were sensitive to treatment with acadesine, in agreement with previously published studies showing that acadesine-induced apoptosis is independent of p53. Only one case showing deletion 17p and VH3-21 usage was not sensitive to acadesine. CONCLUSIONS Acadesine induces apoptosis in B-cells from CLL regardless of ZAP-70 expression, IgVH mutational status and 17p status. Disclosures: Campàs: Advancell: Employment. de Frías:Advancell: Employment.

Abstract 2003 Background: Tumor microenvironment has been shown to play a critical role in the biology and treatment response of some types of non Hodgkin's lymphomas. The significance of the presence of Treg cells (FOXP3+) in MALT lymphomas of the stomach remains unknown. The main aim of this study was to analyze by immunohistochemistry the presence of Treg cells in the microenviroment of gastric MALT (gMALT) lymphomas at the time of diagnosis and during follow-up after treatment, and to evaluate their possible impact in outcome. Methods: Patients were included in the study if they had gMALT lymphoma diagnosed according to the 2008 WHO criteria and diagnostic paraffin-embedded blocks were available for review. Thirty-three patients met the criteria for inclusion in the study. Sections were immunostained for CD20, CD3, CD4, CD8, CD68, FOXP3, PD-1, bcl-10 and Ki67. t(11;18) was studied by FISH and/or PCR and t(1;14) by FISH. Clonality study of the B cell receptor was done according to the BIOMED-2 protocol. The number of CD20+ tumor cells and FOXP3+ infiltrating cells was quantified using a micrometric ocular (WPK 10 × mn) that has a 10 mm linear scale divided to 100 parts. Results: The median age was 63 y (range 32–83) with 52% being male. Stage: I in 66%, II in 25% and IV in 9%; B-symptoms in 6%. Translocation t(11;18) was present in 9 (27%) pts and 2 additional cases had strong nuclear expression of BCL-10 without t(11;18) or t(1;14). At diagnosis, the mean (± standard deviation) number of CD20+ tumor cells and FOXP3+ infiltrating cells was 680±232 cells/cm2 and 30±29 cells/cm2, respectively. The mean number of CD20+ tumor cells and FOXP3+ infiltrating cells was similar between gMALT with or without t(11;18). Number of treatments analyzed was 37 (treatment not available in 2 pts and 5 pts received more than 1 treatment). Treatment regimens included: eradication therapy (n=11); interferon+ribavirin (n=1); single or combined agent chemotherapy without rituximab (n=5); rituximab alone or CHOP-like with rituximab (n=4); fludarabine (n=8); fludarabine or bendamustine with rituximab (n=8). The first response evaluation showed an overall clinical response rate of 84% (CR 76%) with persistence of residual disease by histology or molecular (IGHV) studies in 31% and 56%, respectively. Overall, the mean (±SD) CD20+ tumor cells and FOXP3+ infiltrating cells was significantly reduced after treatment (730±208 vs 285±343, p Conclusion: Based on these preliminary findings, there is a significant change of CD20+ cells and moderate reduction of Treg cells in responding patients after treatment for gastric MALT lymphoma. Interestingly, regimens with fludarabine or bendamustine with or without rituximab result in quicker and deeper reduction of CD20+ tumor cells while infiltrating FOXP3+ cells are only moderately reduced. Disclosures: No relevant conflicts of interest to declare.

Abstract 4113 Introduction: Most of the data related to RARS-T, a rare disorder, involve small cohorts of patients. We aimed to analyze more patients also considering a variety of myelodysplastic or myeloproliferative disorders. Objective: To compare a large cohort of patients with RARS-T to refractory anemia with ringed sideroblasts (RARS), refractory anemia with ringed sideroblasts and multilineage dysplasia (RARS-MD) or essential thrombocythemia (ET) at the time of diagnosis and during disease evolution, in terms of survival and complications. Materials: Data of a European multi-center study was used including 199 cases of RARS-T 173 cases of RARS, 102 cases of RARS-MD and 431 cases of ET. Results: At baseline, compared to RARS and RARS-MD patients, RARS-T patients had similar hemoglobin concentration, but a higher white blood count. The JAK2V617F mutation was observed in 43%, 12% and 5% in RARS-T, RARS and RARS-MD patients, respectively. When separated in 2 groups (450,000600,000 × 109/l), RARS-T patients were comparable for sex, age, hemoglobin level and survival. However, patients with platelet count > 600,000 × 109/l had higher WBC (11 ×109/l versus 7.5 ×109/l, p Conclusion: According to our results, the outcome in RARS-T more closely mimics myelodysplastic syndromes rather than myeloproliferative neoplasms. Our results agree with the WHO 2008 classification that considers RARS-T as a separate disorder. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Gattermann:Novartis: Honoraria, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Information about the expression of some myelomonocytic markers in lymphocytes of patients with B-CLL is scarce. We studied the CD13, CD14, CD11c and CD68 surface antigens in 42 controls and in 38 patients with B-CLL to detect their possible reactivity. Eighty-nine percent of B-CLL expressed very strongly the CD14 antigen; on the contrary, the other myelomonocytic antigens tested were very weakly expressed. Forty-one of 42 controls showed a few CD14-positive lymphocytes with a statistical difference between normal and CLL lymphocytes. No statistical difference was recorded either between CD14 expression and Rai's staging system or Binet's stages, nor between CD14 and bone marrow involvement and doubling time or between CD14 and heavy or light chain expression. A minor B lymphocytic subset in humans coexpresses the CD14 and CD5 antigens, it being increasingly speculated that B chronic lymphocyte leukaemias originate precisely from this B CD5- and CD14-positive cells. Just as the CD5 antigen is regarded as an excellent B-CLL marker, it seems to us that a strong expression of the CD14 antigen might have the same diagnostic relevance.

Abstract 3912 Poster Board III-848 Background The myeloproliferative neoplasms (MPNs) include different diseases presenting several mutations in variable frequency. The JAK2V617F mutation is present in 90-95% Polycythemia Vera (PV), 50-60% Essential Thrombocythemia (ET) and 50-60% Primary Myelofibrosis (PMF) patients. In addition, JAK2 exon 12 mutations are observed in 1-3% of PV patients and mutations in the thrombopoietin receptor gene (c-MPL) (S505N, W515K/L) are present in a 5-9% of PMF and in a 1-4% of ET patients. Recently, acquired mutations of the Ten-Eleven Translocation (TET) 2 gene, have been reported in about 14% of sporadic MPNs. TET2 mutations may precede the acquisition of JAK2V617F predisposing to a MPN development. However, the incidence of TET2 mutations in patients lacking the JAK2V617F mutation has not been extensively studied. Aim To analyze the incidence of TET2 mutations in a cohort of MPN patients negative for JAK2V617F, JAK2 exon 12 mutations and c-MPL exon 10 mutations (S505N or W515K/L). Patients and methods From a whole cohort of 241 patients with MPN (93 PV, 16 PMF and 132 ET) we excluded those patients positive for JAK2V617F (determined by quantitative allele-specific PCR), JAK2 exon 12 mutations or c-MPL exon 10 mutations (S505N or W515K/L) (analyzed by direct sequencing). We analyzed the TET2 gene in 5 PV, 5 PMF and 53 ET patients lacking any of the aforementioned molecular markers. The mutational analysis of the coding sequence of TET2 was performed by direct sequencing using cDNA from granulocytes. In 13 patients, DNA from T lymphocytes was obtained to indentify the presence of single nucleotide polymorphisms (SNPs) in germline DNA. Results Sixty-three patients were screened for mutations in the whole coding sequence of the TET2 gene. Only 3 ET patients (4.7%) presented deleterious mutations in the TET2 gene. The three distinct TET2 mutations were: Q706X, S1848X and V1395_R1400delinsR. In 48 out of 63 (76.1%) patients we observed a total of 13 different missense mutations and 2 silent mutations in the coding sequence of the gene. The most frequent missense mutation was the I1762V which was detected in 27 patients. In 13 patients whose matched normal DNA was available, we analyzed the presence of missense mutations being all of them present in the control DNA suggesting that they were SNPs and not acquired mutations. The two nonsense mutations (Q706X and S1848X), were not present in matched normal tissue indicating that these mutations were somatically acquired in myeloid cells. Conclusions TET2 pathogenic mutations are infrequent ( The role and the biological significance of missense mutations in the coding sequence of TET2 has to be elucidated. Acknowledgments Fellowship FI2008 (AGAUR) to LMM-A, FIS EC07/90791 Disclosures: No relevant conflicts of interest to declare.

Abstract 1912 Poster Board I-935 Background: Essential Thrombocythemia (ET) and Polycythemia Vera (PV) are myeloproliferative neoplasms (MPN) arising from a multipotent hematopoietic stem cell characterized by an unregulated production of platelets, red cells and white cells alone or in combination, a tendency to clonal evolution and an increased risk of thrombohemorrhagic complications. MicroRNAs (miRNA) are negative regulators of genes involved in cellular proliferation, apoptosis and/or carcinogenesis. Aim: To analyze the expression pattern of miRNA between PV and ET patients and to find distinctive signatures in ET patients according to JAK2V617F and c-MPL mutational status. Material and Methods: Total RNA was extracted from peripheral blood granulocytes of 50 ET patients, 10 PV patients and 10 controls. Median age of patients was 57 years (range, 20-88); males 36%. The JAK2V617F mutation was present in 23 (46%) of 50 ET patients and in all PV patients. MPL mutations were present in 5 (18%) of 27 JAK2V617F negative cases (3 cases MPLW515L, 1 case MPLW515K and 1 case MPLS505N). miRNA expression was profiled in 384 miRNA via Taqman Low Density Array in ABI PRISM 7900. Expression data was normalized with RNU48 and relative quantification was calculated with the 2–σσCt method. The data were presented as log10 of relative quantity of target miRNA. Median of normal controls was used as calibrator for all samples. Data were analyzed by means of Significant Analysis of MicroArrays (SAM), Prediction Analysis of MicroArrays (PAM) and Class Comparison methods using BRB array tools version 3.7.0 and TIGR multiexperiment viewer version 4.3. Results: We found a general downregulation of miRNA in ET and PV patients respect to normal controls. A set of 29 miRNA allowed us to discriminate between ET and PV versus normal controls; three of these miRNA were up-regulated and 16 down-regulated in PV and ET vs. normal controls with a >2 fold change and p value 2 fold change and p Conclusions: Our study demonstrates that ET and PV can be defined by specific signatures of miRNA. In ET, some miRNA allow us to discriminate cases according to JAK2V617F mutational status and also between MPL-positive and MPL-negative JAK2V617F-negative patients. Research Funding: FIS EC07/90791 Disclosures: No relevant conflicts of interest to declare.

9569 Background: NT-Pro BNP provides diagnostic and prognostic information in many heart syndromes, but its role in oncologic patients is not established. Methods: We studied the association between NT-ProBNP levels and the risk of major toxicity associated with chemotherapy and death from all causes in 116 consecutive patients with lymphoma treated with curative intent. High resolution ultrasound echocardiography and serum NT-ProBNP levels were prospectively done previous the start of chemotherapy. Charlson Comorbidity Index was retrospectively calculated. A major toxicity event was defined as: when chemotherapy had to be discontinued, when chemotherapy had to be changed to a less intensive regimen, and treatment-related death. Results: High blood levels of NT-proBNP were associated with previous cardiologic history but not with left ventricular ejection fraction. With a median follow-up of 16 moths (0–49 months), 25 patients had a major toxic event with first line chemotherapy and 16 had died at last follow-up. The threshold of NT-ProBNP with better predictive accuracy for major toxicity-free and overall survival was 900 pg/mL. Patients with levels of NT-proBNP greater of 900 pg/mL had an adjusted hazard ratio (HR) for major toxicity after first-line chemotherapy for lymphoma of 6.4 (95%CI, 2.7–15.1). Two additional independent factors predicting higher major toxicity associated with chemotherapy were albumin < 3.5 g/mL (HR 3.5, P=0.008) and number of extranodal sites ≥ 2 (HR 3.1, P No significant financial relationships to disclose.

A case of refractory anemia (RA) with an isochromosome 14q is described. This finding is compared with other hematologic disorders with trisomy 14 as the sole abnormality.

Background. Hydroxyurea (HU) remains the cytoreductive drug of choice in high-risk patients with essential thrombocythemia (ET) and polycythemia vera (PV). There is scarce information about its effect upon the JAK2V617F allele burden in these patients. Aim. To analyze prospectively the JAK2V617F response to HU treatment in newly diagnosed patients with ET or PV and to investigate the clinical and/or biological variables associated to a good molecular response. Material and Methods. Sixty patients (25 PV and 35 ET) consecutively diagnosed in a single institution according to WHO criteria were included in the study. All PV patients were JAK2 V617F positive (14 homozygotes) whereas 21 (60%) ET patients were positive for the mutation (all in heterozigosity). Cytoreductive treatment was started according to standard guidelines with the mean HU daily dose being 894 ± 171 mg and 867±223 mg for PV and ET, respectively. Blood samples were taken immediately before starting treatment, at 3 months and then every 6 months. A mean number of 4.6 samples per patient were analyzed by quantitative real-time PCR assay after a median of 16 months (range: 3.5–57) of cytoreductive treatment. Determination of the relative change of the allelic ratio of JAK2V617F was calculated as follows: (% last V617F minus % first V617F) divided by % first V617F × 100. Major molecular response (MMR) was defined when pre-treatment V617F allele burden was reduced by > 50%. Major hematological response (MHR) was defined in PV when Hct 50% decrease in palpable splenomegaly, > 50% reduction in phlebotomies and normal platelet (< 400 × 109/L) and leukocyte (≤ 10 × 109/L) counts were achieved. MHR was defined in ET when normal platelet and leukocyte counts and > 50% reduction in palpable splenomegaly were obtained. The probability of major hematological and molecular response was calculated by the Kaplan-Meier method, followed by the log-rank test. Results. MHR was seen in 46 (77%) patients. Median time to MHR was 4 months with the probability of response at 6 and 12 months being 55% and 71% respectively in the whole series. MHR was faster and more frequently observed in ET patients than in PV (MHR probability at 6 months: 36% in PV and 69% in ET, p=0.009). Patients with a higher JAK2 allele burden showed a significantly lower probability of response (MHR at 6 months: 36% in patients with 50% of mutated JAK2 alleles versus 63% in patients with < 50% of mutated JAK2 alleles, p=0.01). MMR was observed in 15 out of 46 assessable patients (33%). Median time to MMR was 7.5 months (range 2–57) with the probability of response at 12 and 18 months being 21% and 30%, respectively, in the whole series. JAK2V617F mean decrease was −22.7±45.5% in PV and −31.8±39.1% in ET, respectively. Six out of 25 PV patients switched from homozygous to a heterozygous status. The probability of obtaining a MMR was significantly higher in patients achieving a MHR than in those who did not obtained a MHR (MMR at 18 months: 41% versus 0%, p=0.02). Age, gender, spleen size, leukocyte count, platelet count, serum LDH level and JAK2V617F allele burden prior to cytoreductive treatment were not associated with the likelihood of MMR. ET patients with an Htc 0.45 L/L at diagnosis obtained a MMR more frequently than those patients with Htc < 0.45 L/L (MMR probability at 18 months: 57% versus 16%, p=0.003). Conclusions. Major hematological response is the main factor predictive for attaining a major molecular response in JAK2V617F-positive PV and ET patients treated with HU.

Introduction: High blood levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), a sensitive indicator of cardiac abnormalities, have been associated with poor outcome in patients with amyloidosis. However, its clinical value in lymphoma is unknown. Aim: To assess the value of NT-proBNP as a prognostic variable for survival in patients with lymphoma treated with chemotherapy with curative intent. Patients and methods: Clinical heart examination, evaluation of cardiovascular risk factors, determination of NT-proBNP (ECLIA; Roche) blood levels and high resolution ultrasound echocardiography were done at diagnosis. Clinical variables related with patient characteristics, lymphoma risk and cardiovascular risk were analyzed. Results: 117 consecutive pts have been analyzed: median age: 63 years (range: 18–88); male: 56%; NHL: 80%; HL: 20%; ECOG/PS ≥2: 29%; III–IV: 56%; B-symptoms: 16%; LDH >1 nv: 34%; b2-microglobulin >1 nv: 57%; albumin 900, ECOG/PS ≥2, LDH >1 nv, b2-microglobulin >1 nv, albumin < 35 g/L, haemoglobin 60. In multivariate analysis, factors predicting for survival were NT-proBNP >900 (HR 15, 95%CI: 3.9–57.8) (p Conclusions: NT-proBNP >900 pg/mL is the strongest prognostic factor for survival in patients with lymphoma following treatment with curative intent. NT-proBNP is an easy, fast, widely available and well-standardized parameter that could be useful in addition to other well-established prognostic indexes.

Introduction. Exon 12 mutations of the JAK2 gene have been described in polycythemia vera (PV) and idiopathic erythrocytosis (IE) patients. These patients display a clinical phenotype different to that observed in V617F-positive PV patients, but no information is available on their clinical outcome. Patients and methods. Twenty JAK2 V617F-negative PV or IE patients and 86 V617F-positive PV patients were assessed for exon 12 and exon 14 mutations. Analysis of cell lineage mutational status was assessed following cell sorting. Aim. To analyze the presence of JAK2 mutations at exon 12 and 14 in a cohort of V617F-negative PV and IE patients and to correlate them with the patient clinicohematological and evolutive data. Results. Exon 12 mutations were detected in 4 (20%) V617F-negative patients (K539L, two cases, N542_E543del and H538_K539delinsL, one case each). In 16 patients, no mutations were found in either exon 12 or exon 14. Additional mutations in exon 14 were found in two V617F-positive patients. Two patients with exon 12 mutations developed thrombosis after diagnosis, with the probability of thrombosis-free survival being 75% at 5 years. Another patient with exon 12 mutation developed myelofibrosis at 20 years of PV diagnosis. JAK2 mutations were present in granulocytes, platelets and monocytes, but not in lymphocytes or NK cells. Conclusions. Patients harboring exon 12 mutations represent a subset of JAK2 V617F-negative PV or IE patients. These patients have initial hematological data different from V617F-positive patients, but they do not differ with regard to thrombosis development and myelofibrotic transformation. Table 1: JAK2 exon 12 and exon 14 mutations in blood cell subpopulations. Mutation Granulocytes Platelets CD14+ CD3+ CD19+ CD56+ * exon 12; # exon 14; + indicates the presence of the mutation; − indicates no detectable mutation ; ND not determined Patient 1* K539L (AAA →TTA) + + − − − − Patient 2* K539L (AAA →CTA) + + + − − − Patient 3* H538_K539delinsL + + ND − − ND Patient 4* N542_E543del + + + − − − Patient 5# V617F (GTC →TTT) + + − − − ND C618R (TGT →CGT) + + − − − ND Patient 6# V617F (GTC →TTC) + + + − − ND C616C (TGT →TGC) + + + − − ND

We performed a retrospective analysis of our Spanish database of patients with ET in order to assess the role of different response predictive potential factors to anagrelide treatment. 411 ET patients from 54 sites from February 2005 to August 2006 were included in a retrospective chart review. ET was diagnosed according to PVSG criteria (1997). All patients had started treatment with anagrelide before December 2004, either as a first line or as a second line therapy. The population was divided itself in three groups of risk at diagnosis: of high Risk: those patients older than 60 years or with previous history of thrombosis (40.6%); of low Risk: patients younger than 60 years with thrombocytosis lower than 1.5· 109/L, without cardiovascular risk factors and previous history of thrombosis (35.8%); intermediate risk: those patients who did not reunite the criteria of previously mentioned groups (23.6%). The response to the treatment was defined as complete remission (CR), when a reduction of platelets′ count were equal or less than 400×109/L, or a reduction higher than 50% with respect to the basal numbers; partial remission (PR), when the platelets′ count was between 400× 109/L and 600×109/L with respect to the basal numbers; and no response, those with a lower reduction than PR or increasing their platelets′count. CR was obtained in 219 patients (53.6%; 95%CI = 48.6–58.5) and PR in 113 (27.6%; 95%CI = 23.4–32.2), giving an overall response (OR) rate of 81.2% [95%CI = 77.0–84.9]. The influence of certain factors (such as age, gender, risk group, platelets levels before treatment with anagrelide, previous cytoreductive untreated patients and maximum anagrelide dose), in the objective response was explored. A chi-square test and a multivariate analysis (logistic regression) were performed. A worse response (p = 0.021) was associated with a higher dose (≥ 2.5 mg per day). A trend (p = 0.103) to better response in patients without previous treatment (anagrelide as a first line) was detected. Additionally a significant association (p = 0.02) between the previously treated with hydroxyurea and the presence of evolutive hematological transformations (myelofibrosis and acute myeloid leukemia/myelodysplastic syndrome) was observed. It is difficult to set a cause effect relation in all these findings due to the retrospective design of the analysis. As a conclusion, the response to anagrelide in ET patients was demonstrated to be independent of the age, gender, platelets′ count and level of previous risk.

Background: The molecular response in patients with gastric MALT lymphoma achieving histological remission after chemotherapy with or without surgery is not well established. Patients and Methods: 19 patients with gastric MALT lymphoma achieving complete histological remission after treatment with chemotherapy and/or surgery was studied retrospectively. IgVH gene rearrangements were studied by PCR in gastric biopsies obtained at diagnosis and during follow-up. Presence of t(11;18)(q21;q21) was studied by FISH or PCR. Sequencing analysis of PCR products of patients with t(11;18)(q21;q21) was performed. Results: t(11;18)(q21;q21) was present in 3 patients (15.7%) at diagnosis. Median follow-up was 73 months (13–240). In patients with complete histological remission, monoclonality was demonstrated in 11 out of 19 (58%) and maintained monoclonality in 8 out of 10 (80%) who had sequential studies. All 3 t(11;18)(q21;q21) positive patients had maintained monoclonality and sequencing analyses revealed the presence of the same mutated IgVH alleles in diagnostic and in follow-up samples. In contrast, in 2 cases without t(11;18)(q21;q21) the monoclonal rearrangements corresponded to unrelated alleles. Persistent monoclonality was detected after a median of 49 months (6–134) and did not condition histological relapses, either in negative or positive t(11;18)(q21;q21) patients. Conclusions: More than half of patients with gastric MALT lymphoma achieving histological response after chemotherapy and/or surgery have long term persistent monoclonality. t(11;18)(q21;q21) determines long term persistence of the initial lymphoma population. However, the presence of maintained monoclonality did not determine relapse.

Background: The treatment of gastric mucosa-associated lymphoid tissue (MALT) lymphoma in the absence of H. pylori infection or when the lymphoma fails to regress after adequate antibiotic treatment remains controversial. Fludarabine (F) is an active agent for indolent lymphoma, however, its clinical activity in gastric MALT lymphoma has not been studied. The aim of the study is to assess the efficacy and safety of single-agent fludarabine in gastric MALT lymphoma and to analyze the molecular response (MR) after this treatment. Methods: Treatment consisted of fludarabine (25 mg/m2 IV) given on days 1–5, every 4 weeks, for 6 cycles; after the first cycle, oral fludarabine was allowed to be given orally at 40 mg/m2 with the same schedule. Molecular response (MR) was assessed by RT-PCR analysis of t(11;18) or by PCR assays for analysis of IgH gene rearrangements analyzing FR1, FR2 and FR3 in endoscopic biopsies. Results: Eight consecutive patients were included. Median age: 60 years (range: 45–77); 3 pts were in stage I, 2 stage II-1 and 3 stage IV according to Lugano system. Four out of 5 (80%) pts achieved a CR after three cycles and all eight cases (100%) achieved a CR after six cycles, for an overall response rate of 100%. After a median follow-up of 44 months (range 14.5–58 mo) no patient has shown clinical or endoscopic relapse. Hematological toxicity occurred in 75% of pts, mainly mild neutropenia and generally after the third cycle. Three cases received G-CSF (after the 2nd, 3rd and 6th cycle) and three patients required dose modification or delay (3–7 days) in the delivery of the following cycle. No blood transfusions were required. Only one patient had to be admitted because of non-neutropenic fever. None case of myelodisplasia has been detected at last follow-up. Four out of 8 pts (50%) achieved MR during the study-period (see figure). Four out of 5 (80%) pts without t(11;18) achieved MR. In contrast, no patient carrying t(11;18) achieved MR. Sequencing analysis of monoclonal PCR products will be presented. Conclusions: Fludarabine, either intravenous or oral, is safe and achieve a high response rate when given in gastric MALT lymphoma, with many pts achieving MR. In those pts carrying t(11;18), residual disease can be detected by PCR but do not determine relapse at present follow-up. Figure Figure

Introduction. The JAK2 V617F mutation has been detected in 23%–57% of ET patients by direct sequencing or allele-specific (AS) PCR. It remains unknown, however, if the mutation detected in the granulocyte population, may be equally detected in platelets from these patients. Objective. To compare the detection of the JAK2V617F mutation in granulocytes and platelets from ET patients by real time AS RT-PCR. Patients and methods. Platelets and granulocytes from 50 ET patients from a single institution were studied. Patients were diagnosed according to the WHO criteria. At the time when JAK2 mutation was analyzed 16/50 patients were receiving platelet-lowering therapy ± ASA, 14/50 patients only received ASA and 20/50 received no specific treatment. JAK2 mutation was analyzed by real-time AS RT-PCR with probes specific for the mutated and the wild type form. Results. The V617F JAK2 mutation was detected in 18 out of 50 patients in both granulocytes and platelets by real time AS RT-PCR, and was negative in both cell populations in the remaining 32 patients. In the V617F JAK2 positive cases, the mean Ct(V617FJAK2)/Ct(wild type JAK2) ratio was 1.074±0.062 for granulocytes and 1.038±0.039 for platelets (p=0.048). These values corresponded to a 17.79 ±7.4% of mutated population when granulocytes were analyzed, whereas, a significantly higher percentage of mutated population was observed, 23.45±7.78 %, when platelets were analyzed (p=0.032). Conclusions. The results of V617FJAK2 mutation detection by AS RT-PCR were the same in granulocytes and platelets (either positive or negative). The percentage of clonal population detected in ET patients was significantly higher in platelets than in granulocytes.

Introduction: Infusion-related toxicity is frequent after the administration of Rituximab despite the fact that strict guidelines have been recommended. Recently, a rapid rituximab infusion schedule in combination with a steroid containing chemotherapy regimen was well tolerated and safe. Purpose: To investigate the feasibility of a rapid infusion of rituximab with or without steroid containing chemotherapy. Methods: Inclusion criteria: lymphoproliferative disorder CD20+ and having been treated with a first infusion of rituximab according to the product monograph. Exclusion criteria: lymphocytosis > 5 x 109/L, toxicity grade 3/4 in the previous infusion of rituximab or dose > 375 mg/m2. Schedule: First infusion of rituximab according to the product monograph; Further infusions over a total time of 90 minutes (20% in the first 30 minutes and the remaining 80% over 60 minutes). Premedication: acetaminophen and diphenhidramine, plus methylprednisolone only in those patients receiving steroid containing chemotherapy. Results: A total of 50 patients were treated for a total of 165 infusions. Patient characteristics: median age 62 yr (range 28–83), 49 % males, DLBCL 36%, follicular 47%, mantle 6%, MALT 7%, other 4%. Chemotherapy regimens: R-CHOP 28%, R-EPOCH 9%, R-ESHAP 4%, R-other chemotherapy 8%, rituximab monotherapy 10%, rituximab maintenance 41%. Number of rituximab administrations with and without steroids: 68 and 97 infusions, respectively. Median time from previous rituximab infusion was 29 days (range 7–272). Sixteen rapid infusions were administered with an interval greater than 90 days from the previous standard infusions. This rapid rituximab administration schedule was very well tolerated. No grade 3/4 adverse events were seen. Two patients referred symptoms during rituximab infusion (both grade 1): abdominal discomfort that disappeared spontaneously and sore throat that required to slow down the infusion speed. All these reactions occurred in patients who did not receive premedication with steroids. Conclusions: Rituximab administration in a 90-minute infusion schedule is well tolerated and safe in this group of patients. This approach is beneficial, both in patients who are administered steroids and in patients who are not.

We studied the behaviour in culture of erythroid and megakaryocyte progenitor cells (BFU-E, CFU-MK) obtained from peripheral blood (PB) in 38 patients: 15 with essential thrombocythaemia, 3 with reactive thrombocytosis, 16 with polycythaemia vera and 4 with secondary polyglobulia. Clonal erythroid growth without added erythropoietin was observed in all patients with polycythaemia vera and in 5 out of 15 with essential thrombocythaemia, but in none of the patients with reactive thrombocytosis or secondary polyglobulia or in controls. When the CFU-MK were cultured without phytohaemagglutinin-stimulated medium (PHA-LCM), all patients with essential thrombocythaemia and 7 out of 16 with polycythaemia vera showed circulating CFU-MK but none of those with reactive thrombocytosis or secondary polyglobulia or controls did so. This study indicates that the growth in vitro of megakaryocytic and erythroid progenitors from such a readily available source as peripheral blood can be valuable in the diagnosis of certain borderline cases of thrombocytosis or erythrocytosis.

Young patients (age < 60 years) with essential thrombocythemia (ET) and no history of thrombosis are considered at low risk of thrombosis and therefore managed on a conservative approach with antiplatelet therapy or even without any treatment. JAK2 V617F and CALR exon 9 mutations are the most frequent molecular alterations observed in ET, with CALR-positive ET being considered a distinct clinical entity due to its higher platelet counts and lower incidence of thrombosis as compared with JAK2 V617F-positive ET. There is some evidence supporting a role for antiplatelet therapy in JAK2 V7617F-positive neoplasms. However, the role of antiplatelet therapy in CALR-positive ET has not been studied. The aim of the present study was to assess the effect of antiplatelet therapy in the primary prevention of thrombosis in patients with CALR-positive ET without indication of cytoreductive therapy. For such purpose, 240 patients (107 males, 133 females) diagnosed with ET at a median age of 42 years (range 13-59) were included in a multicenter retrospective study. Initial treatment consisted of antiplatelet therapy (n=109) or careful observation (n=108), whereas 23 patients received cytoreduction since diagnosis and were excluded. During a median follow up of 8 years, 137 patients were started on cytoreductive therapy because of the following indications: age > 60 years (n=10), thrombosis (n=10), bleeding (n=2), microvascular symptoms (n=18), extreme thrombocytosis (n=89), and others (n=8). Median time free of cytoreductive therapy was 3.2 years. Thrombosis-free survival restricted to the time of cytoreductive therapy abstention was calculated using the Kaplan-Meier method. Variables attaining a significant level at the univariate analysis were included in a Cox proportional hazard model. During the period of abstention of cytoreductive therapy, a total of 10 thrombotic events and 8 major bleeding episodes were registered. The probability of thrombosis at 3 years was 5% in patients managed with careful observation and 1% in those receiving antiplatelet therapy (p = 0.2). At multivariate analysis, antiplatelet therapy did not result in a lower risk of thrombosis after correction for age, sex and presence of cardiovascular risk factors. Interaction studies did not identify any subgroup of patients that benefited from antiplatelet therapy in thrombosis prevention. Regarding major bleeding, patients receiving antiplatelet therapy experienced a higher rate than those managed on observation (3-year probability of major bleeding, 5.5% and 0%, respectively, p=0.05). At multivariate analysis, antiplatelet therapy was associated with a tendency towards and increased risk of major bleeding (HR: 7.7, 95%CI: 0.9-66.2, p=0.06) independently of platelet count at diagnosis, age and gender. In conclusion, CALR-mutated low-risk ET patients under cytoreductive therapy abstention may not obtain a clear benefit from antiplatelet therapy since the increase in the rate of bleeding may offset the reduction in the rate of thrombosis Disclosures García-Gutierrez: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Harrison:CTI Biopharma: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Gilead: Honoraria; Sanofi: Honoraria, Speakers Bureau; Shire: Speakers Bureau. Cervantes:Sanofi-Aventis: Consultancy; Novartis: Consultancy, Speakers Bureau; CTI-Baxter: Consultancy, Speakers Bureau. Vannucchi:Shire: Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Idiopathic myelofibrosis (IMF) induces dramatic changes in bone. Bone remodeling and densitometric alterations in a series of nine patients with IMF and their relationship with the histologic stage of the disease were assessed. Patients were included at diagnosis and a bone marrow biopsy, dual-energy X-ray absorptiometry, and transiliac bone biopsy for histomorphometric analysis were performed. Five cases were classified as IMF histologic stage 1, one as stage 2, and three as stage 3. Compared with 40 age- and sex-matched controls, the following histomorphometric parameters were significantly higher in our patients: bone volume (BV/TV), osteoblast surface (Ob.S/BS), eroded surface (ES/BS), osteoclast surface (Oc.S/BS), osteoclast number (N.Oc/TA), mineralizing surface (MS/BS), reversal period (Rv.P), and remodeling period (Rm.P). Mineral apposition rate (MAR) and erosion depth (E.Depth) were significantly decreased (P < 0.05 for all comparisons). Bone mineral density (BMD) measurements showed high values for patient age and sex both at femur neck (Z score range +0.19 to +7) and total femur (Z score range -0.09 to +6.48). When densitometric values were analyzed according to IMF histologic stage, patients in stages 1 and 2 had significantly lower BMD values than to those in stage 3 (P = 0.024). In conclusion, patients with IMF present a characteristic bone histomorphometric pattern with increased bone volume and bone cells but low apposition and decreased erosion depth, suggesting a positive balance in bone remodeling units. This balance would produce the increase in bone mass observed in this disease. Given the increase in BMD observed with more advanced stages of IMF, this noninvasive method could be useful tool for assessing IMF progression.