Prefibrotic Primary Myelofibrosis Research Articles

Subcellular mislocalization of the transcription factor NF-E2 in erythroid cells discriminates prefibrotic primary myelofibrosis from essential thrombocythemia

AbstractThe World Health Organization (WHO) classification of myeloproliferative neoplasms (MPNs) comprises several entities including essential thrombocythemia (ET); primary myelofibrosis (PMF); and MPN, unclassifiable (MPN,U). Differential diagnosis between ET and early, prefibrotic PMF can be challenging but is critical because clinical course and outcome vary considerably between these entities. We have previously shown that the transcription factor nuclear factor erythroid 2 (NF-E2) is aberrantly expressed in MPN patients. Here we demonstrate that NF-E2 is mislocalized in PMF cells and that aberrant NF-E2 localization discriminates statistically highly significantly between ET and PMF. A threshold of 20% nuclear NF-E2 staining was cross-validated by “.682+ bootstrapping.” Moreover, this cutoff correctly classifies diagnostic bone marrow biopsies of MPN,U patients specified upon follow-up as ET or PMF with 92% accuracy. Because interobserver concordance between independent pathologists was high (Spearman's rank correlation coefficient, 0.727), we propose that quantitative NF-E2 immunohistochemistry represents a diagnostic tool that can reliably support a differential diagnosis between ET and PMF.

Myelofibrosis in Philadelphia chromosome–negative myeloproliferative neoplasms is associated with aberrant karyotypes

In Philadelphia chromosome-negative myeloproliferative neoplasms (Ph¯ MPN), non-specific chromosomal defects are detectable and essential thrombocythemia (ET) has the lowest rate of aberrations, whereas primary myelofibrosis (PMF) and post-polycythemia vera (PV) myelofibrosis have the highest rates of aberrations. The frequency of cytogenetic defects in pre-fibrotic stage PMF has not been characterized thus far and the underlying molecular defects of chromosomal instability are unknown. In this study, histopathological findings were correlated with cytogenetic data (n = 249). The expression of DNA repair factors ERCC1 and LIG4 were determined in Ph¯ MPN with and without cytogenetic aberrations. Pre-fibrotic PMF and ET have similarly low frequencies of karyotype anomalies. The expression of ERCC1, but not LIG4, is increased in fibrotic stage PMF but is not associated with accumulation of cytogenetic defects. In conclusion, aberrant karyotypes in Ph¯ MPN reflect the chromosomal instability in these diseases and, in comparison with pre-fibrotic stages, Ph¯ MPN with fibrosis has the highest frequency of cytogenetic aberrations.

Primary myelofibrosis: 2013 update on diagnosis, risk‐stratification, and management

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by stem cell-derived clonal myeloproliferation, abnormal cytokine expression, bone marrow fibrosis, anemia, splenomegaly, extramedullary hematopoiesis (EMH), constitutional symptoms, cachexia, leukemic progression, and shortened survival. Diagnosis is based on bone marrow morphology. The presence of fibrosis, JAK2/MPL mutation, or +9/13q- cytogenetic abnormality is supportive but not essential for diagnosis. Prefibrotic PMF mimics essential thrombocythemia in its presentation and the distinction is prognostically relevant. Differential diagnosis of myelofibrosis should include chronic myeloid leukemia, myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia. The Dynamic International Prognostic Scoring System-plus (DIPSS-plus) prognostic model for PMF can be applied at any point during the disease course and uses eight independent predictors of inferior survival: age >65 years, hemoglobin <10 g/dL, leukocytes >25 × 10⁹/L, circulating blasts ≥ 1%, constitutional symptoms, red cell transfusion dependency, platelet count <100 × 10⁹/L, and unfavorable karyotype (i.e., complex karyotype or sole or two abnormalities that include +8, -7/7q-, i(17q), inv(3), -5/5q-, 12p-, or 11q23 rearrangement). The presence of 0, 1, "2 or 3," and ≥ 4 adverse factors defines low, intermediate-1, intermediate-2, and high-risk disease with median survivals of approximately 15.4, 6.5, 2.9, and 1.3 years, respectively. A >80% two-year mortality is predicted by monosomal karyotype, inv(3)/i(17q) abnormalities, or any two of circulating blasts >9%, leukocytes ≥ 40 × 10⁹/L or other unfavorable karyotype. Most recently, mutations involving ASXL1, SRSF2, EZH2, and IDH1/2 or increased plasma IL-2R, IL-8, or serum-free light chain levels have been shown to adversely affect survival. Observation alone is adequate for asymptomatic low/intermediate-1 risk disease. Allogeneic stem cell transplantation (ASCT) is often considered for high risk disease. Conventional or experimental drug therapy is reasonable for symptomatic intermediate-1 or intermediate-2 risk disease; however, ASCT is an acceptable treatment option for such patients in the presence of ASXL1 or other prognostically adverse mutations. Splenectomy and low-dose radiotherapy are used for drug-refractory splenomegaly. Radiotherapy is also used for the treatment of non-hepatosplenic EMH, PMF-associated pulmonary hypertension, and extremity bone pain.

Decanucleotide Insertion Polymorphism of F7 Influences Significantly the Risk of Thrombosis in Patients with Essential Thrombocythemia

Abstract 1730Thrombosis is one of the main complications observed in patients with myeloproliferative neoplasm (MPN). The underlying mechanisms of thrombosis are not entirely clarified. The incidence of, and risk factors for, thrombosis appear to differ in distinct and accurately diagnosed sub-entities of MPN. Similar incidences have been reported for essential thrombocythemia (ET) and prefibrotic primary myelofibrosis (PMF) diagnosed according to the WHO 2008 criteria. However, the two sub-entities differ in terms of their risk factor profiles (Carobbio et al, Blood 2011; Buxhofer-Ausch et al, Am J Haematol 2012). A gain of function in procoagulant pathways is believed to be the reason for an inherited hypercoagulable state (Anderson et al, Crit Care Clin 2011). There is strong evidence indicating that certain thrombophilic single nucleotide polymorphisms account for increased risk of thrombotic complications (Di Castelnuovo et al, Thromb Res 2000; Shimasaki et al, J Am Coll Cardiol 1998; Elbaz et al, Stroke 2000; Mollaki et al, J Thromb Haemost 2004). Data concerning the impact of an inherited risk in patients with underlying MPN are scarce (Shetty, Thromb Res 2011). We aimed to investigate the influence of three pre-selected thrombophilic single nucleotide polymorphisms (SNPs) on the risk of thrombosis in patients diagnosed with ET or prefibrotic PMF according to the WHO 2008 criteria.In 167 patients with a valid consensus diagnosis of ET (n= 105) or prefibrotic PMF (n= 62), the thrombophilic SNPs of NOS3 (Glu 298 Asp), F7 (10 nucleotide insertion-deletion) and FCGR2A (His 131 Arg) were determined by AS-PCR or sequencing. These variables and certain disease-specific and laboratory parameters were correlated with the incidence of major arterial and venous thrombosis by using Cox-regression analysis. The latter was performed in the entire population as well as separately for ET and prefibrotic PMF.The frequency of SNPs of F7, NOS3 and FCGR2A did not differ significantly in ET and prefibrotic PMF. Notably, the homo- or heterozygous insertion variant of F7 was found to be a significant (multivariate analysis) risk factor for total thrombosis and arterial thrombosis in the entire population (HR 3.06, p= 0.0082 and HR 3.94, p= 0.0007, respectively) as well as on separate analysis of those patients with ET (HR 5.94, p= 0.001 and HR 9,47, p= 0.00024, respectively). The homozygous 298 Asp NOS variant was significantly (univariate analysis) associated with total thrombosis in the entire population (HR 3.29, p= 0.0257) as well as on separate analysis of the ET cohort (HR 4.739, p= 0.0161). These significances were lost on multivariate analysis. No significant associations were established with any of the tested thrombophilic SNPs and the risk of thrombosis in the prefibrotic PMF cohort.Our data show that the risk of thrombosis in ET diagnosed according to WHO 2008 is increased many-fold by the inherited insertion polymorphism of F7. This is very interesting because, in patients without an underlying pathological condition, this polymorphism appears to protect the organism against thrombosis by reducing FVII activity (Humphries et al, Thromb Haemost 1996). Moreover, the different impact of thrombophilic SNPs in ET compared to prefibrotic PMF might provide further evidence of the presumed difference in risk profiles between these two sub-entities of MPN. These data emphasize the importance of considering the inherited as well as acquired hypercoagulable state in patients with MPN. Larger studies are needed to confirm these findings. If proven true, we would be able to more accurately define the risk of thrombosis in an individual patient and subsequently tailor treatment and anticoagulation strategies. Disclosures:No relevant conflicts of interest to declare.

Gene expression profiling with principal component analysis depicts the biological continuum from essential thrombocythemia over polycythemia vera to myelofibrosis

The recent discovery of the Janus activating kinase 2 V617F mutation in most patients with polycythemia vera (PV) and half of those with essential thrombocythemia (ET) and primary myelofibrosis (PMF) has favored the hypothesis of a biological continuum from ET over PV to PMF. We performed gene expression profiling of whole blood from control subjects (n = 21) and patients with ET (n = 19), PV (n = 41), and PMF (n = 9) using DNA microarrays. Applying an unsupervised method, principal component analysis, to search for patterns in the data, we demonstrated a separation of the four groups with biological relevant overlaps between the different entities. Moreover, the analysis separates Janus activating kinase 2-negative ET patients from Janus activating kinase 2-positive ET patients. Functional annotation analysis demonstrates that clusters of gene ontology terms related to inflammation, immune system, apoptosis, RNA metabolism, and secretory system were the most significantly deregulated terms in the three different disease groups. Our results yield further support for the hypothesis of a biological continuum originating from ET over PV to PMF. Functional analysis suggests an important implication of these gene ontology clusters in the pathogenesis of these neoplasms and in disease evolution from ET over PV to PMF.

European Bone Marrow Working Group trial on reproducibility of World Health Organization criteria to discriminate essential thrombocythemia from prefibrotic primary myelofibrosis. Haematologica 2012;97(3):360-5 - Comment

Any study of myeloproliferative neoplasms (MPNs) that lacks adequate clinical input is doomed to cause diagnostic uncertainty and increased controversy. In the paper by Buhr et al. published in Haematologica,[1][1] the authors studied 102 cases of essential thrombocythemia (ET) and early primary

European Bone Marrow Working Group trial on reproducibility of World Health Organization criteria to discriminate essential thrombocythemia from prefibrotic primary myelofibrosis. Haematologica 2012;97(3):360-5 - Reply

We are grateful for the critical comments of Thiele et al. because they give us the opportunity to underline the difficulties in applying the current World Health Organization (WHO) classification of thrombocytic myeloproliferative neoplasms and to eliminate potential misunderstandings. 1. The

Risk Factors for Thrombosis in WHO-Defined Early/Prefibrotic Myelofibrosis: An International Study of 264 Patients,

Abstract 3846▪▪This icon denotes a clinically relevant abstract INTRODUCTIONThere is strong evidence indicating that the clear-cut separation of prefibrotic primary myelofibrosis (PMF) from essential thrombocythemia (ET) by consequent application of the World Health Organization (WHO) 2008 criteria is reflected in different, well defined clinical pictures and divergent prognoses (Barbui et al, JCO 2011,Thiele et al, Blood 2011). Very recently the specific risk profile for arterial and venous thrombosis in WHO- diagnosed ET was published (Carobbio et al, Blood 2011). In PMF so far all published data on vascular events were exclusively based on overt disease manifestations until now there are no data available regarding prefibrotic stages. Consequently, we aimed to evaluate the corresponding risk profile of patients with WHO-diagnosed prefibrotic PMF. METHODSA total number of 264 patients with WHO-defined prefibrotic PMF derived from either the Medical University of Vienna or an International Database (Barbui et al, JCO 2011) were studied. Nonfatal thrombotic events considered in this study were reported as rates per 100 patient-years and included transient ischemic attacks, thrombotic cerebrovascular accidents, coronary artery disease, myocardial infarction, peripheral arterial disease, deep vein thrombosis of peripheral vasculature, pulmonary embolism, and abdominal large vein thrombosis. Cardiovascular risks factors considered, comprised of arterial hypertension, diabetes mellitus and tobacco use. To evaluate risk factors for total thrombosis and for arterial and venous events in particular multivariate Cox- regression analysis including the co-variables sex, age, previous thrombotic event, laboratory parameters measured at diagnosis and need for cytoreductive and/or antiplatelet therapy during follow-up was calculated. P values less than 0.05 were considered as statistically significant. RESULTSAfter a median follow- up of 5.8 years (range0.0 – 27.2), the total rate of non fatal thrombotic events was 2.1% patient-years (95% CI, 1.5–2.8); the incidence of arterial events was higher (1.7% patient- years) than of venous events (0.6% patient-years).Considering thrombosis in general a higher white blood cell (WBC) count enhances the risk significantly (p=0.005; HR 1.15). This is also true in arterial events in particular (p=0.047; HR 1.12). A lower platelet count is associated with a higher risk for thrombotic events; for thrombosis in general this association is of borderline significance (p=0.056; HR 0.99), for arterial thrombosis in particular of significance (p= 0.042; HR 0.99). A lower hemoglobin level is associated with a higher risk for venous thrombosis (p=0.007; HR 0.59). CONCLUSIONLeukocytosis appears as a risk factor for thrombosis in general and also for arterial thrombosis in particular in WHO-diagnosed prefibrotic PMF. Moreover, higher platelet counts seem to decrease significantly the risk for thrombotic events in general and arterial thrombosis in particular. Anemia is associated with a higher risk for venous thrombosis. These observations are partly in line with recently published findings in WHO-diagnosed ET (Carobbio et al,2011) and might indicate the existence of a specific risk profile for thrombotic events in prefibrotic PMF. This is the first study reporting data on the risk profile for thrombosis in WHO-diagnosed prefibrotic PMF. Certainly these findings ask for validation in a larger patient population. Disclosures:Gisslinger:Novartis: Speakers Bureau; Celgene Austria: Research Funding, Speakers Bureau; AOP-Orphan Pharmaceuticals AG: Speakers Bureau. Vannucchi:Novartis: Honoraria.

Primary myelofibrosis: 2012 update on diagnosis, risk stratification, and management

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by stem cell-derived clonal myeloproliferation, abnormal cytokine expression, bone marrow fibrosis, anemia, splenomegaly, extramedullary hematopoiesis (EMH), constitutional symptoms, cachexia, leukemic progression, and shortened survival. Diagnosis is based on bone marrow morphology. The presence of fibrosis, JAK2/MPL mutation or +9/13q- cytogenetic abnormality is supportive but not essential for diagnosis. Prefibrotic PMF mimics essential thrombocythemia in its presentation and the distinction is prognostically relevant. Differential diagnosis of myelofibrosis should include chronic myelogenous leukemia, myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia. The Dynamic International Prognostic Scoring System-plus (DIPSS-plus) prognostic model for PMF can be applied at any point during the disease course and uses eight independent predictors of inferior survival: age >65 years, hemoglobin <10 g/dL, leukocytes >25 × 10(9) /L, circulating blasts ≥1%, constitutional symptoms, red cell transfusion dependency, platelet count <100 × 10(9) /L, and unfavorable karyotype (i.e., complex karyotype or sole or two abnormalities that include +8, -7/7q-, i(17q), inv(3), -5/5q-, 12p- or 11q23 rearrangement). The presence of 0, 1, "2 or 3," and ≥4 adverse factors defines low, intermediate-1, intermediate-2, and high-risk disease with median survivals of ~15.4, 6.5, 2.9, and 1.3 years, respectively. A >80% two-year mortality is predicted by monosomal karyotype, inv(3)/i(17q) abnormalities, or any two of circulating blasts >9%, leukocytes ≥40 × 10(9) /L or other unfavorable karyotype. Observation alone is adequate for asymptomatic low/intermediate-1 risk disease. Allogeneic stem cell transplantation or experimental drug therapy is considered for intermediate-2/ high risk disease. Conventional or experimental drug therapy is reasonable for symptomatic intermediate-1 risk disease. Splenectomy and low-dose radiotherapy are used for drug-refractory splenomegaly. Radiotherapy is also used for the treatment of non-hepatosplenic EMH, PMF-associated pulmonary hypertension, and extremity bone pain.

European Bone Marrow Working Group trial on reproducibility of World Health Organization criteria to discriminate essential thrombocythemia from prefibrotic primary myelofibrosis

The World Health Organization classification of myeloproliferative neoplasms discriminates between essential thrombocythemia and the prefibrotic phase of primary myelofibrosis. This discrimination is clinically relevant because essential thrombocythemia is associated with a favorable prognosis whereas patients with primary myelofibrosis have a higher risk of progression to myelofibrosis or blast crisis. To assess the reproducibility of the classification, six hematopathologists from five European countries re-classified 102 non-fibrotic bone marrow trephines, obtained because of sustained thrombocytosis. Consensus on histological classification defined as at least four identical diagnoses occurred for 63% of the samples. Inter-observer agreement showed low to moderate kappa values (0.28 to 0.57, average 0.41). The percentage of unclassifiable myeloproliferative neoplasms rose from 2% to 23% when minor criteria for primary myelofibrosis were taken into account. In contrast, the frequency of primary myelofibrosis dropped from 23% to 7%, indicating that the majority of patients with a histological diagnosis of primary myelofibrosis did not fulfill the complete criteria for this disease. Thus, over 50% of cases in this series either could not be reproducibly classified or fell into the category of unclassifiable myeloproliferative neoplasms. World Health Organization criteria for discrimination of essential thrombocythemia from prefibrotic primary myelofibrosis are poorly to only moderately reproducible and lead to a higher proportion of non-classifiable myeloproliferative neoplasms than histology alone.

Incidence and risk factors for bleeding in 1104 patients with essential thrombocythemia or prefibrotic myelofibrosis diagnosed according to the 2008 WHO criteria

In an international study of 1104 patients with essential thrombocythemia (ET), a histological review according to the 2008 World Health Organization (WHO) criteria confirmed ET in 891 patients (WHO-ET, 81%), and revised the diagnosis to prefibrotic primary myelofibrosis (PMF) in 180 patients (PMF, 16%). Major bleeding during follow-up occurred in 55 (6%) WHO-ET and 21 (12%) PMF patients (P = 0.009), at a rate of 0.79 and 1.39% patients per year, respectively, (P = 0.039). In a multivariable analysis, predictors of bleeding included diagnosis of PMF (P = 0.05; hazard ratio (HR) 1.74), leukocytosis (P = 0.04; HR 1.74), previous hemorrhage (P = 0.025; HR 2.35) and aspirin therapy (P=0.001; HR 3.16). The analysis restricted to patients with WHO-ET confirmed previous hemorrhage (P = 0.043; HR 1.92) and aspirin (P=0.027; HR 2.24) as independent risk factors. The current study reveals that major bleeding associated with thrombocytosis might be relatively specific to PMF, as opposed to WHO-defined ET. Furthermore, it shows that low-dose aspirin exacerbates these hemorrhagic events of PMF. In contrast, thrombocytosis per se was not a risk factor for bleeding; however, low-dose aspirin had a synergistic hemorrhagic effect unmasking the bleeding tendency of patients with extreme thrombocytosis. These observations carry significant therapeutic implications in these two WHO entities.

In Vitro Megakaryocyte Differentiation and Proplatelet Formation in Ph-Negative Classical Myeloproliferative Neoplasms: Distinct Patterns in the Different Clinical Phenotypes

BackgroundPh-negative myeloproliferative neoplasms (MPNs) are clonal disorders that include primary myelofibrosis (PMF), polycythemia vera (PV) and essential thrombocythemia (ET). Although the pathogenesis of MPNs is still incompletely understood, an involvement of the megakaryocyte lineage is a distinctive feature.Methodology/Principal FindingsWe analyzed the in vitro megakaryocyte differentiation and proplatelet formation in 30 PMF, 8 ET, 8 PV patients, and 17 healthy controls (CTRL). Megakaryocytes were differentiated from peripheral blood CD34+ or CD45+ cells in the presence of thrombopoietin. Megakaryocyte output was higher in MPN patients than in CTRL with no correlation with the JAK2 V617F mutation. PMF-derived megakaryocytes displayed nuclei with a bulbous appearance, were smaller than ET- or PV-derived megakaryocytes and formed proplatelets that presented several structural alterations. In contrast, ET- and PV-derived megakaryocytes produced more proplatelets with a striking increase in bifurcations and tips compared to both control and PMF. Proplatelets formation was correlated with platelet counts in patient peripheral blood. Patients with pre-fibrotic PMF had a pattern of megakaryocyte proliferation and proplatelet formation that was similar to that of fibrotic PMF and different from that of ET.Conclusions/SignificanceIn conclusion, MPNs are associated with high megakaryocyte proliferative potential. Profound differences in megakaryocyte morphology and proplatelet formation distinguish PMF, both fibrotic and prefibrotic, from ET and PV.

Mutations with epigenetic effects in myeloproliferative neoplasms and recent progress in treatment: Proceedings from the 5th International Post-ASH Symposium

Immediately following the 2010 annual American Society of Hematology (ASH) meeting, the 5th International Post-ASH Symposium on Chronic Myelogenous Leukemia and BCR-ABL1-Negative Myeloproliferative Neoplasms (MPNs) took place on 7–8 December 2010 in Orlando, Florida, USA. During this meeting, the most recent advances in laboratory research and clinical practice, including those that were presented at the 2010 ASH meeting, were discussed among recognized authorities in the field. The current paper summarizes the proceedings of this meeting in BCR-ABL1-negative MPN. We provide a detailed overview of new mutations with putative epigenetic effects (TET oncogene family member 2 (TET2), additional sex comb-like 1 (ASXL1), isocitrate dehydrogenase (IDH) and enhancer of zeste homolog 2 (EZH2)) and an update on treatment with Janus kinase (JAK) inhibitors, pomalidomide, everolimus, interferon-α, midostaurin and cladribine. In addition, the new ‘Dynamic International Prognostic Scoring System (DIPSS)-plus' prognostic model for primary myelofibrosis (PMF) and the clinical relevance of distinguishing essential thrombocythemia from prefibrotic PMF are discussed.

Gene Expression Profiling with Principal Component Analysis Depicts the Biological Continuum From Essential Thrombocythemia Over Polycythemia Vera to Myelofibrosis

AbstractAbstract 4115 Introduction:The classical Philadelphia-negative chronic myeloproliferative neoplasms (CMPNs) encompass polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). After several years, a proportion of patients with PV may develop increasing splenomegaly and bone marrow fibrosis to terminate in postpolycythemic myelofibrosis, which then has a very poor prognosis with median survival of only a few years. A high proportion of the ET-population has been shown to have prefibrotic PMF or a “PV-like” phenotype, which after a variable time may terminate in myelofibrosis with myeloid metaplasia (MMM). Although the concept of prefibrotic myelofibrosis as a separate disease entity to be distinguished from ET by distinct histopathological features has been questioned, it seems logical that a continuum exists from “early” myelofibrosis with no or minimal bone marrow fibrosis to the advanced stage of myelofibrosis with myeloid metaplasia. Accordingly, a new concept of these diseases as a biological continuum from ET over PV to PMF has emerged. Indeed, this biological continuum reflects the various clinical stages as also described for chronic myelogenous leukaemia (CML) – a chronic stable phase (ET-PV), an accelerated phase (transitional phase), and a terminal myelofibrosis or blast phase of the disease (PMF-BP).It is evident that the JAK2 V617F mutational load is an important determinant of phenotype, but other genetic and epigenetic events contribute to the phenotypic presentation. Several gene expression studies have identified genes which might be of pathogenetic relevance for the development of CMPNs. However, no study has applied an unsupervised method as Principal Component Analysis (PCA) to discover unknown trends in the data. We have carried out gene expression profiling with PCA to ascertain if this analysis might unravel distinct grouping of disease entities supporting the biological continuum from early ET over PV to the advanced myelofibrosis phase. Patients and Methods:Using HG-U133 Plus 2.0 microarray from Affymetrix, recognizing 54675 probe sets (38500 genes), gene expression microarray studies have been performed on control subjects (n=21) and patients with ET (n =19), PV (n=41), and PMF (n=9). An unsupervised statistical method, PCA, which is an exploratory tool used to uncover unknown trends in the data based on gene expression profiles, was applied to the data. Results:The results from the PCA confirm the hypothesis of a biological continuum from control subjects over ET to PV and finally PMF (figure 1). The figure also reveals that patients with PV in a transitional stage (PVtrans) with pancytosis, huge splenomegaly, and bone marrow fibrosis have a gene profile between PV and PMF. Furthermore, single gene analysis revealed that several genes are highly dysregulated in patients with ET, PV, and PMF compared to control subjects even after correcting for multiple testing. Discussion and Conclusions:Using global gene expression profiling with PCA in patients with different CMPNs (ET, PV, PMF), we have yielded support to the concept of a biological continuum from early ET to the advanced myelofibrosis stage. Studies are in progress to identify single genes or clusters of genes accounting for the biological continuum and accordingly the progression from ET over PV to myelofibrosis. Disclosures:No relevant conflicts of interest to declare.

Survival and Risk of Leukemic transformation in Essential Thrombocythemia Are Significantly Influenced by Accurate Morphologic Diagnosis: An International Study on 1,104 Patients

AbstractAbstract 457 Introduction-The 2008 World Health Organization (WHO) diagnostic criteria for myeloproliferative neoplasms (MPN) underscore the role of bone marrow (BM) morphology in distinguishing essential thrombocythemia (ET) from early, and at times prefibrotic, primary myelofibrosis (PMF) that clinically presents like ET. The value of such a distinction in predicting clinical outcome has been questioned by some investigators. Study Design-Clinician scientists and hematopathologists from seven international centers of excellence for ET convened to design a clinicopathologic database of patients locally diagnosed and treated as ET. Study eligibility criteria included availability of treatment-naïve BM specimens obtained within one year of diagnosis. All bone marrows subsequently underwent a central blinded re-review by the author of the pertinent WHO chapter (JT) who applied strict WHO guidelines to identify cases of early PMF that were spuriously diagnosed as ET. Results-Revision of the BM biopsies according to the WHO morphological criteria confirmed ET in 891 patients (81%). Diagnosis was revised to early, prefibrotic PMF in 180 patients (16%). The remaining 33 (3%) BM specimens were qualitatively inadequate for accurate classification or represented unclassified cases.i) At presentation, early PMF, as opposed to true ET, was characterized by higher leukocyte (p<0.0001) and platelet (p=0.002) counts, lower hemoglobin (p=0.01) and hematocrit (p=0.001) levels, higher serum lactate dehydrogenase level (p<0.0001), higher circulating CD34+ cell count (p=0.03) and higher rate of palpable splenomegaly. JAK2V617F mutational frequency was 60% in each of the two groups.ii) Median follow-up was 6.2 years for ET and 7.0 years for early PMF. During this period, the rates of arterial (1.2-1.4% patients/year) and venous (0.6 patients/year) thrombotic complications were similar in the two groups. However, patients with early PMF, as compared to those with ET, were more likely to develop overt myelofibrosis (1% vs. 0.5% patients/year; RR=2.0, p=0.04) or acute leukemia (0.6% vs. 0.1% patients/year; RR=5.2, p=0.001). Cumulative leukemic transformation rate at 10 and 15 years was 0.7% and 2.1% in true ET versus 5.8% and 11.7% in PMF Similarly, survival was significantly worse in patients with early PMF (mortality rate: 2.7% vs. 1.3% patients/year; RR=2.1, p=0.002).The 10 and 15-years overall survival rates were: 89% and 80% in true ET versus 76% and 59% in PMF. On multivariable analysis, other risk factors for poor survival were age > 60 years (HR=6.3, p<0.0001), a leukocyte count of > 11×109/L (HR=2.13, p<0.0001) and history of thrombosis (HR=3.0, p<0.0001). Conclusions-The current study validates the clinical relevance of strict adherence to WHO criteria in the diagnosis of ET and provides seminal information on the survival and risk of leukemic transformation in strictly WHO-defined ET, which appears to be substantially better than previously assumed. Disclosures:No relevant conflicts of interest to declare.

Study of Megakaryopoiesis and Proplatelet Formation In Myeloproliferative Disorders

AbstractAbstract 3075 Introduction.Myeloproliferative neoplsms (MPNs) are clonal disorders that include primary myelofibrosis (PMF), polycythemia vera (PV) and essential thrombocytemia (ET). Several abnormalities that could explain the myeloproliferation have been reported, and most important is a striking involvement of the megakaryocyte (Mk) lineage, with hyperplasia and dysplasia resulting in an excessive production of several cytokines and chemokines. Whereas molecular defect(s) associated with the development of MPNs have been described, their pathogenesis is not thoroughly elucidated. Hypothesis.The general objective of this research was to study the process of megakaryopoiesis in MPNs in order to understand whether the pathological mechanisms underlying MPNs are intrinsic defects of Mk function or depend on an altered regulation by the bone marrow microenvironment. Patients and Methods.We analyzed in vitro the Mk differentiation and proplatelet formation in 25 PMF patients, 13 ET/PV patients, and 19 healthy controls (HCs). Mks were differentiated from peripheral blood CD34+ or CD45+ cells in the presence of TPO for 14 days. Mature Mks were, then, grown in suspension or plated onto glass coverslips coated with collagen I or fibrinogen for additional 16 hours. Mk differentiation-maturation and proplatelet formation were evaluated by phase contrast and fluorescence microscopy upon cell staining with anti alpha-tubulin and CD41 antibodies. Measurement of Mk diameters was performed on acquired images, at least one hundred Mks were analyzed for each sample. Mk ploidy was evaluated by flow cytometry after staining with propidium iodide. Controls were analyzed in parallel with each patient sample. Results.Mk output was higher in patients with MPNs than HCs. Patients with PMF and PV had the highest Mk output, and in PMF the number of Mks was strictly associated with the V617F JAK2 genotype. PMF Mks presented smaller size and increased nuclear/cytoplasm ratio with respect to the other MPNs and HCs. Further, the measurement of Mk diameters revealed that PMF-derived Mks rarely reached the diameter of 40 μm, while HCs and ET/PV presented larger Mks. Finally, PMF produced decreased numbers of polyploid Mks (>8N) with respect to HCs. In order to explore if defects in Mk development were associated to altered Mk function, we investigated the competence to generate proplatelet by MPN-derived and HC-derived Mks. In HC samples, a 7.5% median of the total Mks formed proplatelets, while a significant defect in extending proplatelets was observed in PMF-derived Mks with respect to other MPNs and HCs. On the contrary, patients with PV or ET showed increased numbers of Mks forming proplatelets with respect to PMF and HCs. No differences were observed between pre-fibrotic and fibrotic PMF, and pre-fibrotic PMF-derived Mks formed a decreased number of proplatelet as compared to both ET/PV and HCs. Most importantly, platelet counts correlated with Mk ability of extending proplatelets of different MPN patients. Finally, we found that the proplatelets extended by patients with PMF presented peculiar morphological features with respect to the normal counterpart and patients with PV and ET. Their more evident alteration was a significantly reduced number of bifurcations in secondary processes despite a normal length of the individual proplatelet shafts, thus indicating a defect in proplatelet branching. On the opposite, ET- and PV-derived proplatelets presented an enormous increase of bifurcations with respect to PMF and HCs. Summary and Conclusions.Mks from patients affected by MPNs presented high proliferative capacity and defects in proplatelet formation. Thus, platelet production in MPNs may not depend only on an increased number of Mks, but also on intrinsic defects in Mk function. Interestingly, our results also showed that Mk of pre-fibrotic and fully fibrotic PMF present similar biological characteristics and different from those of ET, providing a rationale for considering pre-fibrotic as a different entity from ET. In conclusion, this study proposes important new elements in the understanding of the biology of Mk and proplatelet formation in MPNs, and open a new perspective into the understanding of the pathophysiology of defects of platelet production in MPNs. Disclosures:No relevant conflicts of interest to declare.

Aberrant proplatelet formation in chronic myeloproliferative neoplasms

Proplatelets represent pseudopodia of megakaryocytes which extend into bone marrow sinuses to release platelets. Proplatelets were visualized by immunohistochemical and confocal microscopy. In trephines from essential thrombocythaemia (ET, n = 10), fibrotic ( n = 10) and pre-fibrotic ( n = 10) primary myelofibrosis (PMF) there was a significant increase of proplatelet density compared with normal bone marrow samples ( n = 10; p < 0.001). Manifest fibrosis exhibited the highest density and volume ratio with significant differences to non-fibrotic PMF ( p < 0.001) and ET ( p < 0.001). This study demonstrates that besides megakaryocytic proliferation extensive pseudopodial proplatelet formation provides a hallmark of MPN. Fibrosing differ from non-fibrosing MPN by density and size of aberrant proplatelets.

Practical application and clinical impact of the WHO histopathological criteria on bone marrow biopsy for the diagnosis of essential thrombocythemia versus prefibrotic primary myelofibrosis

To evaluate the feasibility of the histopathological diagnosis of prefibrotic-early primary myelofibrosis (PM) as described in the World Health Organization (WHO) classification and to evaluate the clinical implications of prefibrotic-early PM in a series of patients previously diagnosed as having essential thrombocythemia (ET) according to the Polycythemia Vera Study Group criteria. WHO criteria were applied to bone marrow biopsy specimens by two pathologists who then reclassified 127 cases as 102 ET (80.3%), 18 prefibrotic-early PM (14.2%) and seven fibrotic PM (5.5%). In 45 cases (35%), the final diagnosis was only reached by consensus. The megakaryocytic criteria that best discriminated between ET and prefibrotic-early PM were an increased nucleo-cytoplasmic ratio, presence of cloudlike nuclei, hyperchromatic-dysplastic nuclei, paratrabecular megakaryocytes and tight clusters. A histological score discriminated between ET (score < or =3) and PM (score > or =6), but 21 cases showed an intermediate ambiguous score. No significant differences were observed at diagnosis and at follow-up (median time 93 months) for thrombosis, major haemorrhage, laboratory data, transformation into overt myeloid metaplasia and survival. The distinction between ET and prefibrotic-early PM is impaired by subjectivity in pathological practice and is of questionable clinical relevance, at least when considering individual patients.

High Frequency of Circulating Endothelial Colony Forming Cells (ECFCs) in Myeloproliferative Neoplasms (MPNs) Is Associated with Diagnosis of Prefibrotic Myelofibrosis, History of Splanchnic Vein Thrombosis, and Vascular Splenomegaly.

Abstract 309> Background.High mobilization into peripheral blood of putative endothelial progenitor cells (EPCs) has been proposed as a characteristic of primary myelofibrosis (PMF) among the myeloproliferative neoplasms (MPNs). Endothelial colony forming cells (ECFCs) are a distinct colony type with robust proliferative potential and vessel forming capacity in vivo, and candidates to represent true EPCs. Low number of circulating ECFCs, however, imposes analytical work out in order to use this measurement as a feasible biological indicator of EPC mobilization. Here we report the assessment of ECFCs in patients with MPNs, based on a high number of patients in order to characterize the MPN categories and patients' clinical phenotype that better correlate with such measurement. Materials and methods.One hundred thirty nine patients with PMF, 32 patients with essential thrombocythemia (ET) or polycythemia vera (PV) and 22 normal subjects were studied. ECFCs were grown in vitro by plating mononuclear cells, obtained from 40 ml of peripheral blood, onto collagen treated culture plates in the presence of EGM-2MV medium, according to Ingram et al (Blood, 2004). Medium was changed every 3 days. Plates were scored for the presence of ECFC every 2 days, starting from 10 days of culture and up to 28 days. JAK-2V617F mutation was assessed on patients granulocytes by semi-nested PCR. Results.Patients with PMF had a significant higher (p<0.5) median frequency of ECFCs in peripheral blood (0.48 ECFC/10 ml, range 0–6) with respect to normal subjects (0.14, range 0–0.5) and patients with ET (0.25, range 0–1) or PV (0.13, range 0–0.57). Frequency was higher (p<0.05) in patients with prefibrotic PMF (0.86 ECFC/10 ml; n=36) than in patients with fibrotic PMF (0.41/10 ml; n=103). A history of previous splanchnic vein thrombosis (SVT) was associated to statistically higher (p<0.01) ECFC frequency (1.45 ECFC/10 ml; n=21) compared to patients without thrombosis (0.46/10 ml; n=118), as well as to healthy subjects, either with or without history of thrombosis (0.29 and 0.14, respectively, p<0.05 for both). At univariate analysis, increased expression of circulating ECFCs in PMF was associated with younger age (r = − 0.22, p<0.02), diagnosis of prefibrotic myelofibrosis (r = 0.72, p<0.01) and history of splanchnic vein thrombosis (r = 0.68, p<0.001). No correlation was found with the JAK-2 mutational status. At multivariate analysis all the three characteristics maintained an independent association. Since there was no association between the frequency of circulating ECFCs and the time from the onset of SVT, and since patients with an idiopathic SVT had marginal increase in ECFCs frequency compared to healthy subjects, we favored the hypothesis that an increased frequency of circulating ECFC is associated with the myeloproliferative phenotype more than with thrombosis per se. Finally, in the whole population of patients, a direct relationship (r= 0.71, p< 0.02) between the frequency of circulating ECFCs and the index of vascular splenomegaly (spleen index / number of circulating hematopoietic CD34+ cells) was evidenced. Conclusion.Increased mobilization into peripheral blood of ECFCs is a distinctive and new biomarker of prefibrotic myelofibrosis. In addition, this study addresses to the hypothesis that mobilization of ECFCs in MPNs is associated with high risk of SVT and with increased vasculature in the spleen, suggesting a potential role for EPCs in neoangiogenetic processes in this organ. Disclosures:No relevant conflicts of interest to declare.

JAK2 V617F allele burden discriminates essential thrombocythemia from a subset of prefibrotic-stage primary myelofibrosis

Among Philadelphia chromosome-negative myeloproliferative neoplasms (Ph(-) MPN), essential thrombocythemia (ET) and the prefibrotic phase of primary myelofibrosis (PMF) represent two subtypes with considerable overlap. In this study, histopathological classification of 490 MPN cases was correlated with the allelic burden of JAK2(V617F) and MPL(W515L). Ph(-) MPN entities largely overlap with regard to JAK2(V617F) and MPL(W515L) allele burden, but ET displayed mutant allele burden <50%. PMF with different stages of myelofibrosis all yielded similar JAK2(V617F) allele burden. At initial presentation one-quarter of prefibrotic PMF cases exhibited an allele burden exceeding 50% (38% median JAK2(V617F) alleles, n=102). In ET, its main differential diagnosis, not a single case was found with >40% JAK2(V617F) alleles (median, 24% JAK2(V617F) alleles; n=90; p<0.001). Increase in JAK2(V617F) alleles during follow-up could not be linked to fibrosis or blastic progression but was related to polycythemic transformation in ET. MPL(W515L) was found in 3% of ET and 8% of PMF, with a significantly higher percentage of mutated alleles in fibrotic than prefibrotic PMF (median, 78% MPL(W515L) alleles; p<0.05). Histopathological categories ET and prefibrotic PMF correlate with significant differences in mutant allelic burden of JAK2(V617F), but not of MPL(W515L) which, by contrast to JAK2(V617F), shows a higher percentage of mutated alleles in fibrotic than in prefibrotic cases. Thus, for Ph(-) MPN in which ET and prefibrotic PMF represent the most probable diagnoses, a JAK2(V617F) allele burden >50% favors a diagnosis of prefibrotic PMF.