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: 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 3631 Introduction: The erythroid differentiation defect observed in 5q– syndrome has been attributed to the RPS14 gene located within the CDR of the long arm of chromosome 5. We have recently demonstrated that RPS14 expression increases during lenalidomide treatment. However, haploinsufficiency of RPS14, which encodes ribosomal protein S14, does not explain clonal dominance. The expression of miRNAs, miR-145 (5q33.1) and miR-146a (5q33.3), in CD34+ bone marrow (BM) cells of individuals with MDS with deletion of the long arm of chromosome 5 (del(5q)) is lower compared to normal controls (Starczynowski et al, Nature Medicine, 2010). miRNAs are small noncoding RNAs that post-transcriptionally repress specific messenger RNA targets through interaction with the 3′ untranslated region (UTR). Loss of noncoding transcripts encoding miRNAs within the CDR may result in haploinsufficiency by loss of inhibition of their targets. Concurrent loss of both miR-145 and miR-146a resulted in activation of innate immune signalling through elevated expression of their respective targets, TIRAP and TRAF6. Furthermore, knockdown of miR-145 and miR-146a or overexpression of TRAF6 in mouse HSPC (Hematopoietic stem and progenitor cells) recapitulated features of 5q– syndrome, such as bone marrow dysplasia, anemia and thrombocytosis. We present preliminary results of changes in miRNA expression in IPSS lower-risk MDS with del(5q) during treatment with lenalidomide. Methods: A prospective single-arm trial investigating the efficacy and safety of lenalidomide in 46 patients with MDS with del(5q) with/without additional cytogenetic abnormalities and Hb 〈 10 g/dL. Lenalidomide was administered orally at a starting dose of 10 mg/day for a maximum of 12 months. When necessary, dosing was reduced to 5 mg/day or 5 mg on alternate days. Bone marrow assessments were performed at baseline and every 3 months, thereafter. For the evaluation of miRNA-145 and miRNA-146a in patient samples, 300 ng/μl of miRNAs were isolated in each purified BM sample by using mirVana™ miRNA Isolation Kit-Ambion and TaqMan miRNA Array Analysis was performed to determine the expression of miRNAs (7900HT Sequence Detection System Applied Biosystems). Patient BM-miRNAs were calibrated with miRNAs from BM of healthy volunteer donors. It was assumed that BM expression value of each calibrator miRNA was 1 unit. RPS14 gene 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: Four patients have been evaluated (1 M, 3 F; ages 65, 66, 73 and 76 years, respectively) at baseline and after 12 weeks. At baseline, 2 patients were RBC-transfusion dependent. One patient had one additional cytogenetic abnormality (+8 in 15% metaphases). All patients obtained an erythroid response by week 12: mean Hb values significantly increased from 8.4 ± 0.9 at baseline to 11.6 ± 0.9 g/dL (p=0.01). All patients obtained a cytogenetic response, 2 of which were complete. miRNA-145 and miRNA-146a expression were both low at baseline and significantly increased by week 12 (Table). Conclusions: Preliminary results confirm that, in IPSS lower-risk MDS with del(5q), miRNA-145 and miRNA-146a expression is low. Lenalidomide treatment is associated with erythroid responses and cytogenetic remissions concurrent with significant increases in miRNA-145 and miRNA-146a expression. Disclosures: Oliva: Celgene: Consultancy.

Description: BACKGROUD: Nilotinib, a potent 2nd generation tyrosin kinase inhibitor, is efficacious in the treatment of chronic myelogenous leukemia (CML). Impaired glucose metabolism represents one of the most frequently observed adverse events and several clinical trials, reported a high diabetes incidence during treatment with this drug. The mechanism of this side effect is poorly understood, but recently has been hypothesized an increased postreceptorial insulin resistence. Moreover, "in vitro"results indicated that c-ABL is involved in the insulin receptor signaling pathway. A large number of genetic variants associated with Type 2 diabetes (T2D) are implicated in beta-cell function but the role of common genetic variants associated with insulin resistance in the etiology of T2D, remains poorly documented. Scott R. and al. (Diabetes 2014) recently identified 10 multiple single nucleotide polymorphisms (SNPs) associated with insulin resistance and created a genetic risk scores (uGRS) as complementary tool to for insulin resistance. AIM of the study was to identify whether this uGRS could be a valid prognostic tool in identifying patients developing diabetes during Nilotinib therapy PATIENTS AND METHODS:45 patients (19 males) were included in the study. Twenty-four were treated with Nilotinib in first-line and 21 as second line (10 for resistance, 8 intolerance and 3 for other reasons). None of the subjects studied had abnormal blood glucose or took any antidiabetic drug before Nilotinib treatment. We genotyped all patients with the GRS created by Scott R. including those in, or near, the IRS1, GRB14, ARL15, PPARG, PEPD, ANKRD55/MAP3K1, PDGFC, LYPLAL1, RSPO3, and FAM13A1 genes that have primary effects on subcutaneous adipocyte function. Also we added 2 new variants, one for TCF7L2 gene, associated with insulin secretion and another in FTO gene, whose effect on insulin levels is mediated by BMI. The uGRS was calculated, as previously reported, by summing the number of risk alleles across the 12 variants (0 for risk allele absent, 1 for heterozygosity and 2 for homozygosity for risk allele). A cut-off point for uGRS, maximizing predictivity and sensitivity of the score was calculated using Youden's index. Diabetes and impaired fasting Glycaemia were defined using the American Diabetes association (ADA) criteria. Data were reported as median and range, RESULTS:Age at diagnosis was 45(18-82) years, the Sokal was low in 16 (42%), intermediate in 12 (26%) and high in 17 (32%) patients. During treatment and subsequent follow up of 45(7-127) months, 28 patients remained euglycemic, 5 (2 treated in the first line) developed diabetes after 14(1-32) months and 12 (6 treated as first line) developed IFG in 6 (1-12) months. No IFG patients developed an overt diabetes in the subsequent follow-up of 60.3 (33-102) months. We calculated a cut-off point of 12 for uGRS. When the 28 euglycemic patient were compared with 5 patients becoming diabetics after Nilotinib, uGRS showed a sensibility of 100% and a specificity of 46% in predicting diabetes. Consequently, the positive predictive valuewas 25% where the negative predictive values 100%. When the 28 euglycemic patients were compared with the 12 patients developing IFG after Nilotinib, the sensibility and specificity of uGRS was low: 50% and 46% respectively. CONCLUSIONS: A Genetic risk score for insulin resistance showed high specificity and a strong negative predictive values when used to identify CML patients treated with Nilotinib developing an overt diabetes. Further studies in lager populations are needed to confirm this predictivity that can be used in clinical practice to tailor the best anti TKI therapy. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2622 Poster Board II-598 Introduction: Genetic alterations reported in myelodysplastic syndromes (MDS) are not disease-specific and the underlying molecular causes of the disease remain poorly understood. It has been suggested that one or more of the genes mapping within the commonly deleted region of the 5q syndrome, together with other distant genes, may be critical to the development of the 5q syndrome. Potential candidate genes have been identified including the tumor suppressor gene SPARC, and the ribosomial protein gene, RPS14. Haploinsufficiency of RPS14 has been demonstrated and recent evidence indicates RPS14 as a causal gene for the 5q syndrome. Lenalidomide has proven efficacy in MDS patients with del(5q). Rapid and durable responses include transfusion-independence, with a rise in Hb, suppression of the 5q-deletion clone and improvement in bone marrow morphologic features. Methods: In a multicenter Italian phase II trial to evaluate safety, changes in quality of life and efficacy of lenalidomide in primary MDS patients with del(5q) and low or Int-1 risk IPSS, we investigate changes in bone marrow cytogenetics and gene expression patterns during treatment. The starting dose of lenalidomide is 10 mg p.o once daily on a continuous daily schedule for a maximum of 12 months. Dosing is based upon clinical and laboratory findings. Bone marrow cytogenetics and gene expression profiling are performed on study entry and every 12 weeks up to end of study (week 52). Gene expression assays of 51 candidate genes from the published literature and genomic databases have been selected and are carried out with TaqMan® Low Density Array Fluidic card (TaqMan® Human Array, Applied Biosystems, Foster City, CA, USA) based on Applied Biosystems PRISM® 7900HT comparative dd CT method, according to manufacturer's instructions. Using an 18S mRNA gene pre-designed assay from Applied Biosystems to detect the expression of the housekeeping gene 18S in each sample, target gene expression is normalized with 18S gene expression derived from a bone marrow pool of normal healthy subjects and for each sample the ratio between the target and 18S are expressed. Results: Baseline values for 23 patients (mean age 73 ± 10 years) are available and 16 have been re-evaluated after 12 weeks. Mean Hb was 8.6 ± 0.9 g/dL and 20 patients were transfusion-dependent. Seven patients had additional cytogenetic abnormalities. At baseline, RPS14 was under-expressed in 19 out of 21 patients evaluated. After 12 weeks RPS14 was re-evaluated in 13 patients: all had erythroid responses and RPS14 increased significantly from 0.07 (IQ Range 0.03–0.13) to 76.1 (0.73– 304.0, p=0.002). SPARC expression was under-expressed in 15/23 patients and variations during treatment were not significant. Baseline FAS gene was under-expressed in all patients and increased above reference values (p=0,006) after 12 weeks in 7/14 cases. IL7R was over-expressed in all patients at baseline (median 3263.3, IQ range 1998.3–5027.1) and was significantly reduced after 12 weeks (median 0.17, IQ range 0.05–2.20, p

Description: Background: Diagnosis of a prefibrotic state (MF0) presents histological diagnostic difficulties. MF0 has a worst prognostic impact than Essential Thrombocythemia (TE) as regard the thrombotic risk and a higher risk towards Idiopathic Myelofibrosis (MI) and acute leukemia evolution. For this reason it is very useful to identify this group of patients. Aims: The aim of this study was to evaluate clinical and hematological impact of the mutational status in patients with an MF0 diagnosis. Methods: A retrospective chart review was performed from January 2010 to July 2015 in a single center in 317 patients affected by Ph negative chronic myeloproliferative neoplasms. Polycythemia vera cases were excluded. Thirty-three patients have been classified as MF0 on the base of the bone marrow histological examination. Onset features include cytogenetics, blood counts, peripheral CD34-positive cells, spleen and liver size, thrombotic events (prior to and post-diagnosis) and thrombotic risk. Fragment analysis was performed to study exon 9 CALR mutation, Real-time PCR was applied for exon 14 JAK2 V617F mutation and Sanger sequencing was used to identify exon 10 MPL mutations for 505 and 515 codons. According to genetic results, patients were identified into four groups according to a positivity for JAK2, MPL, CALR and for triple negativity. Results: The 33 patients with MF0 were: 11 (33%) JAK2 positive, 10 (30%) CALR positive, (10) 30% triple negative and 2 (7%) MPL positive. The latter group was not further considered for analysis due to the low number of cases. Eighty percent of CALR positive patients had a deletion on the exon 9 of the gene (8 del52bp and 2 del46bp), while 20% had the type 2 mutation (ins5bp). The average of JAK2 allelic burden was 20%. The 3 groups of patients were comparable for age, white blood counts and hemoglobin values. There was a female prevalence (23 vs 10 males). Platelet count was higher (median 875.500 103 µl, interquartile range 303.000 103 µl , p = 0.008) in CALR positive patients compared to JAK2 positive (median 569.000 103 µl, interquartile range 433.000 103 µl) and to triple negative patients (median PLT count 629.500 103 µl, interquartile range 378.250 103 µl). Noteworthy, bone marrow cytogenetic exam was normal in all patients. JAK2 positive patients had a larger spleen compared to the other two groups. Pre-diagnosis thrombotic events were exclusive for JAK2 positive patients and absent in the other groups. Triple negative patients do not have a negative prognostic impact for the thrombotic risk. Conclusions: CALR deletion could be considered as an MF0 marker and should be included in diagnostic work-up. JAK2 positivity in MF0 is associated with a high thrombotic risk. Disclosures No relevant conflicts of interest to declare.

Description: Budd-Chiari syndrome (BCS) and Non-Cirrhotic Extra-Hepatic Portal Vein Obstruction (EHPVO) are two rare vascular diseases of the liver. Inherited and acquired disorders including Myeloproliferative Disorders (MPD) have been frequently reported associated with these diseases, although the large majority of cases is still defined as “idiopathic disease”. The somatic mutation in the tyrosine kinase JAK2 has been described in MPD, specifically in the majority of patients with polycythemia vera (PV), in about half of cases with essential thrombocythemia (ET) and in one third of idiopathic myelofibrosis (IMF), suggesting its pathogenetic role in these diseases. Since the diagnosis of MPD is often difficult in patients with BCS or EHPVO because of spleen enlargement and secondary pancitopenia that can mask erythrocytosis and thrombocytosis, recent observations have included in their diagnostic work-up the analysis of JAK2 mutations. In fact, recently, a high prevalence of JAK2 mutations has been described in splanchnic vein thrombosis. The aim of this study was to evaluate the prevalence and the levels of JAK2 mutation in the patient population affected by BCS and EHPVO followed at our Hepatology Division. The JAK2 mutation was evaluated by allele-specific real-time TaqMan polymerase chain reaction. We enrolled in this study 18 patients (median age: 38 ± 9.8 years) affected by BCS (7 patients), EHPVO (10 patients), or both (1 patient). In 9 of them the JAK2 mutation was absent, while it was demonstrated in the remaining cases. Among these, an heterozygous deletion (JAK2 mutation ranging between 2.7% and 48%) was detected in 5 and an homozygous deletion (55.7%–88%) in 4. A diagnosis of PV was made in 2 patients with JAK2 heterozygous deletion affected by BCS and EHPVO. The 3 IMF were all characterized by homozygous deletions: 1 had BCS and 2 EHPVO. In the remaining 3 patients, in the absence of other diagnostic criteria, only the bone marrow biopsy revealed the presence of initial marrow fibrosis: grade 0–1 (2 patients both affected by EHPVO with heterozygous and homozygous deletion) and grade 1 (in a heterozygous patient affected by EHPVO). In summary, the JAK2 mutation was demonstrated in half of the cases with vascular liver diseases, regardless of the JAK mutation levels. The marrow fibrosis was, in these cases, frequently associated with both BCS and EHPVO, suggesting a common potential pathogenetic role in these vascular diseases of the liver.

Description: Abstract 50623 Background: Bone marrow karyotype in myelodysplastic syndromes (MDS) is essential to define the prognosis and to guide treatment decisions, including targeted therapies. Due to the lack of an extensive national MDS registry in Italy, epidemiological data on MDS, including cytogenetics, throughout the territory is unknown. Objective: We evaluated the incidence of cytogenetic abnormalities amongst newly diagnosed MDS patients in in 2 southern Italian regions (Calabria and Puglia). Methods: A pilot project, denominated ANDROMEDA (ANalysis of cytOgenetics alteRatiOn in the MyEloDysplAstic syndromes) was developed in 17 centers to offer a service of conventional cytogenetic analysis for all consecutive patients undergoing diagnostic evaluation for cytopenia and suspected MDS between January 1 and December 31, 2011. The study conformed to the ethical standards set out in the Declaration of Helsinki and was approved by institutional review boards at each participating center. Patients were required to provide their written informed consent. Clinical characteristics of patients and bone marrow morphology, iron staining and histology were registered. Bone marrow samples were centralized for standard cytogenetic studies and fluorescence in situ hybridization to two dedicated genetics laboratories (one for each region), blind to patients' data. Results: Two hundred and thirty-five patients were evaluated and MDS diagnosis was confirmed in 220 cases (88. 3%), according to WHO criteria. The overall incidence of clonal chromosome abnormalities detected by conventional analysis was 36. 9%. Single abnormalities included +8 (13 cases, 5. 8%), del(5q) (12 cases, 5. 4%), –Y (11 cases, 5. 0%) and del(7)/-7 (4 cases, 1. 8%). Complex karyotypes were detected in 18 (8. 1%) cases. Among all cases only 10 (4. 5%) bone marrow samples were not evaluable for cytogenetic analysis. FISH revealed additional abnormalities not identified by conventional analysis only in 3 (1. 3%) out of 72 cases. Patients were classified in WHO subtypes: 39. 2% refractory cytopenia with unilineage dysplasia (RCUD), 1. 5% refractory anemia with ring sideroblasts (RARS), 32. 5% refractory cytopenia with multilineage (RCMD), 10. 8% refractory anemia with excess of blast-1 (RAEB-1), 9. 3% refractory anemia with excess of blast-2 (RAEB-2), 4. 1% MDS with deletion 5q (MDS 5q-) and 2. 6% MDS unclassifiable (MDS-U). Conclusions: These preliminary results demonstrate that the incidence of abnormal karyotype patterns and WHO subgroups in MDS patients in Southern Italy is comparable with that described in other geographical areas. It is confirmed that conventional cytogenetic analysis is a standard in the diagnostic workup of MDS of patients with a suspected myeloid malignancy in order to identify primary abnormalities and prognostic models. Disclosures: No relevant conflicts of interest to declare.