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To identify prognostic factors affecting thrombosis-free survival (TFS) and overall survival (OS), we report the experience of a Regional cooperative group in a real-life cohort of 1,144 patients with essential thrombocythemia (ET) diagnosed from January 1979 to December 2010. There were 107 thrombotic events (9.4%) during follow-up [60 (5.3%) arterial and 47 (4.1%) venous thromboses]. At univariate analysis, risk factors for a shorter TFS were: age >60 years (P 60 years (P 15 × 109/l (P = 0.0370) were independent risk factors. Previous thrombotic events in ET patients are crucial for TFS but their importance seems related not to the occurrence per se but mainly to the interval between the event and the diagnosis. Am. J. Hematol. 89:542–546, 2014. © 2014 Wiley Periodicals, Inc.

NADP-dependent enzyme isocitrate dehydrogenase (IDH) mutations, IDH1 and IDH2, have been described in acute myeloid leukemia (AML) using next generation sequencing approaches. IDH2 mutations are heterozygous; they alter a single arginine residue at position 140 or 172 and have distinct prognostic significance. The current detection methods of IDH2 mutations are laborious and time consuming as they require DNA sequencing. Herein, we report a new allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) method to detect the IDH2 mutations. Analysis of leukemic DNA samples from 120 AML patients enabled to identify IDH2 mutations in 22 cases which were confirmed by direct DNA sequencing. Of these, 17 harbored IDH2 (R140Q) and 5 IDH2 (R172K) mutations. Serial dilution experiments showed that the assay enable to detect mutations in 10⁻³ dilutions. Our ASO-PCR method appears useful for routine diagnostic screening of these prognostically relevant alterations in AML and may be conveniently included in the diagnostic workup.

Overexpression, polymorphisms, and mutations of the WT1 gene have been reported in several human tumors including acute myeloid leukemia (AML) and variably correlated with prognosis. Acute promyelocytic leukemia (APL) represents the AML subset disclosing higher WT1 expression levels; however, no WT1 studies specifically focused on APL have been conducted. We screened for the presence of mutations, SNP rs16754, and expression levels of WT1 gene in 103 adult patients with newly diagnosed APL. Fms-like tyrosine kinase (FLT3) mutations were analyzed as well. WT1 mutations were identified in four (4 %) patients. At least one copy of the minor SNP rs16754 allele (WT1 AG or WT1 GG) was detected in 30 (29 %) patients. Six patients (6 %) were homozygous for the minor allele (WT1 GG ) and this genotype was associated with higher WT1 mRNA copies (p = 0.018). FLT3 mutations were found in 37 % of patients and correlated with high WT1 mRNA expression (p = 0.004). Patients heterozygous or homozygous for the minor allele and patients homozygous for major (WT1 AA) allele did not differ in terms of presenting features. In adult APL, WT1 gene mutational and polymorphic profile shows similarities with pediatric AML rather than with adult AML.

Thrombotic events are major complications in patients (pts) affected by Essential Thrombocytemia (ET) and Polycytemia Vera (PV). To compare thrombotic risk in these 2 groups, we evaluated retrospectively our database of 1249 ET and 623 PV pts diagnosed and followed in 11 hematological centers in the Latium region between 1/1980 and 12/2010: the diagnosis was done according to PVSG, WHO 2001 and 2008criteria based on the time of first observation. Baseline features of ET pts: 797F/452M,median age 62.9 yrs (range 19-96),median WBC count 8.8 x 109/L (range 1.2-57.7), median PLT count 812 x 109/L (range 457-3582), median Hb level 14.0 g/dl (range 6-20.5), JAK-2V617F positivity 59.7% with a median allele burden of 19,6% (range 0.2- 99.9), spleen enlargement in 18.7% of pts, previous thrombosis223/1239 evaluable pts (17.9%) [arterial 176/223 (14.1%), venous 47/223 (3.8%)]. Baseline features of PV pts: 289F/334M, median age 63.0yrs (range 21-91), median WBC count 10.1 x 109/L (range 3.5-37.6), median PLT count 457 x 109/L (range 169-1790), median Hb level 18.2 g/dl (range 10.5-24.8), JAK-2V617F positivity 94.3% with a median allele burden of 59.1% (range 0.3-99.9), spleen enlargement in 42% of patients, previous thrombosis 146/617 evaluable pts (23.7%)[arterial 114/617 (18.5%), venous 32/617 (5,2%)].in the ET cohort, after a median follow-up of 7.7 yrs, thrombotic complications were seen in 107/1141 evaluable pts (9.4%) [arterial60 (5.25%), venous 47 (4.11%)]; in the PV cohort, after a median follow-up of 8.5 yrs, thrombotic complications were seen in 107/623pts (17.2%) [arterial 67 (10.8%),venous 40 (6.4%)].All common risk factors for thrombosis were evaluated in multivariate analysis, searching the cut-off number for continuous variables with ROC curves. The significant variables at multivariate analysis for ET and PV pts are shown in the table; age, previous thromboses and spleen enlargement were risk factors in ET pts, while previous thromboses and JAK-2V617F allele burden were risk factors in PV pts. PLT count above ROC value seemed to be a protective factor in both cohorts. In conclusion, in contrast with the tendency to evaluate in a similar manner the thrombotic risk of PV and ET, data from our retrospective database showed that these 2 groups should be considered populations with different risk factors for thrombosis. Table 1.Putative prognostic factorsPolycythemia VeraEssential ThrombocythemiaHR95% C.I.pHR95% C.I .pPrevious thromboses2,311,13 - 4,740,021,871,08 -3,230,026Age ≥ 60 y1,540,79 - 2,990,211,901,18 - 3,060,009JAK2V617FPV: allelic burden ≥ 81% ET: pos1,951,03 - 3,710,040,760,48 - 1,210,25Plt countPV ≥ 452.109/L ET ≥ 944.109/L0,490,25 - 0,950,040,520,31 - 0,890,017Spleen enlargement0,670,34 -1,310,241,711,02 - 2,890,04CV risk factors (at least 1)0,920,41 - 2,030,830,870,51 - 1,490,62WBCPV ≥ 10,175.109/L ET ≥ 9,630.109/L1,090,57 - 2,080,801,410,89 -2,260,15 Disclosures No relevant conflicts of interest to declare.

The protective effect of higher platelet count at diagnosis of Essential Thrombocythemia (ET) was reported in some papers (Carobbio A. 2011, Palandri F. 2012, Montanaro M., 2014). As at our knowledge, there is no study specifically addressing this point; in this retrospective analysis we have examined 1201 ET patients (pts) followed in 11 Hematological centers of our region from 1/1978 to 12/2010. The diagnosis of ET was made with PVSG, WHO 2001 and WHO 2008 criteria, respectively, according to the period of 1st observation. The main features of our cohort were as follows: median age 62,9 yrs (19-96), male/female 435/766 (36.2%/63.8%), median WBC count 8,8 x 106/L (1.2-57.7), median Hb level 14.0 g/dl (6.0-20.5), median platelet count 813 x 106/L (457-3582), JAK-2V617F mutation in 498/834 performed pts (59,7 %) with a median allele burden of 19.6% (0.2-99.9%), spleen enlargement in 226 pts (18.7%), previous thrombosis in 17.9% of pts (arterial 14.1%, venous 3.8%). The median follow-up of the entire cohort was 7.75 yrs. Thrombosis-free survival curves were plotted according to Kaplan-Meier method and independent risk factors were identified with the Cox proportional-hazards method. At the multivariate analysis, negative prognostic factors for TFS resulted: previous thrombotic events (p= 0.012), age ≥60 yrs (p= 0.008) and spleen enlargement (p= 0.039): on the contrary, platelet count ≥ 944.109/L resulted a protective factor for TFS [p= 0.031 with an HR 0,57 (C.I. 95% 0,35-0,95)]. Receiver operating characteristic (ROC) analyses based on thrombotic events during follow-up were used to identify the baseline platelet count of 944 x 109/L as the best threshold for predicting thrombotic events. Thrombotic events according to this cutoff were 40/384 (10.3%) in pts with platelet count ≥ 944 x 109/L and 109/817 (13.3%) in pts with platelet count < 944 x 109/L. The sites of thrombosis are reported in the table. A comparison of the main features in these two populations showed that pts with PLT count < 944 x 109/L were older (median age 60.4 yrs vs 57.1 yrs, p= 0.016), had a lower median WBC count (8.8 x 109/L vs 10.6 x 109/L, p< 0.0001), an higher median Hb level (14.1 g/dL vs 13.6 g/dL, p< 0.0001) and an higher rate of JAK-2V617F mutation (67.2% vs 41.6%, p< 0.0001); no differences were observed between the two groups as to thrombotic events before diagnosis, spleen enlargement and cardiovascular risk factor (p=NS). As to the treatment, both groups resulted equally treated with anti-aggregant agents (84,6% vs 87,4%, p= 0,76) while in pts with platelet count Disclosures No relevant conflicts of interest to declare.

Polycythemia Vera (PV) is a myeloproliferative neoplasm characterized by trilinear marrow expansion and an increased susceptibility to thrombo-embolic complications. Data of 623 patients (pts) followed in 11 Hematological centers of our region from 1978 to December 2010 were collected in our database. The diagnosis was made according to PVSG criteria, WHO 2001 and 2008 criteria, respectively, based on the year of diagnosis. The main epidemiological and clinical features of all pts are reported in table. Of 623 pts, 161( 25,8%) died, 87 (13,9%) were lost to follow up and 375 (73,1%) were alive at the time of evaluation. The median follow up was 8.5 years. The thrombotic events during follow-up were 107 (17,2% of 622 evaluable pts): the arterious events were 67 (10.8%), the venous were 40 (6,4 %). The rate of thrombosis (patients/year) was 1,71 %. At the univariate analysis, the risk factors for thrombosis-free survival (TFS) at diagnosis that resulted statistically significant were: age (> 60 yrs, p= 0,036), WBC (> 10.2 x 109/L, p= 0,034), previous thrombosis (p< 0,0001). The presence of cardiovascular risk factors, Hb (>18.2 g/dL), PLT count (either > 457 x 109/L or >1000 x 109/L), JAK2V617F allele burden > 59.15% and spleen enlargement, did not reach the cut-off value of significance. At multivariate analysis with the Cox proportional hazards model method, age (> 60 yrs, p= 0,049) previous thrombotic events (p< 0.0001) and platelet count < 457 x 106/L (p= 0.019) maintained an independent prognostic value (p< 0,05), while WBC count (> 10.2 x 109/L, p =0,065) did not. The risk factors significant for overall survival (OS) at univariate analysis were: age > 60 yrs (p 10.2 x 109/L (p< 0,0001), previous thrombosis (p< 0,0001). diabetes (p= 0,0008), platelet count< 457 x 109/L (p= 0.013) and spleen enlargement (p= 0.02); Hb level < 18,2 gr% showed only a trend of significance (p= 0.056), while allele burden of JAK2 > 59.15% and the presence of at least 1 CV risk factor did not reach the cut-off value of significance. At multivariate analysis, age (p< 0,0001), WBC count > 10.2 x 109/L (p< 0,0001), previous thrombosis (p= 0,0004), diabetes (p= 0.0035) and Hb level < 18,2 gr% (p= 0.0078) maintained their independent prognostic value on OS; in contrast platelet count < 457 x 109/L and spleen enlargement lost their prognostic significance. In conclusion our retrospective analysis of a large series of PV confirm the prognostic value of age and previous thrombosis on TFS and OS, while seems to exclude any impact of spleen enlargement and high Hb level. Interesting, PLT count and Hb below median value resulted as independent risk factors for TFS and OS, respectively. Table CARACTERISTICS Evaluable N. VALUE Number of patients 623 Age years: median, (range) 63 (21 - 91) Gender, F/M : number, (%) 289 (46,4) / 334 (53,6) WBC x 109/L: median, (range) 582 10,2 (3.5-37.6) Hb g/dL: median, (range) 580 18,2 (10,5-24,8) Htc ; median, range (%) 581 56 (36-78) Plt x 109/L: median, (range) 587 457 (169-1790) JAK2 V617F : mutated / performed, (%) 386 364/386 (94,3%) JAK2 V617F quantitative (%): median, (range) 252 59,15 (0,3-99,9) Splenomegaly: number, (%) 588 247/588 (42 %) Epatomegaly: number, (%) 606 167/606 (27,5%) Disclosures Breccia: novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy.

Abstract 5062 Thrombotic events occurring before or at diagnosis of Essential Thrombocythemia (ET) are a worldwide recognized prognostic factor for the incidence of thrombosis during the follow-up of ET patients: however, these previous thrombotic events have been considered on the whole in the vast majority of studies, without further characterization. Among 1063 ET patients collected in the retrospective database of the “Gruppo Laziale SMPC Ph-”, we revised 186 cases with a previous thrombosis and a known data of occurrence, to evaluate the role of the interval from previous thrombotic episode and the diagnosis of ET. In 95 patients (51. 1%) previous thrombotic event occurred < 24 months before diagnosis of ET (group A) while in 91 patients (48. 9%) thrombosis occurred ≥ 24 months before diagnosis of ET (group B). Clinical features of patients at diagnosis are shown in the Table: GROUP A GROUP B p Gender (M/F) 40/55 42/49 0.636 Median age (yrs) (Interquartile range) (IR) 64.1 (52.7–71.8) 70.9 (61.0 – 78.0) 0.001 Hb median (g/dl) (IR) 13.9 (12.5 – 14.7) 14.2 (13.0 – 15.2) 0.136 PLT median (x 109/l) (IR) 800 (669 – 1066) 778 (652 – 926) 0.453 WBC median (x 109/l) (IR) 9.2 (7.8 – 11.3) 8.6 (7.1 – 10.8) 0.121 Median interval diagnosis – CHT (mos) (IR) 0.9 (0 – 7.0) 1.7 (0.4 – 5.6) 0.194 *CV risk factors (n°/%): 0.454 0 20 (21.0) 17 (18.6) 1 42 (44.2) 44 (48.3) ≥ 2 33 (34.8) 30 (33.1) Type of previous thrombosis (n°/%): 0.873 Arterial 78 (82.1) 67 (73.6) Venous 17 (17.9) 24 (26.4) * Cardiovascular (CV) risk factors at diagnosis were considered the presence of arterial hypertension, diabetes, smoking attitude, and hypercholesterolemia. In the group A, 9 out 95 patients (9. 4%) reported thrombotic episodes (5 arterial and 4 venous) during follow-up compared to 23 out 91 patients (25. 2%) (13 arterial and 10 venous) in the group B (p=0. 004). Consequently, patients of group B had a significantly higher cumulative risk of thrombosis compared to patients of group A (p=0. 0029, CI95% 1. 5 – 6. 1, RR 3. 04). In addition, it is worth of note that there was no difference in the cumulative risk of thrombosis between the patients of group A and the 877 patients without previous thrombotic events (p=0. 303, CI95% 0. 64 – 3. 21, RR 1. 24) In conclusion, the prognostic role of a previous thrombotic event in ET patients seems to be related not to the occurrence per se of the event but mainly to the interval between the event and the diagnosis of ET. Disclosures: Tafuri: Sigma Tau Pharmaceuticals: Research Funding.

Abstract 3854 Increasing age is a well-recognised risk factor for thrombotic events in patients with Essential Thrombocythemia (ET): however, few data exist on the role of other clinical and biological features in different age groups. To address this issue, we analysed retrospectively 1090 ET patients (M/F 403/687, median age 63 years, IR 17 – 96) diagnosed at 11 Hematological Institutions in the Lazio region from 1980 to 2010 and with a median period of follow-up of 84 months (IR 1 – 371). Based on the commonly adopted age threshold, 480 patients (44 %) were < 60 years (Group A) and 610 (56 %) were ≥ 60 years (Group B). Clinical and biological features as well as cardiovascular risk factors analyzed for the impact on the thrombotic risk in the two age groups are reported in the Table.Group A < 60 yearsGroup B ≥ 60 yearsPutative risk factorsRisk ratio (95% CI)P valueRisk ratio (95% CI)P valueM/F167/3132.68 (1.03–6.94)0.0029236/3741.12 (0.17–2.59)0.73WBC median (range) x 109/l8.9 (4.29–22.35)0.387 (0.149–1,004)0.06458.9 (1.2–57.7)0.79 (0.41–1.47)0.445PLTS median (range) x 109/l837 (451–3582)0.37 (0.258–1.70)0.66802 (450–3104)0.52 (0.28–0.99)0.0052Hb median, g/dL (range)14.1 (6.0–18.4)0.86 (0.33–2.24)0.76914.0 (7.0–17.8)0.87 (0.45–1.67)0.674*JAK-2 mutational status: wild type/mutated (%)53.2/46.81.57 (0.50–4.87)0.4434.1/65.90.498 (0.17–1.48)0.209Previous thrombotic events: n° (%)· All events72 (15)2.18 (0.59–7.96)0.12149 (24.4)3.01 (1.38–6.57)0.0004· within 24 months from diagnosis48 (10)1.43 (0.19–10.4)0.7464 (10.5)0.506 (0.18–1.39)0.189· within 60 months from diagnosis60 (12.5)NA0.5191 (14.9)0.323 (0.11–0.95)0.023Cardiovascular risk factors: Y/N %○ Arterial hypertension41.7/58.31.68(0.64–4.36)0.2880.7/19.30.96 (0.36–2.57)0.935○ Diabetes10.2/89.81.11 (0.23–5.15)0.8925.0/75.01.09 (0.38–3.11)0.86○ Smoking attitude45.6/54.42.78 (1.01–7.65)0.06758.3/41.71.04 (0.35–3.09)0.94○ Hyperlipidemia31.0/69.03.11(0.917–10.592)0.03951.6/48.42.31 (0.70–7.55)0.203 In Group A, 39 patients (8.1%) had at least one thrombotic event during follow-up; there were 20 (51.3%) arterial thrombosis and 19 (48.7%) venous thrombosis. In Group B, 63 patients (10.3%) had at least one thrombotic event during follow-up; there were 38 (69.4%) arterial thromboses and 25 (39.6%) venous thromboses. In group A univariate analysis for thrombosis-free survival performed by Kaplan-Meier method, disclosed a significant impact of male gender (p=0.0029, CI 1.03–6.94, HR 2.68), > 2 cardiovascular risk factors (p=0.0002, CI 1.87 – 190, HR 18.94) and isolated hyperlipidemia (p=0.039, CI 0.917 – 10.59, HR 3.11), while previous thrombotic events had no significant impact (p=0.27). By contrast, the presence of a previous thrombotic event was the only feature with a significant impact on thrombotic risk in Group B (p=0.0004, CI 1.38 – 6.55, HR 3.01). WBC and PLTS values at different cut-off levels as well as JAK-2 mutational status did not have any impact on thrombosis in either age groups. However, in group B, we observed a trend (p=0.052, CI 0.28–0.99, HR 0.52) towards a protective effect of higher PLTS values (> 800 × 109/l). In conclusion, our data seem to reinforce the need of a different thrombotic risk assessment in distinct age groups: in particular, younger patients could benefit from early recognition and treatment of well-known cardiovascular risk factors. Disclosures: No relevant conflicts of interest to declare.