Anagrelide Research Articles

Leukemic transformation and second cancers in 3649 patients with high-risk essential thrombocythemia in the EXELS study

EXELS, a post-marketing observational study, is the largest prospective study of high-risk essential thrombocythemia (ET) patients, with an observation time of 5 years. EXELS found higher event rates of acute leukemia transformation in patients treated with hydroxycarbamide (HC). In the current analysis, we report age-adjusted rates of malignant transformation from 3460 EXELS patients exposed to HC, anagrelide (ANA), or both. At registration, 481 patients had ANA treatment without HC exposure, 2305 had HC without ANA exposure, and 674 had been exposed to both. Standard incidence ratios (SIRs) were calculated using data from the Cancer Incidence in Five Continents database to account for differences in age-, gender-, and country-specific background rates. SIRs for acute myelogenous leukemia (AML) were high in ET patients. SIRs for AML were high in HC-treated patients, but AML was rare in ANA-treated patients; no cases of AML were found in patients only treated with ANA. No statistically significant difference was seen between SIRs for ANA and HC treatment for AML or skin cancer. SIRs for other cancers were similar in the HC and ANA groups and close to 1, indicating little difference in risk. Although statistically inconclusive, this study strengthens concerns regarding possible leukemogenic risk with HC treatment. (NCT00202644)

Role of treatment on the development of secondary malignancies in patients with essential thrombocythemia.

Aim of this study is to explore the role of different treatments on the development of secondary malignancies (SMs) in a large cohort of essential thrombocythemia (ET) patients. We report the experience of a regional cooperative group in a real‐life cohort of 1026 patients with ET. We divided our population into five different groups: group 0, no treatment; group 1, hydroxyurea (HU); group 2, alkylating agents (ALK); group 3, ALK + HU sequentially or in combination; and group 4, anagrelide (ANA) and/or α‐interferon (IFN) only. Patients from groups 1, 2, and 3 could also have been treated either with ANA and/or IFN in their medical history, considering these drugs not to have an additional cytotoxic potential. In all, 63 of the 1026 patients (6%) developed 64 SM during the follow‐up, after a median time of 50 months (range: 2–158) from diagnosis. In univariate analysis, a statistically significant difference was found only for gender (P = 0.035) and age (P = 0.0001). In multivariate analysis, a statistically significant difference was maintained for both gender and age (gender HR1.7 [CI 95% 1.037–2.818] P = 0.035; age HR 4.190 [CI 95% 2.308–7.607] P = 0.0001). The impact of different treatments on SMs development was not statistically significant. In our series of 1026 ET patients, diagnosed and followed during a 30‐year period, the different therapies administered, comprising HU and ALK, do not appear to have impacted on the development of SM. A similar rate of SMs was observed also in untreated patients. The only two variables which showed a statistical significance were male gender and age >60 years.

Observational retrospective study of vascular modulator changes during treatment in essential thrombocythemia

Essential thrombocythemia (ET) patients are at risk of developing thrombotic events. Qualitative platelet (PLT) abnormalities and activation of endothelial cells (ECs) and PLTs are thought to be involved. Microparticles (MPs) can originate from PLTs (PMPs), ECs (EMPs), or red cells (RMPs). Previous studies have indicated that MPs contribute to ET pathophysiology. Endothelial modulators (eg, nitric oxide [NO], adrenomedullin [ADM], and endothelin-1 [ET-1]) are also involved in the pathophysiology of this condition. We hypothesized that treatments for reducing PLT count might also indirectly affect MP generation and endothelial activity by altering endothelial modulator production. The rationale of this study was that hydroxyurea (HU), a cytostatic drug largely used in ET, induces the production of a potent vasoactive agent NO in ECs. An observational retrospective study was designed to investigate the relationship between MPs, NO, ADM, and ET-1 in ET patients on treatment with HU, anagrelide (ANA), aspirin (ASA), and a group of patients before treatment. A total of 63 patients with ET diagnosis: 18 on HU+ASA, 15 on ANA+ASA, 19 on ASA only, and 11 untreated patients, and 18 healthy controls were included in this study. Blood samples were analyzed for MP (absolute total values) and functional markers (percentage values) by flow cytometry. PLT-derived MPs were studied using CD61, CD62P, CD36, and CD63, whereas endothelial-derived MPs were studied using CD105, CD62E, and CD144. Endothelial modulator markers (NO, ADM, and ET-1) were measured by ELISA. Total MP count was higher in the group treated with ANA+ASA (P<0.01). MP markers modified in ET patients returned to levels of healthy controls following treatment, in particular, in patients on ANA treatment. NO and ADM values were higher in the HU group (P<0.001). HU and ANA treatment also affected MP production in a cell origin-specific manner. HU and ANA, although acting via different pathways, have similar final effects. For instance, HU causes vasodilatation by increasing NO and ADM levels, whereas ANA impairs vasoconstriction by reducing ET-1. In conclusion, therapy with HU cytostatic drugs and ANA can reduce PLT count in ET, and also affect endothelial modulatory agents, with HU sustaining vasodilation and prothrombotic MP concentration, whereas ANA decreases vasoconstriction.

Low-Burden TP53 Mutations Occur in Chronic Phase of Myeloproliferative Neoplasms Regardless of Hydroxyurea Administration, Disease Type, and JAK2 Status

▪Myeloproliferative neoplasms (MPN) are characterized by abnormal proliferation of myeloid lineages and a tendency toward leukemic transformation. Inactivation of tumor suppressor TP53 has been repeatedly associated with MPN transformation into secondary acute myeloid leukemia (sAML), but no fully expanded TP53 mutated clones in chronic phase of MPN were reported. The link between TP53 mutations and widely used cytoreductive treatment by hydroxyurea (HU) still remains controversial. To which extent TP53 mutations represent a risk of disease progression is not known. We aimed to search for low-burden TP53 mutated subclones in chronic-phase-MPN patients and to correlate presence of these clones with therapy, disease course, and other clinical features.In total we analyzed 220 patients by ultra-deep next generation sequencing (NGS). We detected TP53 mutations in 39 (18 %) patients with variant allelic frequency of 0.1-16.3 % at the first examination. The analysis of 136 patients treated with hydroxyurea or other drugs (anagrelide (ANA), interferon α (IFN)) for more than 4 years, as well as a group of 84 patients untreated by cytoreductive drugs, showed that TP53 mutations occurrence in chronic phase is independent of hydroxyurea use, disease type, and JAK2/CALR/MPL status. Mutations were found in 17/72 (24 %) HU treated patients, in 11/64 (17 %) patients treated by other drugs, and in 11/84 (13 %) untreated patients. Median size of mutated clones was 0.5 % and was not influenced by previous treatment. In 10 patients we found more than one mutation.In patients harboring TP53 mutations, retrospective samples were examined if available to explore the clonal evolution of TP53 mutated clones. The respective TP53 mutations were found in 13/20 cases analyzed; out of them, in 4 cases the mutation was found even in a diagnostic sample. Follow-up samples were examined in 28 patients with TP53 mutations and the mutation burden changed during the monitored time in majority of patients; however, the expansion into dominant clone was observed in one patient only. When all data from retrospective and prospective analyses taken together (30 patients), the median follow-up was 7.2 years. TP53 mutation burden tended to increase in 14 patients. In 6 patients the mutation burden remained stable and in 4 patients it fluctuated. In 6 patients the mutated clone size decreased; out of them in 2 originally very low-burden mutations (0.2 %) were not detectable in samples taken 15 and 3.4 years later. We did not observe any correlations of different patterns in TP53 mutation changes with therapy or other clinical characteristics (disease type, driver mutation, time from diagnosis, treatment response).Further, we assessed the TP53 mutation impact on overall survival and leukemic transformation. The mutations did not negatively affect disease progression or overall survival either from diagnosis or from mutation identification. sAML developed in 2 patients with TP53 mutations 17.9 (treated with IFN) and 8.3 (treated with HU) years from diagnosis. In the latter patient, the sAML developed from a different clone as it was TP53-wt, JAK2-wt, although 2 TP53 mutated clones within JAK2 mutated clone were detected in chronic phase. On the other hand, we have observed another interesting case where the TP53 mutation burden grew rapidly from 10 % up to nearly 100 % during the follow-up. In contrast to published data, this patient did not show any clinical signs of disease progression for 2 years after the expansion and died of MPN unrelated cause.In summary, using highly sensitive method we showed that low-burden TP53 mutations are present in MPN chronic phase. Neither their presence nor their size is associated with previous therapy and has impact on overall survival or leukemic transformation. Monitoring of TP53 mutations during the disease course showed that their clonal development is rather variable; nevertheless, TP53 minor mutations may represent a pool for future clonal evolution.Supported by MZ CR-RVO (FNBr, 65269705), MUNI/A/1028/2015, H2020 692298, MZO AZV 15-31834A, 15-30015A, MEYS LQ1601 and LM2015064. DisclosuresGisslinger:Baxalta: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; AOP Orphan: Consultancy, Honoraria. Mayer:AOP Orphan Pharmaceuticals: Research Funding; Novartis: Research Funding. Kralovics:AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees.

The Use of Anagrelide in Myeloproliferative Neoplasms, with Focus on Essential Thrombocythemia.

Anagrelide (ANA) is a drug with specific platelet-lowering activity, used primarily in ET, registered as a second-line drug in essential thrombocythemia (ET) in Europe and in some countries as first-line therapy, in USA licensed by FDA for thrombocythemia in myeloproliferative neoplasms (MPN). The platelet-lowering efficacy is similar to that of hydroxycarbamide (HC), around 70 % complete response and 90 % partial response. Side effects are common, especially headache and tachycardia, but usually subside or disappear within a few weeks. Around 20 % of patients stop ANA therapy due to side effects or insufficient response. Studies of treatment patterns in Europe show that ANA is preferentially given to younger patients, probably because of the concern for a possible leukemogenic effect of the common first-line drug, HC. Only two randomized studies have compared the efficacy of ANA and HC in preventing thrombosis and haemorrhage, the larger of them showing a slightly better efficacy of HC, the other showing non-inferiority of ANA to HC. A recent observational 5-year study of 3600 patients shows a low and basically similar efficacy of ANA and other cytoreductive therapies in ET. ANA does not appear to inhibit fibrosis development, and probably due to its anticoagulation properties, the combination of ASA and ANA produces an increased rate of haemorrhage. Combination of ANA with HC or interferon (IFN) is feasible and effective in patients with insufficient platelet response to mono-therapy.

Treatment of high risk ET – data from the EXELS study

BackgroundThe Evaluation of Xagrid Efficacy and Long-term Safety (EXELS) study (NCT00567502) is the largest prospective observational cohort of high-risk patients with essential thrombocythemia (ET) reported to date. ObjectivesThe primary objective were safety and pregnancy outcomes of anagrelide (ANA) compared with other cytoreductive therapies (CRT). Secondary objectives included efficacy, measured by the incidence of thrombohemorrhagic events and platelet reduction. MethodsHigh-risk patients (≥1 of age >60 years, previous thrombotic event, platelet count >1000 × 109/L) with ET were enrolled across 13 countries in Europe between 2005 and 2009. Pts were required to be receiving CRT. Data, including events predefined in the protocol (PDEs), were collected every 6 months for 5 years for all patients. Event rates are presented as number of patients per 100 patient-years exposure and by treatment at time of event. Event rates are provided rather than p values due to the observational nature of the study. Preliminary final data are presented and final data, including platelet response and pregnancy results, will be available at ASH. Recently, results have remained stable and conclusions are not expected to change. Results3649 patients were categorized according to treatment at registration as follows: ANA (n=804), ANA + other CRT (n=141), other CRT (n=2666) and no CRT (n=38). Over 80% of patients received either hydroxycarbamide (HC) or ANA, and 69.8% of patients received antiaggregatory therapy. At registration, median age was lower in the ANA (55.5 years, range 18–89) and ANA + other CRT (59.0 years, range 22–88) groups vs the other CRT group (70.0 years, range 17–95).The arterial thrombotic event rate was similar in ANA (1.63) and other CRT (1.62) groups, whereas venous thrombotic event rates differed (0.35 vs 0.57). The major hemorrhagic event rate was highest in the ANA group, especially in patients also treated with anti-aggregatory therapy (1.24). 105 patients transformed to myelofibrosis (MF) and 62 to acute leukemia (AL). Transformation to MF rates were similar in the ANA (1.31) and ANA + other CRT (1.27) groups, but lower in the other CRT (0.32) group. Rate of transformation to AL was 0.17, 0.46, and 0.33, respectively. In patients who had only ever received either ANA or HC, rate of transformation to MF was higher in the ANA vs HC group (0.78 vs 0.17) whereas transformation to AL was higher in the HC vs ANA group (0.22 vs 0). All patients who ever received ANA and transformed to AL had also received prior HC.PDEs of greatest interest are displayed in Table 1. Non-hematological malignancy was the most frequent PDE in the other CRT group. 57.4% of deaths were attributed to a PDE; transformation (event rate, 1.9), most frequently to AL (1.3), and non-hematological malignancies (1.6) were the most frequent causes of PDE-related death. No unexpected side effects were noted.There were 54 pregnancies, of which 41 were successful (76%).The proportion of patients with a white blood cell (WBC) count >15 × 109/L at any time was higher in patients who died (12.5%) vs alive patients (6.1%) and in patients who had transformed (15.7%) vs those who did not transform (5.7%). ConclusionPatients receiving ANA were younger than those receiving other CRT. Thrombotic event rates were low; arterial events were similar between ANA and other CRT groups, and venous events were lower in the ANA vs other CRT group. Hemorrhage was most frequent in the ANA + anti-aggregatory therapy group, whereas non-hematological malignancy was most frequent in the other CRT group. Transformation to MF was more frequent in the ANA group, whereas transformation to AL was more frequent in the HC group. The incidence of death and transformation was higher in patients with a WBC count >15 × 109/L.

Pregnancy Outcomes in Patients with Myeloproliferative Neoplasms in the United Kingdom

IntroductionPhiladelphia negative myeloproliferative neoplasms (MPNs) are associated with an increased incidence of haemorrhagic and thromboembolic complications as well as progression to myelofibrosis and acute myeloid leukaemia. MPNs have a peak incidence in 6th and 7th decade of life. However, they can occur in younger age groups and women of child bearing age.Pregnant women with MPN are at an increased risk of maternal thrombosis and haemorrhage and also of impaired uterine blood flow, placental dysfunction, fetal growth restriction, pre-term delivery and fetal loss. There are no prospective studies of pregnancy outcome in the literature.MethodA national prospective cohort was established to describe the maternal and fetal outcomes of pregnancy in women with MPN in the United Kingdom (UK). Data were collected using the UK Obstetric Surveillance System (UKOSS) between January 2010 and December 2012 (UKOSS methodology described by Knight et al, 2005). UK hospitals with a consultant led maternity unit identified pregnant woman with a MPN and provided detailed data on antenatal, maternal and fetal outcomes. Pregnancy outcome data were compared with that reported by the UK Office of National Statistics (ONS) in 2011.Results58 women were identified: 47 with Essential Thrombocythaemia (81%), 5 with Polycythaemia Vera (8.6%), 5 with Myelofibrosis (8.6%) and 1 with JAK2 positive MPN unspecified (1.7%). The incidence of MPN in pregnancy was 6.4/100,000 maternities.The median age was 33 years (range 16-42), compared to average national age during 2011 of 29.7 years. There were 58 live births (56 singleton, 2 twin), one miscarriage and one stillbirth (at 37 weeks) with a miscarriage rate of 1.8% and the perinatal mortality rate of 17/1000 live and stillbirths. The national rates reported by ONS in 2011 were 6.4 miscarriages/100 pregnancies and of 5.2 stillbirths/1000 live and stillbirths. The incidence of pre-eclampsia was 8.8% (n=5/57), compared to 3.4% as reported by Ananth et al 2013. The majority 78.9% (n = 45/57) of women laboured; 42.1% (n = 24/57) underwent induction and spontaneous vaginal delivery occurred in 39% (n = 22/57) of women. Caesarean section (CS) was performed in 42.1% (n = 24/57). Most of the caesareans were performed pre-labour, with an elective CS rate of 24.6% (n = 14/57), and emergency CS rate of 17.5% (n = 10/57). The UK average CS rate in 2011 was 24.8%.Most (86%; 49/57) women delivered at term (>37 weeks). The incidence of preterm (< 37 weeks) and very preterm (<32 weeks) birth was 7.0% (n = 4/59) and 3.5% (n =2/59) respectively; ONS 2011 UK averages were 10.7% and 5.3% respectively. The median birth weight was 3150g (range 720 - 4700g); 13.6% (n = 8/59) of neonates had a low birth weight (<2500g) and 5.1% (n=3) had a very low birth weight (<1500g). 5% of births in 2011 were documented to be below 2500g according to ONS.There was considerable variation amongst clinicians in the management of MPNs. Only 50% (n = 29/58) of women were in receipt of aspirin therapy prior to the index pregnancy. This increased during pregnancy with over 88% (n = 51/58) prescribed aspirin; 38% (n = 22/58) were on both aspirin and low molecular weight heparin. Cytoreductive therapy was used in 43% (n=25/58) of the women prior to index pregnancy (including hydroxyurea (HU), anagrelide (ANA) and interferon). During the index pregnancy, 7 women continued cytoreductive therapy, switching to interferon when prior HU or ANA were used. One woman with ET was initiated on interferon.ConclusionThis first prospective analysis of maternal and fetal outcomes in women showed an incidence of MPN in pregnancy of 6.4/100,000 maternities which is consistent with previous reports. The increased occurrence of pre-eclampsia and fetal growth restriction might explain the higher rates of induction and caesarean section. The majority (87%) of babies were delivered at term.Although this was a small sample, it was reassuring there were no maternal deaths or admissions to intensive care, and only one perinatal death, as previous retrospective data has reported worse outcomes. The clinician variability in the approach to the management of women with MPNs in pregnancy suggests further improvements may be possible. DisclosuresHarrison:Shire: Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Sanofi: Honoraria, Speakers Bureau; Gilead: Honoraria; CTI Biopharma: Consultancy, Honoraria, Speakers Bureau.

Treatment of Essential Thrombocythemia with a Telomerase Inhibitor

Patients with essential thrombocythemia (ET) are at risk for vascular events and transformation to myelofibrosis or leukemia. Cytoreduction in symptomatic patients is usually achieved with hydroxyurea, anagrelide, or interferon, but there are few options for those who are refractory or have unacceptable adverse effects with these agents. Recently, the megakaryocyte telomerase enzyme from patients with ET was found to be more sensitive to inhibition than the …

Development of pathogenesis and therapeutics in myeloproliferative neoplasms

This article introduces the genes related to pathogenesis and prognosis of myeloproliferative neoplasms (MPN) and the current situation of MPN domestic and overseas, which have been reported in the 56th ASH annual meeting. It is emphasized on essential evaluation of the risk score using IPSET in ET, Tefferi in PV and DIPSS-plus in PMF separately. It has assessed advancements in allogeneic hematopoietic stem cell transplantation, phlebotomy, cytoreductive therapy and drug therapy. The article pays more attention to aspirin, anagrelide, JAK2 targeted agent ruxolitinib and thalidomide which is domestically used in the treatment of PMF. Key words: Myeloproliferative neoplasms; Molecular mechanism; Therapy

Advances and challenges in the management of essential thrombocythemia.

The new World Health Organization (WHO) diagnostic criteria for essential thrombocythemia (ET) issued in 2008 made an important distinction between true ET and early myelofibrosis (MF), which has helped to identify a more homogenous population for the diagnosis with longer survival and much less transformation to overt MF. The recent finding of a new mutation (CALR), which is mutually exclusive with JAK2 and MPL mutations, adds to the characterization of ET patients, since there are important phenotypic differences between the mutation types. CALR patients are younger, have lower white blood cell counts (WBC) and a lower thrombosis incidence. A growing field of interest is the state of hypercoagulation due to dysfunction of hemostatic systems, cell-cell interaction and hereditary prothrombotic traits. Activation of platelets, WBC and endothelial cells has been found, making the whole intravascular milieu prothrombotic. Several risk score models, based on retrospective studies, have been developed lately, distinguishing patient groups with graded risk for complications and death. Even if these may be helpful in evaluating patients, they have not been validated in prospective studies and there are not enough data to support their use as a basis for treatment algorithms. The traditional risk factors age, previous thrombosis and platelets >1500 × 10(9)/l are still recommended for the distinction between high risk and low risk ET and the decision to give cytoreductive therapy. However, cardiovascular (CV) risk factors add to thrombosis risk and should be considered both for specific treatment in any risk group and for upgrading low risk patients with high CV risk to an intermediary group where active therapy with aspirin and cytoreduction may be considered. First-line cytoreductive therapy differs with age; in younger patients interferon (IFN) or anagrelide are preferable, in older patients hydroxycarbamide (HC). Second-line therapy for younger patients is HC, for older patients IFN or anagrelide (ANA). JAK2 inhibitors may be suitable in rare cases with symptoms not responding to other therapy.

Anagrelide treatment and cardiovascular monitoring in essential thrombocythemia. A prospective observational study

In this prospective observational single-center study, 55 patients with essential thrombocythemia who were candidates for second line treatment with anagrelide (ANA) received a preliminary cardiovascular (CV) clinical, instrumental and biochemical evaluation (CV history and symptoms, CV risk factors, blood pressure, heart rate, ECG and ECHO-cardio parameters, Troponin I, NT-proBNP). After this in-depth CV screening, 54 out of 55 patients were deemed to be fit for ANA treatment. Thirty-eight of the 55 patients received ANA treatment for a median of 36 months (range 3–48), and were monitored using the same CV evaluation. Fourteen of these 38 patients manifested CV adverse events (10 palpitation, 4 edema, 2 arterial hypertension, 2 acute myocardial infarction) that were not predicted by the in-depth CV evaluation, and that led to ANA withdrawal in only one case (non-cardiac refractory edema). In conclusion, the planned in-depth CV evaluation did not appear to be necessary in ET patients to evaluate their suitability for ANA treatment, and, moreover, was not able to predict the occurrence of CV adverse events during ANA treatment. Nevertheless, the CV adverse events (mostly palpitations and edema) were easily managed by the hematologists, and required the cardiologist involvement in very few selected cases.

Treatment of Essential Thrombocythemia in Europe: An Observational Study of 3649 High-Risk Patients in Exels

BACKGROUND: The Evaluation of Xagrid Efficacy and Long-term Safety (EXELS) study (NCT00567502) is the largest prospective observational cohort of high-risk patients (pts) with essential thrombocythemia (ET) reported to date.OBJECTIVES: The primary objective was safety and pregnancy outcomes of anagrelide (ANA) compared with other cytoreductive therapies (CRT). Secondary objectives included efficacy, measured by the incidence of thrombohemorrhagic events and platelet reduction.METHODS: High-risk pts (≥1 of age >60 yrs, previous thrombotic event, platelet count >1000 x 109/L) with ET were enrolled across 13 countries in Europe between 2005 and 2009. Pts were required to be receiving CRT. Data, including events predefined in the protocol (PDEs), were collected every 6 mo for 5 yrs for all patients. Event rates are presented as number of pts per 100 patient-years exposure and by treatment at time of event. Event rates are provided rather than p values due to the observational nature of the study. Preliminary final data are presented and final data, including platelet response and pregnancy results, will be available at ASH. Recently, results have remained stable and conclusions are not expected to change.RESULTS: 3649 pts were categorized according to treatment at registration as follows: ANA (n=804), ANA + other CRT (n=141), other CRT (n=2666) and no CRT (n=38). Over 80% of pts received either hydroxycarbamide (HC) or ANA, and 69.8% of pts received anti-aggregatory therapy. At registration, median age was lower in the ANA (55.5 yrs, range 18‒89) and ANA + other CRT (59.0 yrs, range 22–88) groups vs the other CRT group (70.0 yrs, range 17‒95).The arterial thrombotic event rate was similar in ANA (1.63) and other CRT (1.62) groups, whereas venous thrombotic event rates differed (0.35 vs 0.57). The major hemorrhagic event rate was highest in the ANA group, especially in pts also treated with anti-aggregatory therapy (1.24).105 pts transformed to myelofibrosis (MF) and 62 to acute leukemia (AL). Transformation to MF rates were similar in the ANA (1.31), ANA + other CRT (1.27) groups, and lower in the other CRT (0.32) group. Rate of transformation to AL was 0.17, 0.46, and 0.33, respectively. In pts who had only ever received either ANA or HC, rate of transformation to MF was higher in the ANA vs HC group (0.78 vs 0.17) whereas transformation to AL was higher in the HC vs ANA group (0.22 vs 0). All pts who ever received ANA and transformed to AL had also received prior HC.PDEs of greatest interest are displayed in Table 1. Non-hematological malignancy was the most frequent PDE in the other CRT group. 57.4% of deaths were attributed to a PDE; transformation (event rate, 1.9), most frequently to AL (1.3), and non-hematological malignancies (1.6) were the most frequent causes of PDE-related death. No unexpected side effects were noted.The proportion of pts with a white blood cell (WBC) count >15 x 109/L at any time was higher in pts who died (12.5%) vs alive pts (6.1%) and in pts who had transformed (15.7%) vs those who did not transform (5.7%).CONCLUSION: Pts receiving ANA were younger than those receiving other CRT. Thrombotic event rates were low; arterial events were similar between ANA and other CRT groups, and venous events were lower in the ANA vs other CRT group. Hemorrhage was most frequent in the ANA + anti-aggregatory therapy group, whereas non-hematological malignancy was most frequent in the other CRT group. Transformation to MF and AL were most frequent in the ANA and HC groups, respectively. The incidence of death and transformation was higher in pts with a WBC count >15 x 109/L.Abstract 1846. Table 1Treatment at time of eventANA N=1127ANA + other CRTN=451Other CRT N=2909No CRT N=645PDEPts(events)nEvent ratePts(events)nEvent ratePts(events)nEvent ratePts(events)nEvent rateTotal thrombohemorrhagic events92 (113)2.7524 (29)2.86270 (326)2.6030 (33)4.91Arterial thrombotic events55 (65)1.6319 (21)2.25171 (200)1.6217 (19)2.74Venous thrombotic events12 (13)0.351 (1)0.1161 (67)0.577 (7)1.13Major hemorrhagic events30 (35)0.876 (7)0.6953 (59)0.497 (7)1.12Transformation to:Myelofibrosis45 (45)1.3111 (11)1.2735 (35)0.3214 (14)2.31Acute leukemia6 (6)0.174 (4)0.4636 (36)0.3316 (17)2.57Non-hematological malignancy17 (18)0.494 (5)0.46143 (161)1.3512 (13)1.95Non-PDE death22 (22)0.647 (7)0.8128 (128)1.1830 (30)4.80ANA, anagrelide; CRT, cytoreductive therapy; PDE, predefined event; Pts, patients DisclosuresBirgegard:Shire Pharmaceuticals: Consultancy, Honoraria, Research Funding. Besses:Shire Pharmaceuticals: honoraria for educational lectures Other. Griesshammer:Amgen: Honoraria; Sanofi: Honoraria; Shire: Honoraria; Novartis: Honoraria; Roche: Honoraria. Gugliotta:Shire Pharmaceuticals: Honoraria, Research Funding. Harrison:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; S Bio: Honoraria; YMBioscience: Honoraria; CTI: Honoraria; Gilead: Honoraria; Sanofi: Honoraria, Speakers Bureau; Shire Pharmaceuticals: Honoraria, Speakers Bureau. Hamdani:Shire Pharmaceuticals: Employment. Achenbach:Shire Pharmaceuticals: Employee Other. Kiladjian:Shire Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Advances in Essential Thrombocythemia

Essential thrombocythemia (ET) is a myeloproliferative neoplasm characterized by high level of megakaryocyte and platelet count.In recent years,the discovery of Janus kinase (JAK)2 V617F mutation has revolutionized the pathogenesis and diagnosis of ET.The mutation of thrombopoietin receptor (MPL),cell-cycle checkpoint kinase (CHEK)2,ten eleven translocation (TET)2,calreticulin (CALR) and other genes replenishes pathogenesis of ET.Traditional therapy which used hydroxyurea or interferon can reduce platelet count of ET patient,and drugs such as aspirin or anagrelide were able to interfere the function of platelet.What's more,breakthrough of ET therapy has been made in a number of clinical trials of JAK2 inhibitors.This article reviews the literatures on the pathogenesis,diagnosis,risk factor and therapy of ET. Key words: Thrombocythemia, essential; Etiology; Diagnosis; Drug therapy

Successful Long-Term Treatment of Philadelphia Chromosome-Negative Myeloproliferative Neoplasms With Combination of Hydroxyurea and Anagrelide

Resistance or intolerance to two favored therapeutic choices for myeloproliferative neoplasms (MPN) is a common clinical challenge. Hydroxyurea (HU) is a common culprit for granulocytopenia and causes poorly reversible side effects including mucocutaneous toxicity. The second line agent, Anagrelide (AG), has poor tolerability. Thus, single-drug regimen is limited in dose escalations and effective cytoreduction in long-term management of MPN.

The characteristics and treatment of patients with primary myelofibrosis at the University medical centre Ljubljana in the period 2008–2012

Background: Primary myelofibrosis (PMF) is a rare hematologic condition belonging to the group of classical myeloproliferative neoplasms. For the diagnosis of PMF, bone marrow histology examination and fibrosis evaluation is mandatory. No recurrent cytogenetic abnormality has been identified and JAK2 mutation is present only in approximately half of the patients. Treatment is limited mainly to alleviation of B symptoms, splenomegaly and anemia. Only allogeneic hematopoietic stem-cell transplant (HSCT) can offer a cure from the disease. A new drug, JAK2 inhibitor ruxolitinib, that has been available since recently, will change treatment options mainly for patients not eligible for HSCT. Methods: Hipokrat medical database was searched for patients diagnosed with PMF between 2008–2012. Data were collected retrospectively and analysed using basic statistics. Results: PMF was newly diagnosed in 28 patients (18/64.3 % men). Two (7.1 %) patients presented with systemic symptoms and ten (35.7 %) had enlarged spleen. Their median blood counts and lactate dehydrogenase activity (LDH) results were: leukocytes 11.25 x 109/l (3.00–72.00), hemoglobin 117 g/l (68–173), platelets 420 x 109/l (111–1477), LDH 7.45 μkat/l (3.00–20.71). In six (21.4 %) patients only bone marrow cytology examination was performed without histologic examination. Cytogenetics was performed in 11 (39.3 %) patients with three abnormalities detected: complex, +9 and del(13q14). JAK 2 mutation was identified in 12 (60.0 %) of 20 examined cases. Patients were treated with acetylsalicylic acid (n = 12/42.9 %), hydroxyurea (n = 12/42.9 %), blood transfusions or epoetins (n = 12), anagrelide (n = 4/14.3 %), methylprednisolone (n = 4/14.3 %), immunomodulatory drugs (n = 3/10.7 %), interferon (n = 2/7.1 %), spleen irradiation (n = 2/7.1 %) and venipunctures (n = 2/7.1 %). At the end of 2011, first eligible patients were treated with ruxolitinib. Conclusions: PMF is a rare disorder with various presentations and treatment suited specifically to patients’ complaints. The treatment is symptomatic with HSCT being the only curable option available. Ruxolitinib will play an importatnt role in PMF treatment in the future.

Blood Cells Derived Microparticles and Vascular Markers Study in Essential Thrombocythaemia Patients

AbstractAbstract 5193Essenthial thrombocythemia (ET) patients are at risk of developing an arterious or venous thrombotic event. Recent studies have shown that there is no clear association between platelet (PLT) count and thrombotic events, but that white cell count plays a role. Qualitative PLT abnormalities, activation of endothelial cells (EC) and PLT may be involved in these processes. Following activation of blood cells, plasma membrane vescicles are released, these are known as microparticles (MP) (<1. 5I′¼m of size). They may originate from PLT (PMP), EC (EMP) or red cells (RMP). MP may affect endothelial modulators such as vasorelaxant nitric oxide (NO), adrenomedullin (ADM) and endothelin-1 (ET-1), a potent vasocostrictor agent. Previous studies [Trappenburg MC et al 2009] demonstrated that MP are present in elevated numbers in ET. However, the relationship between MP with treatment and MP with endothelial modulators is unknown. We investigated the relationship between MP, NO, ADM and ET-1 in ET patients on treatment with hydroxyurea (HU), anagrelide (AN) and aspirin(ASA) only. 52 patients with ET diagnosis were studied: 18 on HU, 15 on AN, and 19 on ASA. Platelet-poor-plasma aliquotes were stored at −80A′°C and subsequently processed. Samples were analysed for MP numbers (absolute total values) and functional markers (percentage values) by flow cytometry, as previously described [Jy W et al JTH 2004]. PMP was identified using CD62P, CD36, and CD63, EMP using CD105 and RMP using CD235a. Endothelial modulator markers NO, ADM and ET-1 were measured by ELISA. Statistical analysis included Kruskal-Wallis test, Bonferroni test, correlation matrix, principal component (PCA) and partial least square regression analysis (PLSR).Total-MP were increased in all patients groups compared to controls (p<0. 01). Strong correlations were seen for tot-MP and %MP CD62P (R=0. 89, p<0. 05), tot-MP and %MPCD36 (R=0. 97, p<0. 05), tot-MP CD62P+ and CD36+ (R=0. 85, p<0. 05) and for tot -MP TF+ and %MP CD105+ (R=0. 66, p<0. 05). Bonferroni test, showed higher MP total number in the AN group compared to the HU and the ASA group (p <0. 05). The percentage of CD63 MP was higher than control both in the HU and in the AN group (p <0. 05), while CD36 MP% was higher in the AN group compared to the HU group (p <0. 05). NO and ADM values were higher in the HU group, while ET-1 values were lower in the AN group (p <0. 05). The principle components (PC) responsible for value variations were PC1 [positive loading for WCC, PMP and EMP] reflecteing PLT and EC activation, and PC4 [positive loadings for PLT, PMP; negative loadings for RMP, NO]. Both PC1 and PC4 were statistically significant for treatment.This study confirms previous findings that MP are present in ET. We also showed that MP are affected by treatment and that MP are mainly PMP and express CD62P and CD36, a marker of immature PLT, which suggests that reticulated PLT partecipate in these processes. We also showed that HU increase NO and ADM levels, while AN reduces ET-1. As NO and ET-1 have antagonistic actions on endothelial cells, we suggest that HU and AN may have overlapping effects on endothelium, although using different biochemichal pathways. [Display omitted] The finding that treatment influences PC1 (PMP and EMP) supports the idea that in ET, treatment impacts directly on MP. The finding that NO and RMP have a negative loading within PC4, suggests that they have an antagonist-action to PMP and EMP in ET. Disclosures:Pizzolo:Hoffmann-La Roche: Consultancy, Honoraria.

Impaired apoptosis of megakaryocytes and bone marrow mononuclear cells in essential thrombocythemia: correlation with JAK2V617F mutational status and cytoreductive therapy

Essential thrombocythemia (ET) is a clonal myeloproliferative disorder characterized by overproduction of megakaryocytes (MKCs) and platelets. The recent discovery of the JAK2 mutation has shed a new light on the development of ET but its pathogenesis still remains unknown. One of the possible mechanisms can be deregulation of apoptosis, resulting in accumulation of bone marrow MKCs. In this study, we investigated the apoptotic profile, as well as the expression of apoptosis-regulating protein in MKCs and bone marrow mononuclear cells (BMMCs) in 43 patients with ET. We found significantly lower percentages of apoptotic MKCs and BMMCs, as measured by the rate of annexin-V+ and caspase-3+ (Cas-3+) cells in relation to healthy volunteers. Additionally, the expression of Bax protein in ET patients naïve to cytoreductive treatment, as well as their Bax/Bcl-2 ratio, was significantly lower than in controls (p = <0.05 and p < 0.001, respectively). Patients positive for the JAK2V617F mutation had markedly higher activation of Cas-3, as well as higher Bax expression (p = 0.02 and p = 0.04, respectively) than JAK2V617F negative cases. There were no marked differences between patients already treated with anagrelide (ANA) or hydroxyurea (HU), although tendency toward the higher apoptosis rate was observed in the HU-treated group. In conclusion, these results demonstrate the inhibition of caspase-dependent apoptosis of both MKCs and BMMCs in untreated ET. This is associated with upregulation of Bcl-2 and downregulation of Bax proteins, predominantly in JAK2V617F negative cases. Patients treated with HU showed slightly higher pro-apoptotic Bax/Bcl-2 index than patients on ANA therapy, which may influence the better efficacy of HU therapy in ET.

Secondary malignancies in essential thrombocythemia and polycythemia vera: A retrospective analysis of 437 patients from a single institution.

6621 Background: Essential thrombocythemia (ET) and polycythemia vera (PV) are chronic myeloproliferative neoplasms (MPN). Patients (pts) are at risk of thrombohemorrhagic complications for which they are often treated with different agents. Previous studies suggested that the risk of secondary malignancy (SM) may be increased in pts with PV or ET treated with Hydroxyurea (HU) or Alkylating agents (ALK). We report the incidence of SM in pts with ET and PV at our institution and treatment received. Methods: We performed a retrospective chart review of all pts with ET and PV seen at MD Anderson (diagnosis according to WHO) between January, 1960 and September 2010, recorded the pathological diagnosis of SM, and performed an analysis to look for differences in risk of SM according to treatment received. Results: A total of 437 pts charts (ET 263, PV 174) were evaluated. 29 pts developed SM [12 ET (4.6%), 17 PV (9.8%)] with median time to diagnosis of 68 months (14-167) in ET pts and 76 months (1-247) in PV pts. 6 pts developed 2 SM. 23 Pts had 29 solid tumors [7 Skin cancers (ET 2, PV 5), 5 breast (ET 2, PV3), 4 lung (ET 3, PV1), 4 genitourinary (ET 2, PV 2), 2 Thyroid (PV 2) and 7 other cancers]. 6 pts had hematologic malignancy [4 AML (ET 2, PV 2), 1 CLL (ET) and 1 NHL (ET)]. 341 (78%) of the 437 pts [222 (84%) ET; 119 (68%) PV] received one or combination of: 1- HU: 259 (60%) pts [162(62%) ET; 97 (56%) PV]; 2- Anagrelide (AG): 145 (33%) pts [112 (43%) ET; 33 (19%) PV]; 3- interferon (INF): 69 (16%) pts [47 (18%) ET; 22 (13%) PV]; 4- ALK (busulfan, melphalan or chlorambucil): 23 (5%) pts [16 (6%) ET; 7 (4%) PV]. Median follow up (MFU) was 69 months (1-396) with 27% of pts followed > 10 y. Risk of SM was 7% in pts treated with HU (ET 6%; PV 8%), 5% with AG (ET 4%; PV 9%), 4% with INF (ET 6%, PV 0%), and none of the pts receiving ALK developed SM (perhaps due to low number of pts and short MFU). 8 (8%) of the 96 pts not receiving any treatment developed SM. No statistical difference in SM risk was observed among treated and non treated groups. Conclusions: Our analysis confirmed inherited risk of developing SM in pts with ET and PV. Contrary to previous reports, the type of therapy received did not increase that risk, perhaps due to a relatively short MFU.

Acute Myeloid Leukemia (AML) or Myelodysplastic Syndrome (MDS) Transformation From Essential Thrombocythemia (ET) In Patients Treated with Hydroxyurea or Anagrelide: A Single Center Series of 17 Cases

AbstractAbstract 4102 Background:ET is an acquired clonal myeloproliferative neoplasm rarely transforming into AML or MDS. Such transformations have been reported to occur in patients treated with alkylating agents primarily. Recently, concern has arisen about the long term safety of the 2 mainstream cytoreductive therapies, i.e. hydroxyurea (HU) and anagrelide (AG), in terms of leukemogenesis and myelofibrosis. This large series of 17 cases of transformed ET aims to compare clinicopathologic features and outcomes of MDS or AML that developed in patients treated with HU or AG. Design:Cases of secondary MDS or AML evolved from ET from January 2000 to February 2010 were retrieved from institutional databases. Results:Eighteen patients with MDS or AML transformed from ET in our institute were evaluated. One patient who was previously treated with an alkylating agent was removed from the study. General characteristics and clinical outcomes of 17 cases are summarized in Table 1. Ten out of 17 patients were diagnosed with AML at the time of transformation while 7 were diagnosed as high grade MDS. Results showed that the median time to MDS/AML development in 17 cases was 87 months; the median survival from the time of ET diagnosis was 124 months and the median survival after transformation was 12 months. There were 16 patients that were mainly treated with long term HU or AG alone. One patient treated with a combination of HU and AG, was excluded from the comparative studies. All patients were founded to have moderate (3/16) to severe (13/16) reticulin fibrosis (2+ to 3+), regardless of treatment. Dyspoiesis including MDS or AML with myelodysplasia related changes (AML-MRC) were more commonly noted in patients treated with AG than in those with HU (6/8 vs. 2/8, p=0.046). The time to transformation was similar in the 2 groups: 76 months (AG) vs. 82 months (HU). The survival since ET diagnosis also was not significantly different, with median survival of 146 months in AG group vs. 96 months in HU group (p= 0.51). However, although not statistically significant (p=0.24), the AG group had a median survival after transformation of 21 months vs. 10 months in the HU group (Figure 1). Thirteen of 14 available karyotypes were abnormal. Complex cytogenetic abnormalities were found in 8/14 cases (5 in AG group vs. 3 in HU group). The common abnormalities, listed in the order of their frequencies, were: -20 or 20q- (5), 5q-(4), 17p- (2), -7 (2), 15q-(2),-18(2), +8(1). Conclusion:There were no differences in degree of fibrosis or time to onset of secondary MDS and/or AML between HU and AG therapy in ET. There was a trend that the median survival after transformation was longer in AG group, which was thought to be related to a predominance of MDS at the time of transformation in AG group. We attributed the shorter median duration of survival from the time of ET diagnosis in our study, in comparison to the literature, to our focus on selection of transformed cases. Complex cytogenetic abnormalities with loss of chromosomal materials in 20, 5, 7, and 17, could play a critical role in disease transformation and progression independent of the specific drug use (HU or AG) in ET. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Fibroblast Growth Factor-2 and Lactate Dehydrogenase Levels in Patients with Essential Thrombocythemia Treated with Anagrelide.

Abstract 4981Essential thrombocythemia (ET) is characterized by platelet-coagulant-endothelial activation. Fibroblast growth factor-2 (FGF-2) is released and upregulated by platelet-fibrinogen(Fg)-endothelial activation and induces vascular and myeloid proliferation. Anagrelide (ANA) is a platelet-lowering drug that inhibits platelet activation. Therefore, we evaluated platelets, platelet factor 4 (PF4) and fibrinogen (Fg), as markers of platelet and coagulant activation, tissue factor pathway inhibitor (TFPI), tissue factor (TF) and von Willebrand factor (vWF), as markers of endothelial activation, FGF-2, vascular endothelial growth factor (VEGF), as indicator of angiogenesis, and white blood cell (WBC) count and haemoglobin (HB), as myeloproliferative indexes. As lactate dehydrogenase (LDH) is a cell cytolysis-associated marker, we determined LDH. To ensure that FGF-2 elevation did not associate with prefibrotic myelofibrosis, we evaluated the CD34 levels. We recruited 21 patients with ET (13 males and 8 females, mean age 52 years) who fulfilled PVSG and WHO. Their mean duration of disease was 9 years (range, 5-21 years). ANA was administered in dose of 0.5 mg/day, with increases of 0.5 mg/day every 7 days until the platelets decreased below 400×109/L and with a average maintenance dosage of 2.1 mg/day. PF4, Fg, FGF-2, VEGF, platelets, WBC, HB, TFPI, TF, vWF and LDH were measured before cytoreduction and to complete response defined as platelets <400×109/L. PF4, FGF-2, VEGF, TFPI, TF and vWF and Fg were assayed by ELISA and immunoturbidimetric and Clauss assay, respectively. LDH was determined by enzymatic method. CD34 levels were measured by flow cytometric assay. Considering that FGF and VEGF may be produced by platelets, we adjiusted FGF and VEGF per platelet (FGFPLT /106 and VEGFPLT/106). Platelets, WBC and HB were measured by automated analyser. Before ANA all patients had marked platelets (1005±314×109/L), high PF4 (127±45 IU/ml vs 4.1±2.4 IU7ml) (p<.0001) and Fg (369±106 mg% vs 237±42 mg%) (p<.0001), elevated TFPI (156±64 ng/ml vs 94±10 ng/ml) (p<.0001), TF (235±287 pg/ml vs 4.4±2.7 pg/ml) (p=<.0001) and low vWF ( 23±7.8 % vs 77±18%) (p<.0001), elevated FGF-2PLT (0.08±0.08 pg/106 vs 0.01±0.001 pg/106) (p<.0001) and VEGFPLT ( 1.8±1.1 pg/106 vs 0.5±0,5 pg/106) (p<.0001), leukocytosis (10.5±2.4×109/L vs 5.2±1.1×109/L) (p<.0001) and high HB (14±1.4 g/dL vs 12±0.4 g/dL) (p<.0001), elevated LDH (529±326 UI/L vs 173±37 UI/L) (p<.0001) and normal CD34 (0.2±0.1%. After ANA, all patients had platelets <400×109/L (386±55×109/L) and normal PF4 (8.5±3 IU/ml), Fg (295±46 mg%), TFPI (100±49 ng/ml), TF (8.7±0.8 pg/ml), vWF (91±35 %), FGF-2PLT (0.01±0.0 pg/106), VEGFPLT ( 1.1±0.6 pg/106), WBC (7.4±1.4×109/L), HB (12.6±1.1 g/dL), and LDH (225±23 UI/L). A positive correlation there was between PF4 and platelets and Fg and TFPI and TF and vWF (p<.0001 and p= 0.002 and p=0.005 and p=0.014 and p<.0001, respectively) and FGF-2PLT and LDH (p<.0001 and p<.0001, respectively). A correlation was found between Fg and FGF-2PLT (p=0.010) and FGF-2PLT and LDH and TFPI and TF and vWF (p=0.003 and p<.0001 and p<.0001, respectively, and p=0.002 and p<.0001 and p=0.001, respectively). A correlation there was between FGF-2PLT and VEGFPLT (p=0.011) and WBC and HB (p=0.011 and p=0.016, respectively). A significant correlation there was between FGF-2PLT and LDH (p<.0001). No correlation was found between FGF-2PLT and CD34. These data suggest that FGF-2PLT and LDH are platelet-endothelial activation-associated prognostic factors reversed by ANA. DisclosuresNo relevant conflicts of interest to declare.