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Background The phase 3b, randomised, open-label RESPONSE-2 study in patients with inadequately controlled polycythaemia vera without splenomegaly showed superiority of the Janus kinase (JAK) 1 and JAK2 inhibitor ruxolitinib versus best available therapy for the primary endpoint of haematocrit control at week 28. Here, we present secondary endpoints of the RESPONSE-2 study after 5 years of follow-up. Methods RESPONSE-2 was an open-label, randomised, phase 3b study done at 48 hospitals or clinics across 12 countries in Asia, Australia, Europe, and Canada. Patients (aged >= 18 years) with polycythaemia vera without splenomegaly, who were intolerant of, or resistant to hydroxyurea, with an Eastern Cooperative Oncology Group performance status of 2 or less were randomly assigned (1:1) to receive ruxolitinib or best available therapy for up to 80 weeks. Patients received oral ruxolitinib at a starting dose of 10 mg twice a day or best available therapy. Patients assigned to best available therapy could cross over to ruxolitinib at week 28 if the primary endpoint was not met, or after week 28 and up to week 80 if best available therapy was ineffective or not tolerated. Patients receiving ruxolitinib at week 80, including crossover patients, could continue ruxolitinib treatment up to week 260. We assessed secondary endpoints at week 260, including durable haematocrit control, median duration of haematocrit control, median haematocrit level over time, number of phlebotomies, and overall survival. Analyses were based on the intention-to-treat principle This study is registered with ClinicalTrials.gov , NCT02038036 and was completed on April 7, 2020. Findings Patients were enrolled between March 25, 2014 and Feb 11, 2015. 149 patients were randomly assigned to ruxolitinib (n=74) or best available therapy (n=75). The median follow-up was 67 months (IQR 65-70). At randomisation, best available therapy regimens included hydroxyurea (n=38), interferon or pegylated interferon (n=9), pipobroman (n=5), lenalidomide (n=1), or no treatment (n=22). Between weeks 28 and 80, 58 (77%) of 75 patients in the best available therapy group crossed over to ruxolitinib; no patients continued best available therapy after week 80 per protocol. 97 patients received ruxolitinib until week 260, including 59 (80%) of 74 patients in the ruxolitinib group and 38 (66%) of 58 patients in the crossover groups. At week 260, 16 (22%; 95% CI 13-33) of 74 patients in the ruxolitinib group had achieved durable haematocrit control, with estimated median duration not reached (NR; 95% CI 144 to NR). Median duration of haematocrit control was not reported for patients in the best available therapy group due to the small number of responders by week 80. During the 5-year follow-up, median haematocrit level among patients in the ruxolitinib group remained below 45%. 60 phlebotomies were required among 74 patients in the ruxolitinib group in 260 weeks, and 106 phlebotomies among 75 patients in the best available therapy group in 80 weeks. 5-year overall survival was 96% (95% CI 87-99) in the ruxolitinib group and 91% (80-96) in the best available therapy group. The most common grade 3-4 adverse events (exposure-adjusted per 100 patient-years) in the ruxolitinib group (n=74) and best available therapy group (n=75) were hypertension (eight [2.4%] vs three [5.6%]), thrombocytopenia (one [0.3%] vs three [5.6%]), and thrornbocytosis (0 vs four [7.5%]). Exposure-adjusted rates of any-grade thromboembolic events were 1.5% per 100 person-years (five of 74 patients) in the ruxolitinib group and 3.7% per 100 person-years (two of 75 patients) in the best available therapy group. No treatment-related deaths occurred during the study. Interpretation 5-year results from the RESPONSE-2 study support the use of ruxolitinib as a second-line therapy of choice for patients with inadequately controlled polycythaemia vera without splenomegaly. Copyright (C) 2022 Elsevier Ltd. All rights reserved., Novartis, Novartis.

Systemic mastocytosis (SM) encompasses a heterogeneous group of clonal disorders characterized by abnormal expansion of mast cells (MCs). Beyond KIT and other genes recurrently mutated in myeloid neoplasms, several genetic variants have been described as predisposing to the development of the disease and influencing its clinical phenotype. Increased copy number variants of the TPSAB1 gene were identified as a cause of nonclonal elevated tryptasemia and defined as hereditary α-tryptasemia (HαT). Moreover, HαT is enriched in patients with SM, where it can affect the incidence of mediator-related symptoms.In a multicenter data set of 444 patients with MC disorders, we aimed to investigate the clinical correlates of germline TPSAB1 copy number gains.Droplet digital PCR was performed in all cases to ascertain the presence of HαT. Clinical history along with blood values and bone marrow examination were analyzed.We confirmed a higher incidence of HαTThese findings on a large patient series support and extend previous data, and suggest that knowledge of HαT status may be useful for personalized management of patients with SM.

Summary Background Polycythaemia vera is a myeloproliferative neoplasm characterised by excessive proliferation of erythroid, myeloid, and megakaryocytic components in the bone marrow due to mutations in the Janus kinase 2 (JAK2) gene. Ruxolitinib, a JAK 1 and JAK 2 inhibitor, showed superiority over best available therapy in a phase 2 study in patients with polycythaemia vera who were resistant to or intolerant of hydroxyurea. We aimed to compare the long-term safety and efficacy of ruxolitinib with best available therapy in patients with polycythaemia vera who were resistant to or intolerant of hydroxyurea. Methods We report the 5-year results for a randomised, open-label, phase 3 study (RESPONSE) that enrolled patients at 109 sites across North America, South America, Europe, and the Asia-Pacific region. Patients (18 years or older) with polycythaemia vera who were resistant to or intolerant of hydroxyurea were randomly assigned 1:1 to receive either ruxolitinib or best available therapy. Patients randomly assigned to the ruxolitinib group received the drug orally at a starting dose of 10 mg twice a day. Single-agent best available therapy comprised hydroxyurea, interferon or pegylated interferon, pipobroman, anagrelide, approved immunomodulators, or observation without pharmacological treatment. The primary endpoint, composite response (patients who achieved both haematocrit control without phlebotomy and 35% or more reduction from baseline in spleen volume) at 32 weeeks was previously reported. Patients receiving best available therapy could cross over to ruxolitinib after week 32. We assessed the durability of primary composite response, complete haematological remission, overall clinicohaematological response, overall survival, patient-reported outcomes, and safety after 5-years of follow-up. This study is registered with ClinicalTrials.gov , NCT01243944 . Findings We enrolled patients between Oct 27, 2010, and Feb 13, 2013, and the study concluded on Feb 9, 2018. Of 342 individuals screened for eligibility, 222 patients were randomly assigned to receive ruxolitinib (n=110, 50%) or best available therapy (n=112, 50%). The median time since polycythaemia vera diagnosis was 8·2 years (IQR 3·9–12·3) in the ruxolitinib group and 9·3 years (4·9–13·8) in the best available therapy group. 98 (88%) of 112 patients initially randomly assigned to best available therapy crossed over to receive ruxolitinib and no patient remained on best available therapy after 80 weeks of study. Among 25 primary responders in the ruxolitinib group, six had progressed at the time of final analysis. At 5 years, the probability of maintaining primary composite response was 74% (95% CI 51–88). The probability of maintaining complete haematological remission was 55% (95% CI 32–73) and the probability of maintaining overall clinicohaematological responses was 67% (54–77). In the intention-to-treat analysis not accounting for crossover, the probability of survival at 5 years was 91·9% (84·4–95·9) with ruxolitinib therapy and 91·0% (82·8–95·4) with best available therapy. Anaemia was the most common adverse event in patients receiving ruxolitinib (rates per 100 patient-years of exposure were 8·9 for ruxolitinib and 8·8 for the crossover population), though most anaemia events were mild to moderate in severity (grade 1 or 2 anaemia rates per 100 patient-years of exposure were 8·0 for ruxolitinib and 8·2 for the crossover population). Non-haematological adverse events were generally lower with long-term ruxolitinib treatment than with best available therapy. Thromboembolic events were lower in the ruxolitinib group than the best available therapy group. There were two on-treatment deaths in the ruxolitinib group. One of these deaths was due to gastric adenocarcinoma, which was assessed by the investigator as related to ruxolitinib treatment. Interpretation We showed that ruxolitinib is a safe and effective long-term treatment option for patients with polycythaemia vera who are resistant to or intolerant of hydroxyurea. Taken together, ruxolitinib treatment offers the first widely approved therapeutic alternative for this post-hydroxyurea patient population. Funding Novartis Pharmaceuticals Corporation.

Myelofibrosis (MF) is a clonal hematologic malignancy with progressive bone marrow fibrosis. Clinical manifestations of MF include splenomegaly, constitutional symptoms, and anemia, whose pathogenesis is multifactorial and largely due to ineffective erythropoiesis and is clinically associated with poor quality of life and reduced overall survival. The only curative treatment for MF is allogenic stem cell transplantation; however, few patients are eligible. Disease management strategies for MF-related anemia have limited effectiveness, and Janus kinase (JAK) inhibitors may induce or worsen related anemia. Thus, there is a significant unmet need for the treatment of patients with MF-related anemia. This review summarizes current and emerging treatments for anemia in MF, including luspatercept and KER-050 (transforming growth factor-β ligand traps), momelotinib and pacritinib (JAK inhibitors), pelabresib (a bromodomain extra-terminal domain inhibitor), PRM-151 (an antifibrotic agent), imetelstat (a telomerase inhibitor), and navitoclax (a BCL-2/BCL-xL inhibitor). Therapeutic combinations with ruxolitinib may offer another treatment approach.

Context: Pelabresib (CPI-0610), a first-in-class, oral, small-molecule inhibitor of BET proteins, has the potential to promote disease-modifying activity through altered gene regulation of key oncogenic, fibrotic, and inflammatory factors in MF. Many patients (pts) with MF treated with ruxolitinib (rux) develop worsening anemia and may become RBC transfusion-dependent (TD). Objective: Evaluation of pelabresib monotherapy or as add-on to rux in advanced MF pts. Design: In Arm 1, pts refractory, intolerant, or ineligible for JAKi were treated with pelabresib monotherapy. In Arm 2, pts receiving rux but not deriving adequate benefit were treated with add-on pelabresib. The primary endpoint was achievement of transfusion independence (TI) ≥12 wks in TD cohorts and ≥35% spleen volume reduction at wk 24 in non-TD cohorts. Results: As of 29 Sept. 2020, 19 TD pts and 27 non-TD pts were treated in Arm 1. Seventy-six percent of pts had baseline Hgb Conclusions: Pelabresib monotherapy was associated with a mean increase in Hgb ≥1.5 g/dL in majority of non-TD pts and conversion of one-fifth of TD pts to TI in Arm 1. Pelabresib add-on to rux in Arm 2 resulted in mean increase in Hgb ≥1.5 g/dL in 17% of non-TD pts and conversion to TI in more than one-third of TD pts.

Context: Pelabresib (CPI-0610) is a potent, selective BET bromodomain inhibitor under investigation in the ongoing Phase 2 MANIFEST trial, given as monotherapy to ruxolitinib (rux)-intolerant/refractory myelofibrosis (MF) pts in arm 1, as an “add-on” to rux in MF pts who have suboptimal/lost response to rux in arm 2, and as combination therapy with rux in JAKi-naive MF pts in arm 3. Objective: We assessed activity of pelabresib, including changes in inflammatory cytokines (Ck) in blood, improvement of bone marrow (BM) biology, including fibrosis (BMF), erythroid (Ery) progenitor and megakaryocyte (MK) numbers and clustering, and impact of mutational status on clinical response. Results: Rapid PD response was demonstrated by median blood IL8 mRNA level reduced to 55% of baseline (BL) levels 4 hours post-1st dose of pelabresib in 102 pts, with similar reduction across arms. Elevated blood Ck levels were observed at BL across arms. Reduction of NF-κB and non-NF-κB regulated inflammatory Ck was observed on cycle 1 day 14 (C1D14) across arms and maintained through C9D1 (24 weeks). BMF grading was assessed by local pathologists for BL and post-treatment (most at 24 weeks) biopsies from 116 evaluable pts. Relative BMF improvement of ≥1 grade was observed in 33% (38/116) of all pts, with 21% (6/29) in arm 1, 41% (16/39) in arm 2, and 33% (16/48) in arm 3. BMF grade worsening was observed in only 6% (7/116) pts. Exploratory analyses of Ery and MK lineages by immunohistochemical staining for CD71 and CD61, respectively, on BM biopsy pairs (BL and week 24) revealed an increase in Ery progenitors in 59% (22/37) of pts. Tight clusters of MK, characteristic in MF BM, were observed at BL, and improvement in MK numbers and/or clustering was observed in 65% (24/37) pts. Mutation analysis revealed similar profiles at BL across arms. SVR35 at 24 weeks was similar, regardless of mutational status for each of the 14 genes analyzed. Conclusions: Our data demonstrate a robust PD effect of pelabresib in MF pts, indicate broad clinical responses irrespective of mutational status, and suggest the disease-modifying potential of pelabresib by improving BM fibrosis and function.

Background Ruxolitinib is established as treatment for symptomatic myeloproliferative neoplasm (MPN)-associated myelofibrosis. The strict inclusion and exclusion criteria and dose modification rules that applied to the COMFORTI and II studies that led to the licensing of ruxolitinib are not always applicable to routine clinical practice. Thus physicians now face decisions regarding ruxolitinib use that were not addressed in these pivotal trials. Methods We performed an online survey of hematologists practicing in Europe, Israel, the United Kingdom and the United States. Demographic details regarding the physicians and their practice as relates to MPNs were collected. Management decisions pertaining to the use of ruxolitinib were obtained regarding 10 clinical scenarios relating to anemia, thrombocytopenia, frailty, infection and lack or loss of response to ruxolitnib in MF patients. Results 140 physicians responded to the survey. There were marked differences regarding their decisions for ruxolitinib administration in MF patients with or developing anemia or thrombocytopenia. Similarly there was little consensus regarding management of patients refractory or losing a response to ruxolitinib. There were differences between “MPN-focused” and “non-MPN-focused” physicians in certain areas. Conclusion Physician practices regarding management of MF patients experiencing ruxolitinib-related toxicities or in whom response to the drug is lost was variable. This was true of “MPN-focused” and “non-MPN-focused” physicians in certain cases. Physician education and experience in using ruxolitinib may improve patient management.

The World Health Organization classification system recognizes 4 variants of JAK2 mutation-enriched myeloproliferative neoplasms (for expansion of gene symbols, use search tool at www.genenames.org): essential thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis (PMF), and prefibrotic PMF. All 4 disorders are characterized by stem cell-derived clonal myeloproliferation with mutually exclusive driver mutations, including JAK2, CALR, and MPL. The median survival is approximately 20 years for ET, 14 years for PV, and 6 years for PMF; age is the most important determinant of survival with the corresponding median of 33, 24, and 15 years in patients younger than 60 years. Genetic information is the second most important prognostic tool and includes karyotype, driver mutational status, and presence of specific other mutations. Karyotype has been shown to carry prognostic relevance in PV (abnormal vs normal) and PMF (unfavorable vs favorable abnormalities). Driver mutational status is prognostically most relevant in PMF; type 1/type 1-like CALR vs other driver mutational status has been associated with superior survival. In ET, arterial thrombosis risk is higher in patients with JAK2 or MPL mutations whereas MPL-mutated patients might be at risk for accelerated fibrotic progression. ASXL1 and SRSF2 mutations have been associated with inferior overall, leukemia-free, or fibrosis-free survival in both PV and PMF, and a recent targeted sequencing study has identified additional other adverse mutations in both these disorders, as well as in ET. Further enhancement of genetic risk stratification in myeloproliferative neoplasms is possible by combining cytogenetic and mutation information and developing a prognostic model that is adjusted for age.

Polycythemia vera (PV) is characterized by erythropoiesis and JAK2-activating mutations, with increased risks of morbidity and mortality. Most patients with PV are iron deficient, and treatment often includes hematocrit control with phlebotomy, which may exacerbate iron deficiency-associated complications. The phase 3 RESPONSE trial evaluated the JAK1/JAK2 inhibitor ruxolitinib (n=110) versus best available therapy (BAT; n=112) in patients with PV who were hydroxyurea-resistant/intolerant. Ruxolitinib was superior to BAT for hematocrit control, reduction in splenomegaly, and blood count normalization. This exploratory analysis, the first to evaluate iron status in a prospective study of patients with PV, investigated ruxolitinib effects on 7 serum iron markers and iron deficiency-related patient-reported outcomes (PRO). Among patients with evidence of baseline iron deficiency, ruxolitinib was associated with normalization of iron marker levels, compared with lesser improvement with BAT. Iron levels remained stable in ruxolitinib patients with normal iron levels at baseline. Regardless of baseline iron status, treatment with ruxolitinib was associated with improvements in concentration problems, cognitive function, dizziness, fatigue, headaches, and inactivity, although improvements were generally greater among patients with baseline iron deficiency. The improvements in iron deficiency markers and PROs observed with ruxolitinib are suggestive of clinical benefits that warrant further exploration.

Background: Momelotinib (MMB) is a potent, orally bioavailable, small-molecule inhibitor of JAK1, JAK2, and ACVR1 with demonstrable clinical activity against the three hallmark features of myelofibrosis (MF), namely anemia, constitutional symptoms, and splenomegaly, across the continuum of intermediate-/high-risk MF patients whether JAKi-naive or previously JAKi-treated. Objective: To characterize the long-term safety of MMB in comparison to ruxolitinib (RUX). Methods: SIMPLIFY-1 was a double-blind, randomized, study of MMB vs RUX in intermediate-/high-risk JAKi-naive patients with MF (n=215 MMB, 217 RUX). SIMPLIFY-2 (S2) was a randomized study of MMB vs best available therapy (BAT; RUX in 88% of patients) in prior RUX-treated patients with hematological toxicity (n=104 MMB, 52 BAT). Randomized treatment (RT) for 24 weeks was followed by open-label MMB extended treatment in both studies. Data from RT were compared, and further long-term data were analyzed from 550 subjects receiving MMB, including 336 subjects on MMB for ≥48 weeks and 148 for ≥144 weeks. Results: Despite treatment duration exceeding 3.5 years for >90 subjects, the incidence of grade 3/4 hematological toxicity was very low. During the S1 RT period, grade 3/4 anemia occurred in only 6.1% of MMB subjects compared with -4-fold higher rate (22.7%) for RUX. Dose reductions due to thrombocytopenia were also 4-fold higher for RUX (24.5%) than MMB (6.1%). Subjects randomized to MMB in S1 experienced a rapid, sustained increase in hemoglobin, in contrast to significant hemoglobin decrease on RUX. Following crossover to MMB from RUX, hemoglobin increased rapidly to above baseline. Platelet values were also significantly higher compared to RUX. Similarly, in S2, hemoglobin and platelet values were maintained or improved on MMB. Importantly, no new safety signals and no cumulative toxicity were observed during extended MMB dosing. Rates of peripheral neuropathy remained low. Conclusions: MMB displays very favorable long-term tolerability, including an absence of significant rates of high-grade hematological and other toxicities or evidence of cumulative toxicity, consistent with its differentiated pharmacological and clinical profile. Notably, subjects switching from RUX also experienced favorable tolerability, despite their previous therapy.

Context: Fedratinib is approved in the US for patients with Intermediate-2/High-risk myelofibrosis. Myelofibrosis can evolve from prior PV or essential thrombocythemia (ET). The ARD12042 study assessed fedratinib in patients with PV/ET. There was no response among 36 ET patients who received fedratinib 100-600 mg/day. Objective: Determine fedratinib clinical activity in patients with PV in ARD12042. Methods: Phase II, open-label, dose-ranging and -expansion study. Patients were aged ≥18 years with WHO-defined PV, ECOG-PS score ≤ 2, and were resistant/intolerant to hydroxyurea. Patients received once-daily fedratinib 100 mg, 200 mg, or 400 mg doses during the dose-ranging phase, and 400 mg during dose-expansion, in continuous 28-day cycles. All endpoints were prospectively defined. Response rate (RR) was the proportion of patients with hematocrit Results: 45 PV patients received fedratinib. Median age was 63 years (36-86) and 98% had ECOG-PS 0-1. The mITT population included 15 patients (13 dose-ranging, 2 dose-expansion): fedratinib 100 mg, n=2; 200 mg, n=5; 400 mg, n=8. RRs during dose-ranging were 0%, 40%, and 67% in the fedratinib 100 mg, 200 mg, and 400 mg groups. Both expansion phase patients responded. Clinicohematologic RRs were 20% and 63% with fedratinib 200 mg and 400 mg. Median SVRs from baseline at EOC8 were -16% and -39% in the 200-mg and 400-mg groups, respectively; 3 fedratinib 400 mg patients (38%) had ≥35% SVR. Among all patients, most common AEs were diarrhea (56%), nausea (53%), and vomiting (31%). Grade 3-4 AEs occurred in 21 patients (47%), most commonly lipase increases (11%) and abdominal pain (9%). Most frequent reason for treatment discontinuation was study termination (56%). AEs led to discontinuation for 7 patients (16%). Conclusion: Fedratinib 400 mg/day was manageable and induced hematocrit

Introduction: The JAK2 inhibitor, fedratinib, significantly improved splenomegaly and myelofibrosis (MF) symptoms vs placebo as first-line treatment in the JAKARTA trial and after prior ruxolitinib in the JAKARTA2 trial. We determined PFS and OS with fedratinib 400 mg/day in these studies. Methods: Patients had intermediate- or high-risk MF. JAKARTA patients were randomized to fedratinib 400 mg/day, fedratinib 500 mg/day, or placebo. After six cycles (earlier if disease progression [PD]), placebo-randomized patients could crossover to fedratinib. In JAKARTA2, all patients with MF resistant/intolerant to RUX initially received fedratinib 400 mg/day. PFS was time from randomization to PD/death. PFS/OS in the placebo arm of JAKARTA included time after crossover to fedratinib. Both trials were terminated early due to a clinical hold in 2013; ongoing patients were censored for PFS/OS. Results: In JAKARTA, 96 patients were randomized to fedratinib 400 mg and 96 to placebo; 71 placebo-randomized patients (74%) crossed over to fedratinib. At study termination, survival follow-up halted for 74 fedratinib-randomized pts (77%) and 65 placebo-randomized pts (68%). Median (m) PFS was prolonged with fedratinib vs placebo: 23.2 vs 17.5 months, respectively (HR 0.42 [95%CI 0.23–0.76]; P=0.004); 1-year PFS rates were 83% vs 67%. mOS was not reached (NR) in either arm (HR 0.57 [0.30–1.10]; P=0.094); 1-year survival was 92% with fedratinib and 86% with placebo. Of 97 patients enrolled in JAKARTA2, 79 (81%) were censored for survival at study termination. mPFS was 13.3 months (95%CI 8.4–17.1), and mOS was NR (17.1 months-NR), with a 1-year PFS of 59% and 1-year survival rate of 84%. Conclusions: Fedratinib 400 mg/day significantly improved PFS vs placebo as first-line MF treatment. Separation of PFS/OS curves between fedratinib and placebo in JAKARTA, despite substantial fedratinib crossover for placebo-randomized patients, suggests early fedratinib treatment is better. In JAKARTA2, mPFS, mOS, and 1-year survival compare favorably with findings in similar patients post-RUX (6.0 months, 11.1 months, and 47%, respectively; Mascarenhas 2020). The FREEDOM and FREEDOM2 trials of fedratinib are ongoing. Adapted from CN Harrison, et al., Overall and progression-free survival in patients treated with fedratinib as first-line myelofibrosis (MF) therapy and after prior ruxolitinib (RUX): results from the JAKARTA and JAKARTA2 trials, HemaSphere, 2021; Abstract S203.

Clinical evidence supports the need of changing the diagnostic criteria of the 2008 updated WHO classification for polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). In JAK2-mutated patients who show characteristic bone marrow (BM) morphology, clinical studies demonstrated that a hemoglobin level of 16.5g/dL in men and 16.0g/dl for women or a hematocrit value of 49% in men and 48% in women are the optimal cut off levels for distinguishing JAK2-mutated ET from "masked/prodromal" PV. Therefore BM morphology was upgraded to a major diagnostic criterion. Regarding ET the key issue was to improve standardization of prominent BM features enhancing differentiation between "true" ET and prefibrotic/early primary myelofibrosis (prePMF). These two entities have shown a different epidemiology and clinical outcomes. Concerning prePMF a more explicit clinical characterization of minor criteria is mandated for an improved distinction from ET and overt PMF and accurate diagnosis and outcome prediction.

Context: MF is characterized by anemia, progressive splenomegaly, and debilitating constitutional symptoms. Fedratinib is a selective JAK2 inhibitor approved in the US for adult patients with intermediate-2 or high-risk MF. Fedratinib was assessed in the phase III, placebo-controlled JAKARTA trial in patients with JAK-inhibitor-naive MF, and in the phase II single-arm JAKARTA2 trial in patients previously treated with ruxolitinib. Reduced splenomegaly and decreased symptom burden are each important goals of treatment but may not occur simultaneously. Objective: Assess the overall clinical benefit of fedratinib, as assessed by proportions of patients in JAKARTA and JAKARTA2 who achieved a spleen volume response, symptom response, or both, after 6 fedratinib treatment cycles. Methods: Both studies enrolled patients with intermediate- or high-risk MF, platelet counts ≥50 × 109/L, and ECOG PS ≤ 2. Efficacy endpoints included rates of spleen volume response (≥35% spleen volume reduction from baseline) and symptom response (≥50% reduction from baseline total symptom score [TSS] on the modified MFSAF) at the end of cycle 6 (EOC6). These analyses assess outcomes among patients in both studies who received fedratinib 400 mg/day and JAKARTA patients randomized to placebo, who had spleen volume and MFSAF TSS data available at baseline and EOC6. Results: In JAKARTA, 67/96 (70%) patients randomized to fedratinib 400 mg and 45/96 (47%) randomized to placebo had spleen volume and MFSAF assessments at baseline and EOC6. Overall, 76% (51/67) of fedratinib-treated patients achieved a spleen volume (n=40 [60%]) or symptom (n=35 [52%]) response, including 24 patients (36%) who achieved both responses. In the placebo arm, 8/45 patients (18%) achieved a spleen volume (n=1) or symptom (n=7) response; no patient attained both responses. In JAKARTA2, 42/97 patients (43%) who received fedratinib 400 mg/day (starting dose) had spleen volume and symptom assessments at baseline and EOC6. Of them, 81% (34/42) achieved a spleen (n=24 [57%]) or symptom response (n=20 [48%]), including 10 patients (24%) who achieved both. Conclusion: A majority of patients with MF who completed 6 fedratinib treatment cycles in JAKARTA or JAKARTA2 experienced a clinical benefit in terms of spleen volume reductions or symptom responses, and one-fourth to one-third of these patients achieved both responses.

The BCR-ABL1 negative myeloproliferative neoplasms (MPNs) are associated with an increased risk of both venous and arterial thromboembolic events. Thromboses may be the presenting clinical feature of an MPN or may occur during the course of the disease. Treatment comprises anticoagulant and antiaggregant agents as in non- MPN thromboses, and treatment of the particular MPN. The duration of anticoagulant treatment that is required for MPN thrombosis is unknown. This study was performed to survey the opinion of hematologists who treat patients with MPN regarding the duration of anticoagulation or antiaggregant therapy in patients in whom thrombosis is the presenting feature of MPN. Five clinical scenarios in which thromboembolism (cerebral vein thrombosis, pulmonary embolism, cerebrovascular accident, splanchnic vein thrombosis, portal vein thrombosis) was a presenting feature of MPN were created using a web-based tool and were sent by email to hematologists in Israel, Italy and England and to hematologists identified as key opinion leaders in the field of MPN. Physicians were asked to recommend duration of anticoagulation and/or aspirin use choosing from 4 alternatives provided. Seventy-three physicians responded to the survey. 42 physicians considered MPNs to be their main area of clinical interest, and 31 did not. 21 physicians saw more than 20 MPN patients per week, and 50 physicians had been in hematology practice for more than 10 years. Responses regarding the duration of anticoagulation and/or the use of aspirin varied for all of the clinical vignettes. Neither physician area-of-interest, volume of MPN patients treated nor years in practice were related to the responses obtained. This study demonstrates that hematologists, including those specializing in MPNs, lack consensus in their approach to the long-term treatment of thromboses as the presenting feature of an MPN. Controlled clinical studies are needed to inform appropriate decision making in this area.

The term RBC-transfusion-dependence is widely-used by hematologists to describe a condition of severe anemia typically arising when erythropoiesis is reduced such that a person continuously requires ≥ 1 RBC-transfusions over a specified interval. Defining a person as RBC-transfusion-dependent has important implications in diverse hematological disorders especially because it strongly-correlated with decreased survival. Conversely, becoming RBC-transfusion-independent or receiving fewer RBC-transfusions over a specified interval is defined as improvement or response in many disease- and/or therapy-setting. Whether this correlates with improved survival is controversial. We used a structured expert-panel consensus panel process to define RBC-transfusion-dependence and -independence or improvement. We suggest these definitions may prove useful to persons studying or treating these diseases.

To assess whether alterations in the stromal cell-derived factor-1 (SDF-1)/CXCR4 occur in patients with primary myelofibrosis (PMF) and in Gata1 low mice, an animal model for myelofibrosis, and whether these abnormalities might account for increased stem/progenitor cell trafficking.In the mouse, SDF-1 mRNA levels were assayed in liver, spleen, and marrow. SDF-1 protein levels were quantified in plasma and marrow and CXCR4 mRNA and protein levels were evaluated on stem/progenitor cells and megakaryocytes purified from the marrow. SDF-1 protein levels were also evaluated in plasma and in marrow biopsy specimens obtained from normal donors and PMF patients.In Gata1 low mice, the plasma SDF-1 protein was five times higher than normal in younger animals. Furthermore, SDF-1 immunostaining of marrow sections progressively increased with age. Similar abnormalities were observed in PMF patients. In fact, plasma SDF-1 levels in PMF patients were significantly higher (by twofold) than normal (p0.01) and SDF-1 immunostaining of marrow biopsy specimens demonstrated increased SDF-1 deposition in specific areas. In two of the patients, SDF-1 deposition was normalized by curative therapy with allogenic stem cell transplantation. Similar to what already has been reported for PMF patients, the marrow from Gata1 low mice contained fewer CXCR4 pos CD117 pos cells and these cells expressed low levels of CXCR4 mRNA and protein.Similar abnormalities in the SDF-1/CXCR4 axis are observed in PMF patients and in the Gata1 low mice model of myelofibrosis. We suggest that these abnormalities contribute to the increased stem/progenitor cell trafficking observed in this mouse model as well as patients with PMF.

Purpose The risk of recurrent venous thromboembolism (VTE) in patients with polycythemia vera and essential thrombocythemia has been scarcely addressed, and whether long-term oral anticoagulant treatment or acetylsalicylic acid should be recommended after first occurrence of deep venous thrombosis (DVT) is unknown. This multicenter cohort study was aimed to assess the rate of recurrent VTE in patients with polycythemia vera or essential thrombocythemia in comparison with a control group of individuals with previous VTE and without neoplastic diseases. Patients and Methods We retrospectively estimated the rate of recurrence in 79 patients with myeloproliferative disorders (MPDs; polycythemia vera/essential thrombocythemia, 45/34) and with a previous proximal DVT. Patients were divided into 2 groups. The first comprised 41 patients who received acetylsalicylic acid after 6 months of oral anticoagulant treatment. The second group was composed of 38 patients given long-term oral anticoagulant treatment without acetylsalicylic acid. The majority of patients were treated with cytotoxic drugs. The results were compared with the recurrences seen in 176 patients without cancer with previous proximal DVT given short-term oral anticoagulant treatment. Results In the patients with polycythemia vera and essential thrombocythemia, the rate of recurrent DVT was higher in the group receiving acetylsalicylic acid (32%) compared with the group on oral anticoagulant treatment (16%), although not statistically significant. The rate of recurrent DVT in MPD cases receiving acetylsalicylic acid was quite similar to that of patients without cancer (33%). The cumulative probability of recurrent VTE indicated a trend of fewer events in the MPD cases on long-term oral anticoagulant treatment. In the patients with MPDs, the incidence of major bleeding during oral anticoagulant treatment or acetylsalicylic acid was 1% and 0.5% patient-years (years of observation), respectively. Conclusion This retrospective analysis would suggest a long-term oral anticoagulant treatment after a first DVT in patients with polycythemia vera and essential thrombocythemia. However, this indication should be weighed against the risk of major hemorrhagic events that seems lower during long-term prophylaxis with acetylsalicylic acid. Therefore, a prospective clinical trial comparing acetylsalicylic acid in patients with polycythemia vera and essential thrombocythemia with oral anticoagulant treatment in the prevention of recurrent VTE is warranted.

We studied the expression of the chemokine receptor CXCR4 on circulating CD34+ cells of patients with myelofibrosis with myeloid metaplasia (MMM), and examined its relationship to the severity of disease.Surface and intracellular CXCR4 expression were measured flow cytometrically in 84 consecutive MMM patients, 16 patients with polycythemia vera (PV), and 20 healthy subjects. In 23 MMM patients, CXCR4 gene expression level was also quantitated by real time-RT-PCR in CD34+ cells.The expression of CXCR4 on circulating CD34+ cells was significantly reduced in patients with MMM (P0.001) as compared to normal controls and patients with PV (P=0.01). The levels of CXCR4 mRNA in CD34+ cells were lower in patients with MMM as compared with normal subjects, and were directly correlated with the degree of CXCR4 surface expression, demonstrating that transcriptional defects were the major cause for receptor down-regulation. No statistical association was found between JAK2V617F mutational status and the extent of CXCR4 down-regulation. CXCR4 expression on CD34+ cells inversely correlated with the number of circulating CD34+ cells (R=-0.55; P0.001), and was severely down-regulated in high risk patients and patients with a high "myelodepletion severity index". CXCR4 down-regulation was associated with advanced patient age, the presence of severe anemia, thrombocytopenia, and degree of bone marrow fibrosis.Reduced expression of CXCR4 by CD34+ cells is a characteristic of MMM which is associated with the constitutive mobilization of CD34+ cells and occurs in patients with advanced forms of the disease.

Using thrombopoietin (TPO), as selective pressure, several TPO-dependent clones were isolated from the murine multipotential IL-3-dependent cell line 32D. Four of them were fully characterized. They depended on TPO for survival and proliferation and, although retaining the capacity to grow in IL-3, did not respond to either EPO, G-CSF or GM-CSF. 32D TPO cells were heterogeneous in morphology and ranged from small cells, with a DNA content nearly tetraploid and a modal chromosome no. 66, to cells 50-75 microm in diameter containing multiple (up to 5-6) interconnected nuclei with a clear megakaryocyte (Mk) morphology by electron microscopy. Cell sorter isolation and single cell cloning experiments indicated that the small cells were those capable to proliferate in TPO and to generate the larger ones over time. 32D TPO cells expressed Mk-specific markers by FACS (CD41, CD61 and 2D5) and RT-PCR (acetyl cholinesterase E and platelet factor 4) and their unique profile, by gene array analysis, included expression of urokinase plasminogen activator surface receptor (CD87 or uPAR), plasminogen activator inhibitor and coagulation factor II (thrombin) receptor (Cf2r). In addition, by quantitative RT-PCR, 32D TPO clones expressed levels of Gata1 similar to those expressed by freshly isolated Mks (DeltaCt approximately 4.7 in both cases). In conclusion, the 32D TPO subclones described here are among the few pure Mk cell lines isolated so far and, for their unique properties, may prove themselves as a useful model to study Mk differentiation.

The primary genetic lesion(s), as well as the biological processes responsible for the typical structural changes of the bone marrow microenvironment in idiopathic myelofibrosis, are still poorly understood, although a central role in disease pathogenesis has been attributed to the clonal proliferation and defective maturation of megakaryocytes. Two animal models of the disease have been described, that in the last few years significantly contributed to the elucidation of some of the pathogenetic steps of the human disease; these are represented by mice genetically modified to overexpress thrombopoietin and by knock-down mice with defective GATA-1 expression in megakaryocytes (GATA-1(low) mice). This review will outline these murine models, both characterized by extensive accumulation of megakaryocytes in hematopoietic tissues, and illustrate how they provided insights into the identification of some of the molecules and mechanisms responsible for the development of fibrosis and osteosclerosis that present major similarities with those observed in patients with idiopathic myelofibrosis.

We evaluated single agent pomalidomide for myelofibrosis-associated anemia. First, 21 patients received pomalidomide 3.0mg/day on 21-day-on/7-day-off schedule. Due to poor tolerance the study was quickly suspended. Second, 29 patients received pomalidomide 0.5mg/day continuously. Three patients (10%) experienced clinical improvement in hemoglobin per International-Working-Group criteria (median time to response 1.6 months; median response duration 6.7 months). Ten patients were RBC-transfusion-dependent per Delphi criteria; 2 (20%) achieved RBC-transfusion-independence (time to response 0.9 months in both; response duration of 8.3 and 15 months). One grade 3/4 toxicity (neutropenia) occurred. Pomalidomide at low dose is well tolerated but has modest clinical activity in myelofibrosis.

1Unit of Clinical Epidemiology and Center for the Study of Myelofibrosis, IRCCS Policlinico S. Matteo Foundation, Pavia, Italy 2Department of Internal Medicine, Unit of Clinica Medica 1, IRCCS Policlinico S. Matteo Foundation, Pavia, Italy 3Gastroenterology 3 Unit, IRCCS Ca Granda Ospedale Maggiore Policlinico Foundation, Milano, Italy 4Section of Hematology, Department of Critical Care, University of Florence and Istituto Toscano Tumori, Florence, Italy

The clinical efficacy of JAK2-inhibitors in patients with myelofibrosis, that involves a rapid and massive reduction of spleen enlargement and improvement of clinical symptoms, is not accompanied by significant modifications of hematologic parameters nor of the burden of JAK2V617F allele. Furthermore, clinical improvement has been reported to occur irrespective of patient's JAK2-mutated status. On the other hand, dramatic changes in plasma cytokine levels have been observed. Based on available information about the role of cytokines in the pathogenesis of myelofibrosis, the hypothesis that the clinical efficacy of JAK2-inhibitors could be mainly ascribed to a general down-regulation of cytokine production and cytokine signaling is discussed.

Objective Expression profiling of microRNA (miRNA) was performed in granulocytes isolated from patients with primary myelofibrosis (PMF), with the aim of identifying abnormally expressed miRNAs in comparison with normal subjects or patients with polycythemia vera (PV) or essential thrombocythemia (ET). Patients and Methods Using stem loop–primed reverse transcription and TaqMan quantitative real-time polymerase chain reaction, the expression of 156 mature miRNAs was evaluated using pooled granulocytes from PMF patients, either wild-type or JAK2 617V>F mutant with >51% allele burden, and control subjects. Differentially expressed miRNAs were then validated on additional control and PMF samples, and also on PV or ET granulocytes. Results There was a global downregulation of miRNA expression in PMF granulocytes; 60 miRNAs, of 128 called present , displayed differential expression compared to normal samples. Twelve miRNAs, which had been selected based on statistically different expression level, were finally validated. In PMF granulocytes, levels of miR-31 , -150 , and -95 were significantly lower, while those of miR-190 significantly greater, than control and PV or ET samples; on the other hand, miR-34a , -342 , -326 , -105 , -149 , and -147 were similarly reduced in patients with PMF, PV, or ET compared to controls. Increased expression of miR-182 and -183 correlated with JAK2 617V>F allele burden. Three in silico–predicted putative target genes ( DTR , HMGA2 , and MYB ), showed deregulated expression in PMF granulocytes that correlated with expression level of regulatory miRNA. Conclusions A defined miRNA profile distinguishes PMF granulocytes from those of normal subjects and, partially, also from PV or ET patients.

Fourteen patients diagnosed with neoplastic disease (5 breast cancers, 9 lymphomas) were treated with Ifosphamide 12 g/sqm, Carboplatin 1500 mg/sqm and VP-16 1800 mg/sqm, followed by either PBSC (n = 9) or Bm (n = 5) rescue. In 8 PBSC infused pts, the cells were collected after a 4 days G-CSF course (15 mcg/kg), without any mobilization chemotherapy. The pts infused with BM received G-CSF (5 mcg/kg) starting on +6. All patients were able to complete the regimen: major toxicity was represented by vomiting and mucositis (grade 1–3); 7 required TPN. Transitory increase of hepatic enzymes was also observed. All pts achieved complete hematopoietic reconstitution, with an advantage of PBSC in platelets recovery. Median hospitalization time after the infusion was 14 days (range 11–22).

A dot assay was developed for the detection of membrane receptor(s) for erythropoietin (Ep). A relatively homogeneous population of cells bearing the receptor for Ep was generated in the spleen of mice made anemic with phenylhydrazine and crude membrane extracts were prepared from spleen cell suspensions. Aliquots of the membrane extracts were applied to microdishes of nitrocellulose in a volume of 4 microliters. After free reactive sites were blocked, the microdishes were incubated for 2 h at 37 degrees C with {sup 125}I-labeled human recombinant Ep ({sup 125}I-rEp), and nitrocellulose bound radioactivity was determined thereafter. Reproducible curves were obtained, and a significant correlation between bound radioactivity and the amount of membrane proteins applied to the nitrocellulose dishes was found. Specific binding was saturable, reaching a plateau at 2.5 nM. Binding parameters of nitrocellulose-immobilized receptor were not significantly different from the values calculated using intact cells. No appreciable binding of {sup 125}I-rEp to control membranes at low Ep-receptor content was observed. Among a panel of growth factors, only unlabeled rEp was able to compete for the binding of {sup 125}I-rEp to nitrocellulose-immobilized membrane proteins in a dose-dependent fashion. The technique described herein may be of use in the study of themore » Ep receptor and as an assay for its purification. Moreover, it may also be of general application in the study of receptor-ligand interactions.« less