Rajmonda Fjerza

Long-term survival and blast transformation in molecularly annotated essential thrombocythemia, polycythemia vera, and myelofibrosis

Janus kinase 2 (JAK2) mutations define polycythemia vera (PV). Calreticulin (CALR) and myeloproliferative leukemia virus oncogene (MPL) mutations are specific to JAK2-unmutated essential thrombocythemia (ET) and primary myelofibrosis (PMF). We examined the effect of these mutations on long-term disease outcome. One thousand five hundred eighty-one patients from the Mayo Clinic (n = 826) and Italy (n = 755) were studied. Fifty-eight percent of Mayo patients were followed until death; median survivals were 19.8 years in ET (n = 292), 13.5 PV (n = 267; hazard ratio [HR], 1.8; 95% confidence interval [CI], 1.4-2.2), and 5.9 PMF (n = 267; HR, 4.5; 95% CI, 3.5-5.7). The survival advantage of ET over PV was not affected by JAK2/CALR/MPL mutational status. Survival in ET was inferior to the age- and sex-matched US population (P < .001). In PMF (n = 428), but not in ET (n = 576), survival and blast transformation (BT) were significantly affected by mutational status; outcome was best in CALR-mutated and worst in triple-negative patients: median survival, 16 vs 2.3 years (HR, 5.1; 95% CI, 3.2-8.0) and BT, 6.5% vs 25% (HR, 7.6; 95% CI, 2.8-20.2), respectively. We conclude that life expectancy in morphologically defined ET is significantly reduced but remains superior to that of PV, regardless of mutational status. In PMF, JAK2/CALR/MPL mutational status is prognostically informative.

Impact of mutational status on outcomes in myelofibrosis patients treated with ruxolitinib in the COMFORT-II study.

The JAK1/JAK2 inhibitor ruxolitinib produced significant reductions in splenomegaly and symptomatic burden and improved survival in patients with myelofibrosis (MF), irrespective of their JAK2 mutation status, in 2 phase III studies against placebo (COMFORT-I) and best available therapy (COMFORT-II). We performed a comprehensive mutation analysis to evaluate the impact of 14 MF-associated mutations on clinical outcomes in 166 patients included in COMFORT-II. We found that responses in splenomegaly and symptoms, as well as the risk of developing ruxolitinib-associated anemia and thrombocytopenia, occurred at similar frequencies across different mutation profiles. Ruxolitinib improved survival independent of mutation profile and reduced the risk of death in patients harboring a set of prognostically detrimental mutations (ASXL1, EZH2, SRSF2, IDH1/2) with an hazard ratio of 0.57 (95% confidence interval: 0.30-1.08) vs best available therapy. These data indicate that clinical efficacy and survival improvement may occur across different molecular subsets of patients with MF treated with ruxolitinib.

Calreticulin mutation-specific immunostaining in myeloproliferative neoplasms: pathogenetic insight and diagnostic value

Mutations in the gene calreticulin (CALR) occur in the majority of JAK2- and MPL-unmutated patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF); identifying CALR mutations contributes to the diagnostic pathway of ET and PMF. CALR mutations are heterogeneous spanning over the exon 9, but all result in a novel common protein C terminus. We developed a polyclonal antibody against a 17-amino-acid peptide derived from mutated calreticulin that was used for immunostaining of bone marrow biopsies. We show that this antibody specifically recognized patients harboring different types of CALR mutation with no staining in healthy controls and JAK2- or MPL-mutated ET and PMF. The labeling was mostly localized in megakaryocytes, whereas myeloid and erythroid cells showed faint staining, suggesting a preferential expression of calreticulin in megakaryocytes. Megakaryocytic-restricted expression of calreticulin was also demonstrated using an antibody against wild-type calreticulin and by measuring the levels of calreticulin RNA by gene expression analysis. Immunostaining using an antibody specific for mutated calreticulin may become a rapid, simple and cost-effective method for identifying CALR-mutated patients complementing molecular analysis; furthermore, the labeling pattern supports the preferential expansion of megakaryocytic cell lineage as a result of CALR mutation in an immature hematopoietic stem cell.

JAK2V617F complete molecular remission in polycythemia vera/essential thrombocythemia patients treated with ruxolitinib

To the editor: Polycythemia vera (PV) and essential thrombocythemia (ET) are characterized by JAK2 V617F mutation in 95% and 60% of the patients, respectively.[1][1] Ruxolitinib is a JAK1/JAK2 inhibitor approved for myelofibrosis (MF) and more recently for hydroxyurea resistant/intolerant PV

Prognostic Impact of Bone Marrow Fibrosis in Primary Myelofibrosis. A Study of the AGIMM Group on 490 Patients

The prognostic significance of bone marrow (BM) fibrosis grade in patients with primary myelofibrosis (PMF) is still debated. A fibrosis grade greater than 1 was shown to associate with higher risk of death, and addition of fibrosis grade to IPSS score resulted in a more accurate prediction of survival. The aim of this study was to analyze the prognostic impact of BM fibrosis in 490 patients with PMF, evaluated at diagnosis, molecularly annotated and with extensive follow‐up information. We found that fibrosis grade 2 and greater on a 0–3 scale was associated with clinical characteristics indicative of a more advanced disease, such as anemia, leukopenia, thrombocytopenia, constitutional symptoms, larger splenomegaly and a higher IPSS risk category. Patients with higher grade of fibrosis were also more likely to have additional somatic mutations in ASXL1 and EZH2, that are prognostically adverse. Median survival was significantly reduced in patients with grade 2 and 3 fibrosis as compared with grade 1; this effect was maintained when analysis was restricted to younger patients. In multivariate analysis, fibrosis grade independently predicted for survival regardless of IPSS variables and mutational status; the adverse impact of fibrosis was noticeable especially in lower IPSS risk categories. Overall, results indicate that higher grades of fibrosis correlate with unique clinical and molecular aspects and represent an independent adverse variable in patients with PMF; these observations deserve confirmation in prospectively designed series of patients. Am. J. Hematol. 91:918–922, 2016.

Safety and efficacy of ruxolitinib in splanchnic vein thrombosis associated with myeloproliferative neoplasms

Splanchnic vein thrombosis (SVT) is one of the vascular complications of myeloproliferative neoplasms (MPN). We designed a phase 2 clinical trial to evaluate safety and efficacy of ruxolitinib in reducing splenomegaly and improving disease‐related symptoms in patients with MPN‐associated SVT. Patients diagnosed with myelofibrosis (12 cases), polycythemia vera (5 cases) and essential thrombocythemia (4 cases) received ruxolitinib for 24 weeks in the core study period. Spleen volume was assessed by magnetic resonance imaging (MRI) and splanchnic vein circulation by echo‐Doppler analysis. Nineteen patients carried JAK2V617F, one had MPLW515L, and one CALRL367fs*46 mutation. Eighteen patients had spleno‐portal‐mesenteric thrombosis, two had Budd–Chiari syndrome, and one had both sites involved; 16 patients had esophageal varices. Ruxolitinib was well tolerated with hematological toxicities consistent with those of patients without SVT and no hemorrhagic adverse events were recorded. After 24 weeks of treatment, spleen volume reduction ≥35% by MRI was achieved by 6/21 (29%) patients, and a ≥50% spleen length reduction by palpation at any time up to week 24 was obtained by 13/21 (62%) patients. At week 72, 8 of the 13 (62%) patients maintained the spleen response by palpation. No significant effect of treatment on esophageal varices or in splanchnic circulation was observed. MPN‐related symptoms, evaluated by MPN‐symptom assessment form (SAF) TSS questionnaire, improved significantly during the first 4 weeks and remained stable up to week 24. In conclusion, this trial shows that ruxolitinib is safe in patients with MPN‐associated SVT, and effective in reducing spleen size and disease‐related symptoms.

Impact of JAK2 ( V617F ) mutation status on treatment response to anagrelide in essential thrombocythemia: an observational, hypothesis-generating study

A JAK2(V617F) mutation is found in approximately 55% of patients with essential thrombocythemia (ET), and represents a key World Health Organization diagnostic criterion. This hypothesis-generating study (NCT01352585) explored the impact of JAK2(V617F) mutation status on treatment response to anagrelide in patients with ET who were intolerant/refractory to their current cytoreductive therapy. The primary objective was to compare the proportion of JAK2-positive versus JAK2-negative patients who achieved at least a partial platelet response (≤600×109/L) after anagrelide therapy. Of the 47 patients enrolled, 46 were included in the full analysis set (JAK2-positive, n=22; JAK2-negative, n=24). At 12 months, 35 patients (n=14 and n=21, respectively) had a suitable platelet sample; of these, 74.3% (n=26) achieved at least a partial response. The response rate was higher in JAK2-positive (85.7%, n=12) versus JAK2-negative patients (66.7%, n=14) (odds ratio [OR] 3.00; 95% confidence interval [CI] 0.44, 33.97). By using the last observation carried forward approach in the sensitivity analysis, which considered the imbalance in patients with suitable samples between groups, the overall response rate was 71.7% (n=33/46), with 77.3% (n=17/22) of JAK2-positive and 66.7% (n=16/24) of JAK2-negative patients achieving at least a partial response (OR 1.70; 95% CI 0.39, 8.02). There was no significant change in median allele burden over 12 months in the 12 patients who achieved a response. In conclusion, the overall platelet response rate was high in both JAK2-positive and JAK2-negative patients; however, a larger study would be required to confirm the differences observed according to JAK2(V617F) mutation status.

Long-term follow-up of concomitant treatment with romiplostim and warfarin in a patient with immune thrombocytopenia and severe cardiac comorbidities

Thrombopoietin receptor agonists (TRAs) offer new opportunities for the treatment of chronic immune thrombocytopenia (ITP). However, little is known about their safety and efficacy in combination with vitamin K antagonists in patients who need chronic anticoagulation. In this study, we report of a 68year-old man with chronic ITP and severe cardiac comorbidities who has achieved a 1-year follow-up of combined treatment with romiplostim and warfarin. He was affected by dilated-hypokinetic cardiopathy with chronic atrial fibrillation and was admitted to our hospital in February 2011 for progressive dyspnea and fatigue with diffuse mucocutaneous purpura. Coagulation laboratories were normal except for an international normalized ratio (INR) of 2.6, as he was on treatment with warfarin for the atrial fibrillation, and an ischemic stroke occurred 2 months before. Hematological data showed thrombocytopenia and mild leukocytosis (leukocytes 11.3 10/l with absolute neutrophil count 9.6 10/l; hemoglobin 13.5 g/dl; and platelet count 21 10/l). The patient was affected by ITP since 2008. Diagnosis of ITP was confirmed throughout by the exclusion of other causes of thrombocytopenia and with a bone marrow histological analysis showing an increased number of megakaryocytes with normal erythroid and myeloid lineages. He had received several cycles of prednisone and intravenous immunoglobulins (IVIG) for ITP. However, since about a year, IVIG were no longer effective. At the time of hospital admission, he was taking prednisone 1 mg/kg per day since 1 month, with poor tolerance due to an insulin-dependent type 2 diabetes mellitus. We stopped warfarin and administered intravenous vitamin K, achieving normalization of INR (1.1). An abdomen ultrasound scan showed a mild enlargement of the liver consistent with chronic heart failure but normal size of the spleen, whereas transthoracic echocardiography revealed a severe left ventricular systolic impairment (0.20 residual ejection fraction) and a moderate pulmonary hypertension. As thrombocytopenia prevented the use of warfarin, a transesophageal echocardiography was performed in order to evaluate the feasibility of percutaneous left atrial appendage transcatheter occlusion as a prophylaxis of cardioembolic stroke [1], but a recent left atrial appendage thrombosis was found. Thus, our patient had a chronic ITP with the loss of response to prednisone [2], but further treatment was needed to ensure a long-term control of platelet count in order to restore the anticoagulation. Splenectomy is a highly effective second-line treatment for chronic ITP but its complications also include thrombotic events and may be greater in patients older than 65 [3]. Furthermore, our patient had severe comorbidities that increased the risk of surgical and anesthesiological complications. TRAs are licensed for the treatment of chronic ITP in patients who fail to respond to splenectomy or have contraindications to it. Long-term data with romiplostim show a sustained platelet response and a significant reduction in the use of steroids, with an acceptable safety profile [4]. Nevertheless, patients treated with romiplostim in a multicenter phase III trial had a median weekly platelet count 50 10/l during quite all the treatment period, but the first quartile of the range was often below this threshold. Moreover, although non-splenectomized patients had a higher rate of durable platelet response (at least 6 weeks with platelet counts of 50 10/l or more during the last 8 weeks of treatment), no more than 61% of them achieved such end-point and the percentage only marginally improved by including patients who resorted to rescue therapies [5]. Thus, we choose to restore the anticoagulation at a higher threshold of platelet count (100 10/l) to have a broader safety margin in the case of sudden drops. Besides, as it has been shown that variations of the dosage of romiplostim are more likely to be required during the first 24 weeks of treatment and are substantial in a small number of patients, both platelet count and INR were checked weekly for such period and then every 2 weeks [4]. Romiplostim was started from 1 mcg/kg/week, adjusting dose on the basis of weekly platelet response according to the prescribing information [6]. Despite the loss of response and the poor tolerance to prednisone, we continued it until the