ALBERT

Description: Background: According to recent findings, the management of myeloproliferative neoplasms (MPNs) is highly dependent on presence or absence of thrombotic events. The JAK2 mutation has been identified as a marker of MPNs. It is also an occult marker in several patients with splanchnic venous thrombosis (SVT), but its contribution as an additional thrombotic risk factor in MPNs is still under discussion. Moreover, a pro-thrombotic risk factor, either inherited or acquired (Factor V Leiden mutation, deficiencies in protein C, protein S and Prothrombin mutation 20210) can be identified in these patients. Recently, another milestone in the molecular diagnosis of MPNs, somatic mutations in the CALR gene, has been reported. A total of 36 types of frame-shifting insertions and deletions were detected in the exon 9 of CALR gene, which encodes a Ca2+ binding protein in endoplasmic reticulum called calreticulin. Type-1, 52-bp deletion (p.L367fs*46), and type-2, 5-bp TTGTC insertion (p.K385fs*47) variants constitute more than 80% of these mutations These mutations were reported to have a incidence of over 60% to 80% in JAK2 and MPLmutation-negative Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) patients. Compared to those with JAK2 mutation, CALR-mutated ET patients are younger and have a lower leukocyte count and higher platelet count. CARL mutations have been also reported as a favorable prognostic factor on thrombosis-free survival (TFS) for ET patients. Aims: In this study, we evaluated the incidence of SVT, JAK2 and CALR mutations, and prothrombotic risk factors in patients with MPNs observed in our center from January 2000 to January 2014. Methods: We performed a retrospective review of clinical charts of 466 Ph1 negative MPN patients followed in our center, classified according to the WHO 2008 classification. Patient and disease characteristics, including JAK2V617F, MPL and CALR mutations and thrombotic risk factors were recorded. Results: The median age of patients with diagnosis of MPN was 43 years. Fourteen patients (13 females, 1 male; 3%) of median age 46 years presented a SVT. Three had a Budd Chiari syndrome and 11 a portal venous thrombosis. According to a histological review, these patients were classified as follows: ET, 2 cases, PMF, 3 cases, Polycythemia Vera (PV) 1 case, Myelofibrosis in a prefibrotic phase (MF0) 8 cases. Classification of 11 cases with Myelofibrosis according to the IPSS identified 7 as INT1, 1 as INT2 and 3 as low risk. Among all 14 patients diagnosed with SVT, 12 were JAK2V617F positive with a median allelic burden of 30%, 1 patient was MPL positive, and 1 patient was triple-negative. CALR mutation was not observed in any of the patients. Two cases were diagnosed with MPN 30 months after SVT, 3 patients experienced SVT after a median follow-up of 108 months from MPN diagnosis while in 9 patients the diagnosis of MPN was concomitant to SVT. In the latter patients, median Hb levels were 12.4 g/dL , WBC 8260 /µL, HCT 36.3%, PLT 337.000/ µL and a modest hepatomegaly and splenomegaly were documented. Prothrombotic risk factors were found in 9 of 13 patients. Two patients experienced a thrombotic episode prior to the diagnosis of SVT and two subsequently during the follow-up. Interestingly, 9 (70%) of MPN patients with SVT exhibited at least one prothromobtic risk factor, such as factor V Leiden, Protein C deficiency, hyperhomocystinemia and 50% had two or more associated defects. Thirteen of the 14 patients are currently being treated as follows hydroxyurea (9), interferon (1), and ruxolitinib (3). All patients received oral anticoagulant treatment except for three who are on antiplatelet therapy. MPN patients without SVT had a lower prevalence of prothrombotic risk factors and developed venous thrombosis in different anatomical sites: in these cases WBC count, platelet values and the presence of JAK2V617F mutation correlated with the development of the thrombotic event. Conclusions: Although SVT has a low incidence in MPN patients, a potential benefit of testing for mutations in CALR gene and for additional prothrombotic risk factors is suggested in the whole MPN population for the prevention and treatment of this complication. Disclosures No relevant conflicts of interest to declare.

Description: INTRODUCTION: Quality-of-life (QOL) is a patient-related outcome of increasing interest in onco-haematological setting. However, there are very few reports on QOL in elderly patients with Acute Myeloid Leukemia (AML). Prognosis of AML in elderly patients is still poor with a median survival of 9–12 months and less than 20% survival at 5 years. This is due to age-related factors and also to a higher incidence of poor-risk cytogenetics, multi-drug resistance and treatment-related mortality. The measurement of QOL at diagnosis may provide useful information regarding patient preferences and prognosis while followup measurements may indicate acceptance, adaptation and adverse effects of disease and therapy. METHODS: From 2/2003 and 2/2007, we included 113 elderly AML patients in a multicenter Italian 12-month observational study to evaluate the association of baseline QOL scores and their changes with disease factors, therapy and survival. Patients aged 〉 60 years (M/F 58/55, mean age 71.7 ± 5.9 yrs) from 4 Institutions with “de novo” AML according to WHO classification were evaluated from diagnosis prospectively up to 12 months. Two different questionnaires were employed: the EORTC QLQ-C30 and the QOL-E v.2. Therapeutic choice was not restricted by protocol and was given freely by the individual center. RESULTS: Forty-eight patients (43%) received intensive chemotherapy and 65 (57%) lowdose therapy or supportive care. Both age 〉 70 years (p=0.007) and concomitant diseases (p=0.031) had a significative impact on medical decision for palliative approaches. At diagnosis, general QOL was affected (median QOL-E standardized score 54, IQ range 47– 68, median EORTC global score 50, IQ range 42–67), loss of appetite was perceived by 75% of patients and QOL-E fatigue scores were low, indicating poorer QOL (median 45, IQ range 32–53). There was a significant correlation between fatigue and age, Hb levels and the duration of fever. In a multivariate regression model, both Hb and age indipendently predicted fatigue (linear R2 0.114, p=0.001, and linear R2 0.066, p=0,010). Most patients were given a good ECOG Performance Status (〈 2) that, interestingly, did not correlate with the perception of QOL, in particular physical and functional scores. QOL scores worsened after 1 month from diagnosis but patients surviving thereafter perceived an improvement in the following months. When correcting for the effects of age, concomitant diseases and changes in Hb values, fatigue improved in patients undergoing aggressive chemotherapy while it worsened in those on standard or supportive-care (p=0.004). Median overall survival was 49 weeks (95% CI 35–63). Patients receiving aggressive therapy had a longer survival with the median not reached at 60 weeks and 72% of patients surviving versus patients receiving palliative care (median 39 weeks, 95% CI 16–63, p 70 yrs (HR 2.4, 95%CI 1.2 – 5.0, p=0.02) and concomitant diseases (HR 2.0, 95% CI 1.1 – 3.8, p=0.044) had an independent negative prognostic impact on survival, as previously reported in many other studies. However, in order to evaluate the predictive value of QOL at diagnosis for survival, in a multivariate model corrected for age, concomitant diseases and treatment option, QOL measures that independently predicted survival were fatigue (p=0.003), global QOL (p = 0.004), physical QOL (p = 0.006) and functional QOL (p = 0.002). CONCLUSION, QOL seems to have an important role also in the elderly AML setting: though QOL is a highly subjective measurable value, we outline the role of QOL measures at diagnosis as a prognostic factor for overall survival and, thus, as a potential factor for treatment decision.

Description: Abstract 4974 Introduction: Primary immune thrombocytopenia (ITP) is an acquired immune-mediated disorder characterized by isolated thrombocytopenia, defined as a peripheral blood PLT count 60 years of age, those with systemic symptoms or abnormal signs or in some cases where splenectomy is considered (Provan et al, Blood, 2009). Methods: We performed a retrospective chart analysis between January 2008 and June 2010 of all patients referred to our clinic for isolated thrombocytopenia but with a PLT count 100– 149 Gi/L. According to recent guidelines, these patients are not to be considered thrombocytopenic and do not require further investigation. The aim of the study was to evaluate the validity of omitting BM analysis in these cases. Results: Twenty-three cases (13 males/10 females) of mean age 58 ± SD 19 years were evaluated at our clinic for a PLT count below normal lab range values. At the time of evaluation none had bleeding symptoms. PLT counts ranged from 101 to 149 Gi/L, mean 123 Gi/L. Four patients had an enlarged spleen. After initial screening, 2 patients had a complex autoimmune disorder and 1 case had HCV hepatitis. The remaining 20 patients had a bone marrow (BM) aspirate performed: a diagnosis of myelodysplastic syndrome (MDS, WHO classification refractory thrombocytopenia) was obtained in 13 cases (65%) and BM biopsy was performed in 12, completed by cytogenetics in 9 cases (7 normal, 1 del20q, 1 –Y). Patients diagnosed with MDS were significantly older (66 ± SD 13 vs 47 ± SD 21 years, p = 0.017), but 4 cases (31%) were 〈 60 years of age (44, 49, 51 and 55 years of age, respectively). Conclusions: The most recent guidelines, which lower the PLT threshold to 100 Gi/L from 150 Gi/L for the investigation of causes of thrombocytopenia, reduce the diagnostic rate of MDS. In our retrospective review, 65% of patients would not have had an early diagnosis of MDS. Furthermore, BM aspirate should be considered irrespective of age, since one third of the patients in our case review had MDS with PLT 〉 100 Gi/L as a single cytopenia and age under 60. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2927 Background: Immunological changes have a primary role in the initiation and progression of myelodysplastic syndromes (MDS). Cytokine levels, such as IL-7 and IFN-gamma, are associated with lower-risk disease. Treatment with lenalidomide has proven efficacy in red blood cell (RBC) transfusion-dependent lower-risk MDS patients with del(5q). Lenalidomide exerts anti-angiogenic, anti-proliferative, and pro-erythropoietic effects; in particular, it has been shown that lenalidomide inhibits the proliferation and function of T regulatory cells (Tregs). Finally, MDS patients undergoing lenalidomide treatment experience erythroid responses and suppression of the del(5q) clone. Aims: In a multicenter Italian phase II trial to evaluate safety and efficacy of lenalidomide in primary MDS patients with del(5q) and Low- or Int-1 risk IPSS, we investigated changes in the transcription of cytokines and their receptors during treatment. Methods: The starting dose of lenalidomide was 10 mg p.o once daily on a continuous daily schedule for a maximum of 12 months. Bone marrow (BM) aspirates were obtained on study entry and every 12 weeks. Assays were performed using TaqMan® Low Density Array Fluidic card (TaqMan® Human Array, Applied Biosystems, Foster City, CA, USA) based on Applied Biosystems PRISM® 7900HT comparative ddCT method, according to the manufacturer's instructions. Target gene expression levels were measured in triplicate and normalized against the expression of the 18S housekeeping gene from a BM pool of normal, healthy subjects at all timepoints. Median relative gene expression values in MDS patients were compared to healthy subjects, set as a value of 1. Results: We report data obtained at baseline and after 12 weeks. Informed consent was obtained in all patients. Twenty-seven patients (5 M, 22 F) were evaluated at baseline and after 12 weeks. Mean age was 72 ± 9 years. Mean Hb level was 8.5 ± 0.9 g/dL and 16 patients were RBC transfusion -dependent (requiring at least 4 RBC transfusions in the preceding 2 months). Seven patients had additional cytogenetic abnormalities. Twenty-one patients (80%) experienced erythroid responses by week 12. Significant variations in gene expression of cytokines and receptors were observed during treatment. Genes significantly regulated during lenalidomide treatment (P 〈 0.05) are shown in the Table. In particular, FAS, IL-7 and FOXP3 gene were generally under-expressed at baseline and significantly increased after 12 weeks. Accordingly, IL7R was over-expressed in all patients at baseline and its expression was significantly reduced during treatment. Furthermore, IFN-gamma expression increased during therapy. Summary: The protein encoded by FAS gene is a member of the TNF-receptor superfamily and its interaction with its ligand leads to apoptosis. Interleukin (IL)-7 is an essential cytokine that promotes the proliferation and survival of B- and T-lymphocyte progenitors. The IL7R gene on chromosome 5 (5p13) codifies for the IL7 receptor, which blocks apoptosis during differentiation and activation of T lymphocytes. It functions, in part, through the induction of the expression of the antiapoptotic protein Bcl-2. The protein encoded by the FOXP3 gene is a member of transcriptional regulators. Defects in this gene are the cause of X-linked autoimmunity-immunodeficiency syndrome. The results of the present study indicate that lenalidomide may act through immunological changes. Further detailed analyses in these patients may provide new insights into the pathogenesis of MDS with del(5q) and the long-term effects of lenalidomide treatment on immunological changes in BM cells. Disclosures: Oliva: Celgene: Consultancy.

Description: Metastatic disease of the bone is a rare complication of chronic lymphocytic leukemia (CLL), it may be result from richter's transformation or metastatic from non lymphoid malignancies. CLL is the most common form of adult leukemia, with the median age of 70 years at diagnosis [Siegel et al. 2013]. The diagnosis is established by blood counts, blood smears, and immunophenotyping of circulating B-lymphocytes.The result is the increased number of lymphocytes in the peripheral blood, leukocytosis with absolute lymphocytosis, the increase of the lymphnodes, the increase in size of the spleen. The diagnosis of chronic lymphocytic leukemia B requires the presence of Clonal B cells in the peripheral blood at or above 5,000 / ul for at least 3 months. Typing immunophenotypical pathological lymphocytes are positive for surface antigens CD5, CD19, CD23, weakly positive for CD20 and CD22, generally negative FMC7 and CD79b; also expressing surface immunoglobulins. The Rai and Binet staging systems, which are established by physical examination and blood counts, have been recognized as standards for deciding whether to begin treatment. Patients with active or symptomatic disease or with advanced Binet or Rai stages require therapy. For fit patients, chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab represents the current standard therapy. For unfit patients, treatment with an anti-CD20 antibody (obinutuzumab, rituximab, ofatumumab) plus a milder chemotherapy (Chlorambucil) may be applied. At relapse, if the treatment-free interval exceeds two to three years, the initial treatment may be repeated, if the disease relapses earlier, drugs such as bendamustine (plus rituximab), alemtuzumab, lenalidomide, ofatumumab, ibrutinib, or idelalisib, must be choosen. Patients with a del(17p) or TP53 mutation can be treated with ibrutinib or a combination of idelalisib and rituximab. in relapsing patients with TP53 mutations or del(17p) or patients that are refractory to repeated chemoimmunotherapies, an allogeneic SCT may be considered [Hallek M 2015]. In this article we show a case of a 66-year-old man with CLL and a bone localization. In 2011 diagnosis of CLL, Rai Stage 0, Binet Stage A. Principal characteristics at diagnosis: HB 13.2 g /dl, White Blood Cells 15.800 / mm3, lymphocytes 61%, neutrophils 32%, monocytes 4%, platelets 141.000/mm3; normal hepatic end renal function; flowcytometric immunophenotyping of the peripheral blood revealed B-cell CLL; prognostic factors: CD38 negative, ZAP70 positive, rearrangement of the immunoglobulins mutated; FISH: negative; CT chest / abdomen / pelvis: presence of multiple aorto-pulmonary and axillary adenopathies (max diameter of 2 centimeters); bone marrow biopsy: infiltration of CLL equal to 60% of global cellularity. The patient was only observed until January 2015, when he was hospitalized due to acute anemia, requiring supportive therapy, and right foot pain . So it was decided to re-evaluate the whole disease in order to decide whether to start chemotherapy. The disease was staged again with instrumental and laboratory tests: presence of renal insufficiency, egd and colonoscopy negative, Coombs' test negative, bone marrow biopsy confirmed the diagnosis of chronic lymphocytic with bone marrow infiltration of 90%, abdomen ultrasound showed only moderate splenomegaly. On February, persistence of right foot pain and appearance of swelling, assessed by the orthopedic as a suspected algic and dystrophic syndrome. So he suggested to perform scintigraphy which revealed: pronounced inflammatory osteometabolic reaction of the right tibia/fibula/ankle third distal which could be referred, in the first evaluation, to algic and dystrophic syndrome. However, a local biopsy was performed: localization of chronic lymphocytic leukemia. On March 2015 a total body TC showed 2 nodular calcifications in the right lung lobe, multiple right paratracheal, barety space, aortopulmonary and axillary adenopathies. Prostate size increased. In order to study carefully the liver and prostate lesions, an ultrasound abdomen was performed that documented only enlarged spleen, normal size liver, free of focal disease, increased prostate due to symmetric bilobate hypertrophy . After the second cycle of chemotherapy, prolonged thrombocytopenia, so he continues only with a radiotherapy program. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3078 Chronic myeloproliferative neoplasms (CMNs) include Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF). So far limited studies of familial clusters of CMNs have been reported.Familial chronic myeloproliferative neoplasms are defined when in the same pedigree at least two relatives have CMNs. Familial CMNs should be distinguished from inherited disorders with Mendelian transmission, high penetrance and polyclonal haematopoiesis named ‘hereditary erythrocytosis' and ‘hereditary thrombocytosis'. Recently a 5- to 7-fold higher risk of MPN among first-degree relatives of patients with MPNs was reported. These findings support the limited studies suggesting a familial clustering in MPNs. The analysis of mutations of JAK2 and MPL may improve our ability to identify these conditions. In a consecutive series of patients observed in our Institution from January 2000 to June 2010, we found that among 460 patients with sporadic CMNs and 94 Ph1 positive chronic myeloid leukemia (CML), the prevalence of familial cases was 4%.With 22 pedigrees, 44 patients (8%) were identified with two relatives affected. Familial CMNs were 11 PV,14 ET,7 PMF, 5 CML respectively, while sporadic cases were 96 PV,204 ET,115 PMF and with other 45 CMNS not furtherly classified. As far as the distribution of the different CMNs within the familial cluster, We observed that only in 4 of 22 families (18%) all the affected relatives were diagnosed with the same disease (homogeneous pattern: PV one family and ET three families), whereas 14 families exhibited a mixed distribution among PV, ET and PMF. 8 families exhibited CMNs associated with other hematological disease such as chrocic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodisplastic syndrome (MDS). Among this, 6 families presented a first or second degree of relationship of first and second generation. In 10 cases the relatives were brothers, affected by familial CMNs with a prevalence of PV and TE clinical phenotype at diagnosis.According to JAK2 (V617F) mutational status, analyzed in 30 out of 44 patients, 19 patients showed a positivity pattern, while 18 families showed a heterogeneous pattern; they included both JAK2 (V617F) -positive and JAK2 (V617F)-negative patients. Among the 19 patients with JAK2 (V617F) positivity, the distribution of positivity according to the diagnosis was 100% of PV, 45% of ET and 55%of PMF; homozygosity was present only in PV cases. In our series, only two members of the same family were affected by familial CMNs. Finally it should be noted that in our series of familial cases clinical presentation, therapeutic approach and type and severity of complications were comparable to that of sporadic cases. In conclusion, the present study indicates the relevant possibility of familial CMNs, thus suggesting the opportunity of a detailed family history as part of the initial work-up of patients with CMDs; in addition it also suggests the usefulness of an accurate biological study. Disclosures: No relevant conflicts of interest to declare.

Description: The aim of our perspective observational study was identify a fast molecular biomarker of tumorigenic-proliferative haematological disorders at on set using a non-invasive method. At this end, to better discriminate between myeloproliferative or lymphoproliferative hematological disease, from May to July 2014, we collected peripheral blood mononuclear cells (PBMCs) from patients at the first medical examination and without drug therapy. The patients were divided into groups on the basis of the diagnosis. Group 2 (n=8) included patients suffered from mixed disorders such as myeloproliferative neoplasms (MPD) associated to monoclonal gammopathy of undetermined significance (MGUS). Group 3 (n=8) included patients with only MGUS, group 4 included 9 patients with only primary myelofibrosis (M). Healthy donors (Group 1, n=16) were considered as normal subject or calibrator in molecular analysis. Their PBMCs were used to perform relativegene expression profile of a transcriptome involved in apoptotic control, stem-cell differentiation, immune network, inflammation and leukemogenesis, in order to detect whether these may serve to label the patient groups. A multigene expression assay (47 genes) was carried out with the TaqM,an® Low Density Array Fluidic card. In Group 3 (MGUS), 6 genes were differentially expressed (BMI-1, FLT3, FZD1, FZD5, ICAM1, IMP3). Also, we compared variance, main value for each gene in each group and T-test that showed a significant differential expression pattern (p

Description: Abstract 5057 There is evidence in literature of the clinical and pathogenetic role of JAK2 V617F mutation in MPN, while its contribute as an additional thrombotic risk factor in MPN patients is still under discussion. The jak2 mutation has been identified as occult marker in several patients with splanchnic venous thrombosis (SVT); morover, 45% of patients with Budd-Chiari syndrome (BCS) and 34% of patients with portal vein thrombosis (PVT) have associated MPN. On the other hand, the majority of BCS patients without MPN present additional congenital or acquired thrombosis risk factors. The aim of the present study is to evaluate the incidence of SVT in MPN patients. We also evaluated the presence of other prothrombotic risk factors in MPN patients, in addition to the JAK-2 mutation. Out of the 460 Ph1 negative MPN patients observed in our center from January 2000 to January 2010 and retrospectively evaluated, 9 patients (7 females and 2 males; 2%) presented SVT. Six cases had Essential Thrombocythemia (ET), 2 Primary Myelofibrosis (PMF), and 1 Polycitemia Vera (PV). Five of the 6 cases with ET were females. Among the entire population of ET, SVT incidence was 3%. All the 9 patients diagnosed of SVT were JAK2 V617F positive and they were treated with antiaggregant and anticoagulation therapy; 6 received hydrossiurea. Seven patients had SVT before MPN diagnosis, 2 of them had splenectomy at diagnosis for surgical decision. In these patients developing SVT before MPN diagnosis no other major thrombotic event occurred during follow-up. The remaining 2 MPNs patients presented asymptomatic SVT diagnosed by imaging techniques routinely performed during MPN follow up. Interestingly, 75% of MPNs patients with SVT demonstrated at least one prothromobtic risk factor, such as factor V Leiden, Protein C deficiency, hyperhomocystinemia and 50% had 2 or more associated defects. MPNs patients without SVT (396) had a lower prevalence of prothrombotic risk factors and developed venous thrombosis in different anatomical sites: in these cases white blood cell count, platelets values and the presence of JAK2 V617F mutation correlate with the development of the thrombotic event. Conclusion. Even though SVT has a low incidence in MPNs patients, according to the results of the present retrospective study we suggest the potential benefit of searching for additional prothrombotic risk factors in the whole MPN population in order to prevent and/or properly treat this complication. Disclosures: No relevant conflicts of interest to declare.