miR-146a rs2431697 identifies myeloproliferative neoplasm patients with higher secondary myelofibrosis progression risk.

Myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PV) and essential thrombocythemia (ET), and remarkably shortens survival. Although JAK2V617F and CALR allele burden are the main transformation risk factors, inflammation plays a critical role by driving clonal expansion toward end-stage disease. NF-κB is a key mediator of inflammation-induced carcinogenesis. Here, we explored the involvement of miR-146a, a brake in NF-κB signaling, in MPN susceptibility and progression. rs2910164 and rs2431697, that affect miR-146a expression, were analyzed in 967 MPN (320 PV/333 ET/314 MF) patients and 600 controls. We found that rs2431697 TT genotype was associated with MF, particularly with post-PV/ET MF (HR = 1.5; p < 0.05). Among 232 PV/ET patients (follow-up time=8.5 years), 18 (7.8%) progressed to MF, being MF-free-survival shorter for rs2431697 TT than CC + CT patients (p = 0.01). Multivariate analysis identified TT genotype as independent predictor of MF progression. In addition, TT (vs. CC + CT) patients showed increased plasma inflammatory cytokines. Finally, miR-146a-/- mice showed significantly higher Stat3 activity with aging, parallel to the development of the MF-like phenotype. In conclusion, we demonstrated that rs2431697 TT genotype is an early predictor of MF progression independent of the JAK2V617F allele burden. Low levels of miR-146a contribute to the MF phenotype by increasing Stat3 signaling.

miR-203 and miR-221 regulate SOCS1 and SOCS3 in essential thrombocythemia.

The biological basis of essential thrombocythemia (ET) patients lacking known mutations is still unknown. MicroRNAs (miRNA) regulate hematopoietic differentiation and are deregulated in several hematopoietic malignancies. However, miRNA expression in ET patients has been poorly explored. We performed miRNA profiling in platelets from 19 ET patients and 10 healthy controls. Hierarchical cluster analysis showed two well-separated clusters between patients and controls, indicating that ET platelets had a characteristic 70-miRNA signature (P<0.0001), 68 of which were downregulated. According to the mutational status, three differentially expressed miRNAs, miR-15a (P=0.045), miR-150 (P=0.001) and miR-519a (P=0.036), were identified. A 40-miRNA signature was identified characterizing JAK2V617F-positive ET patients. Eight genes, whose interaction with the miRNAs could activate the JAK/STAT pathway were identified. An inverse correlation was observed between miRNAs expression and their target genes for SOCS1 and miR-221, SOCS3 and miR-221, SOCS3 and miR-203, and PTPN11 and miR-23a. All three miRNAs were upregulated in JAK2V617F-negative ET patients. SOCS1 and SOCS3 were validated as targets of miR-221 and miR-203, respectively. In summary, our study shows that platelets from JAK2V617F-negative ET patients harbor a specific miRNA signature that can participate in the modulation of the JAK/STAT pathway through regulation of key genes as SOCS1 and SOCS3.

Current opinion and consensus statement regarding the diagnosis, prognosis, and treatment of patients with essential thrombocythemia: a survey of the Spanish Group of Ph-negative Myeloproliferative Neoplasms (GEMFIN) using the Delphi method.

The current consensus on the diagnosis, prognosis, and treatment of essential thrombocythemia (ET) is based on experts' recommendations. However, several aspects of the diagnosis of, prognosis of, and therapy for ET are still controversial. The Delphi method was employed with an expert panel of members of the Spanish Group of Ph-negative Myeloproliferative Neoplasms in order to identify the degree of agreement on the diagnosis, prognosis, and treatment of ET. Nine leading experts selected a total of 41 clinical hematologists with well-known expertise in ET. An electronic questionnaire was used to collect the questions rated in a four-step scale. The questions were grouped into four blocks: diagnosis, risk stratification, goals of therapy, and treatment strategy. After the first round consisting of 80 questions, a second round including 14 additional questions focused on the recommendations advocated by experts of the European LeukemiaNet in 2011 was analyzed. The median and mean values for the first and second rounds were calculated. A summary of the conclusions considered as the most representative of each block of questions is presented. The Delphi method is a powerful instrument to address the current approaches and controversies surrounding ET.

Clinical end points for drug treatment trials in BCR-ABL1-negative classic myeloproliferative neoplasms: consensus statements from European LeukemiaNET (ELN) and Internation Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MRT).

The discovery of somatic mutations, primarily JAK2V617F and CALR, in classic BCR-ABL1-negative myeloproliferative neoplasms (MPNs) has generated interest in the development of molecularly targeted therapies, whose accurate assessment requires a standardized framework. A working group, comprised of members from European LeukemiaNet (ELN) and International Working Group for MPN Research and Treatment (IWG-MRT), prepared consensus-based recommendations regarding trial design, patient selection and definition of relevant end points. Accordingly, a response able to capture the long-term effect of the drug should be selected as the end point of phase II trials aimed at developing new drugs for MPNs. A time-to-event, such as overall survival, or progression-free survival or both, as co-primary end points, should measure efficacy in phase III studies. New drugs should be tested for preventing disease progression in myelofibrosis patients with early disease in randomized studies, and a time to event, such as progression-free or event-free survival should be the primary end point. Phase III trials aimed at preventing vascular events in polycythemia vera and essential thrombocythemia should be based on a selection of the target population based on new prognostic factors, including JAK2 mutation. In conclusion, we recommended a format for clinical trials in MPNs that facilitates communication between academic investigators, regulatory agencies and drug companies.

Diarrheic syndrome as a clinical sign of intestinal infiltration in progressive B-cell chronic lymphocytic leukemia.

Gastrointestinal involvement is a rare event in patients with B-cell chronic lymphocytic leukemia (B-CLL) and is usually associated to lymphomatous transformation. However, in autopsy studies the reported incidence of microscopic infiltration can reach up to 50% of cases. Seven B-CLL patients in advanced stage/progressive disease were evaluated by colonoscopy because of continuous diarrhea. Five out of seven patients (71%) presented histological evidence of colonic infiltration. Persistent diarrhea in patients with progressive/advanced B-CLL can be a clinical sign of intestinal infiltration and justifies endoscopic examinations.

Gene expression profiling distinguishes JAK2V617F-negative from JAK2V617F-positive patients in essential thrombocythemia.

To explore the gene expression signature in essential thrombocythemia (ET) patients in relation to JAK2V617F mutational status, expression profiling in circulating granulocytes was performed. Twenty ET were studied by microarray analysis and the results were confirmed by real-time quantitative RT-PCR in 40 ET patients, not receiving cytoreductive treatment. A heterogeneous molecular signature characterized by two main gene expression patterns was found: one with an upregulation of inflammatory genes related to neutrophil activation and thrombosis, and the other with significantly lower expression of these genes. Supervised clustering analysis showed 30 genes differentially expressed between JAK2V617F-negative and JAK2V617F-positive ET patients. Among the JAK2V617F-negative, a set of 14 genes (CISH, C13orf18, CCL3, PIM1, MAFF, SOCS3, ID2, GADD45B, KLF5, TNF, LAMB3, HRH4, TAGAP and TRIB1) showed an abnormal expression pattern. In this group of patients, CISH, SOCS2, SOCS3 and PIM1 genes, all involved in JAK-STAT signalling pathway, presented a lower expression. A two-gene predictor model was built comprising FOSB and CISH genes, which were the best discriminators of JAK2V617F status. In conclusion, JAK2V617F-negative ET patients present a characteristic gene expression profile, different from JAK2V617F-positive patients. Other pathways, besides JAK-STAT, might be implicated in the pathophysiology of JAK2V617F-negative ET patients.

Essential thrombocythemia in young individuals: frequency and risk factors for vascular events and evolution to myelofibrosis in 126 patients.

The frequency of vascular events and evolution to myelofibrosis (MF) in young individuals with essential thrombocythemia (ET) is not well known. The incidence and predisposing factors to such complications was studied in 126 subjects diagnosed with ET at a median age of 31 years (range: 5-40). Overall survival and probability of survival free of thrombosis, bleeding and MF were analyzed by the Kaplan-Meier method and the presence of the Janus Kinase 2 (JAK2) V617F mutation correlated with the appearance of such complications. The JAK2 mutation (present in 43% of patients) was associated with higher hemoglobin (Hb) (P<0.001) and lower platelets at diagnosis. With a median follow-up of 10 years (range: 4-25), 31 thrombotic events were registered (incidence rate: 2.2 thromboses/100 patients/year). When compared with the general population, young ET patients showed a significant increase in stroke (odds ratio 50, 95% CI: 21.5-115) and venous thromboses (odds ratio 5.3, 95% CI: 3.9-10.6). Thrombosis-free survival was 84% at 10 years, with tobacco use being associated with higher risk of thrombosis. Actuarial freedom from evolution to MF was 97% at 10 years. In conclusion, young ET patients have thrombotic events, especially stroke and venous thrombosis, more frequently than generally considered, whereas they rarely transform to MF.

A unified definition of clinical resistance/intolerance to hydroxyurea in essential thrombocythemia: results of a consensus process by an international working group.

A widely accepted definition of resistance or intolerance to hydroxyurea (HU) in patients with essential thrombocythemia (ET) is lacking. An international working group (WG) was convened to develop a consensus formulation of clinically significant criteria for defining resistance/intolerance to HU in ET. To this aim, an analytic hierarchy process (AHP), a multiple-attribute decision-making technique, was used. The steps consisted of selecting the candidate criteria for defining resistance/intolerance; identifying the motivations that could influence the preference of the WG for any individual criterion; comparing the candidate criteria in a pair-wise manner; and grading them according their ability to fulfill the motivations. Every step in the model was derived by questionnaires or group discussion. The WG proposed that the definition of resistance/intolerance should require the fulfillment of at least one of the following criteria: platelet count greater than 600,000/micro l after 3 months of at least 2 g/day of HU (2.5 g/day in patients with a body weight over 80 kg); platelet count greater than 400,000/micro l and WBC less than 2500/micro l or Hb less than 10 g/dl at any dose of HU; presence of leg ulcers or other unacceptable muco-cutaneous manifestations at any dose of HU; HU-related fever.

Relationship among densitometry, bone histomorphometry, and histologic stage in idiopathic myelofibrosis.

Idiopathic myelofibrosis (IMF) induces dramatic changes in bone. Bone remodeling and densitometric alterations in a series of nine patients with IMF and their relationship with the histologic stage of the disease were assessed. Patients were included at diagnosis and a bone marrow biopsy, dual-energy X-ray absorptiometry, and transiliac bone biopsy for histomorphometric analysis were performed. Five cases were classified as IMF histologic stage 1, one as stage 2, and three as stage 3. Compared with 40 age- and sex-matched controls, the following histomorphometric parameters were significantly higher in our patients: bone volume (BV/TV), osteoblast surface (Ob.S/BS), eroded surface (ES/BS), osteoclast surface (Oc.S/BS), osteoclast number (N.Oc/TA), mineralizing surface (MS/BS), reversal period (Rv.P), and remodeling period (Rm.P). Mineral apposition rate (MAR) and erosion depth (E.Depth) were significantly decreased (P < 0.05 for all comparisons). Bone mineral density (BMD) measurements showed high values for patient age and sex both at femur neck (Z score range +0.19 to +7) and total femur (Z score range -0.09 to +6.48). When densitometric values were analyzed according to IMF histologic stage, patients in stages 1 and 2 had significantly lower BMD values than to those in stage 3 (P = 0.024). In conclusion, patients with IMF present a characteristic bone histomorphometric pattern with increased bone volume and bone cells but low apposition and decreased erosion depth, suggesting a positive balance in bone remodeling units. This balance would produce the increase in bone mass observed in this disease. Given the increase in BMD observed with more advanced stages of IMF, this noninvasive method could be useful tool for assessing IMF progression.

Waldenström macroglobulinaemia: presenting features and outcome in a series with 217 cases.

In this report we analyse the presenting features of a series of patients diagnosed with Waldenström macroglobulinaemia (WM) in Spain over the last 10 years. Criteria for diagnosis required a serum monoclonal IgM protein > or = 30 g/l and > 20% bone marrow lymphocytes. Two hundred and seventeen patients were included in the study, with a median age of 69 years and male/female ratio of 2:1. The most common symptoms at diagnosis were anaemia (38%), hyperviscosity (31%), B symptoms (23%), bleeding (23%) and neurological symptoms (22%). Sixty-one patients (27%) were asymptomatic at diagnosis and, to date, 32 of them have not received chemotherapy. Variables predicting a shorter survival free of therapy were haemoglobin < 12.5 g/dl and high beta2microglobulin (beta2M). The 83% of patients who did receive treatment were distributed as follows: chlorambucil/prednisone (43%), intermittent chlorambucil (11%), continuous chlorambucil (26%), cyclophosphamide/vincristine/prednisone (COP, 13.5%) and other (6.5%). Response to therapy was complete in 2%, partial in 48% and minor in 10%. Finally, 28% and 13% of patients presented stable and progressive disease, respectively, which was more common among patients treated with COP. Progression-free survival was 43% at 5 years, with three independent predictors for shorter progression-free survival (PFS): COP treatment, age > 65 and B symptoms at diagnosis. The 10-year projected overall survival (OS) was 55%. The two most frequent causes of death were development of second malignancies (31%), or infections (19%). The two main variables predicting a poor OS were hyperviscosity and high beta2M. In summary, this study favours the use of chlorambucil-based therapy as the standard treatment for WM, and describes a subset of patients who should be considered as suffering a smouldering form and who therefore do not require treatment for a long period of time.