Long-term pharmacodynamic and clinical effects of twice- versus once-daily low-dose aspirin in essential thrombocythemia: The ARES trial.

Patients with essential thrombocythemia (ET) are treated with once-daily low-dose aspirin to prevent thrombosis, but their accelerated platelet turnover shortens the antiplatelet effect. The short-term Aspirin Regimens in EsSential Thrombocythemia trial showed that twice-daily aspirin dosing restores persistent platelet thromboxane (TX) inhibition. However, the long-term pharmacodynamic efficacy, safety and tolerability of twice-daily aspirin remain untested. We performed a multicenter, randomized, open-label, blinded-endpoint, phase-2 trial in which 242 patients with ET were randomized to 100 mg aspirin twice- or once-daily and followed for 20 months. The primary endpoint was the persistence of low serum TXB2, a surrogate biomarker of antithrombotic efficacy. Secondary endpoints were major and clinically relevant non-major bleedings, serious vascular events, symptom burden assessed by validated questionnaires, and in vivo platelet activation. Serum TXB2 was consistently lower in the twice-daily versus once-daily regimen on 10 study visits over 20 months: median 3.9 ng/mL versus 19.2 ng/mL, respectively; p < .001; 80% median reduction; 95% CI, 74%-85%. No major bleeding occurred. Clinically relevant non-major bleedings were non-significantly higher (6.6% vs. 1.7%), and major thromboses lower (0.8% vs. 2.5%) in the twice-daily versus once-daily group. Patients on the twice-daily regimen had significantly lower frequencies of disease-specific symptoms and severe hand and foot microvascular pain. Upper gastrointestinal pain was comparable in the two arms. In vivo platelet activation was significantly reduced by the twice-daily regimen. In patients with ET, twice-daily was persistently superior to once-daily low-dose aspirin in suppressing thromboxane biosynthesis and reducing symptom burden, with no detectable excess of bleeding and gastrointestinal discomfort.

Characterization of genetic variants in the EGLN1/PHD2 gene identified in a European collection of patients with erythrocytosis.

Hereditary erythrocytosis is a rare hematologic disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2,160 patients with erythrocytosis sequenced in ten different laboratories. We focused our study on the EGLN1 gene and identified 39 germline missense variants including one gene deletion in 47 probands. EGLN1 encodes the PHD2 prolyl 4-hydroxylase, a major inhibitor of hypoxia-inducible factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: (i) in silico studies of localization, conservation, and deleterious effects; (ii) analysis of hematologic parameters of carriers identified in the UK Biobank; (iii) functional studies of the protein activity and stability; and (iv) a comprehensive study of PHD2 splicing. Altogether, these studies allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extended to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences from the variants of unknown significance regarding the severity of the developed disease (hematologic parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare disorders in order to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematologic diseases.

Hematologic Neoplasms Associated with Down Syndrome: Cellular and Molecular Heterogeneity of the Diseases.

The molecular basis of Down syndrome (DS) predisposition to leukemia is not fully understood but involves various factors such as chromosomal abnormalities, oncogenic mutations, epigenetic alterations, and changes in selection dynamics. Myeloid leukemia associated with DS (ML-DS) is preceded by a preleukemic phase called transient abnormal myelopoiesis driven by GATA1 gene mutations and progresses to ML-DS via additional mutations in cohesin genes, CTCF, RAS, or JAK/STAT pathway genes. DS-related ALL (ALL-DS) differs from non-DS ALL in terms of cytogenetic subgroups and genetic driver events, and the aberrant expression of CRLF2, JAK2 mutations, and RAS pathway-activating mutations are frequent in ALL-DS. Recent advancements in single-cell multi-omics technologies have provided unprecedented insights into the cellular and molecular heterogeneity of DS-associated hematologic neoplasms. Single-cell RNA sequencing and digital spatial profiling enable the identification of rare cell subpopulations, characterization of clonal evolution dynamics, and exploration of the tumor microenvironment's role. These approaches may help identify new druggable targets and tailor therapeutic interventions based on distinct molecular profiles, ultimately improving patient outcomes with the potential to guide personalized medicine approaches and the development of targeted therapies.

A randomized double-blind trial of 3 aspirin regimens to optimize antiplatelet therapy in essential thrombocythemia.

Essential thrombocythemia (ET) is characterized by abnormal megakaryopoiesis and enhanced thrombotic risk. Once-daily low-dose aspirin is the recommended antithrombotic regimen, but accelerated platelet generation may reduce the duration of platelet cyclooxygenase-1 (COX-1) inhibition. We performed a multicenter double-blind trial to investigate the efficacy of 3 aspirin regimens in optimizing platelet COX-1 inhibition while preserving COX-2-dependent vascular thromboresistance. Patients on chronic once-daily low-dose aspirin (n = 245) were randomized (1:1:1) to receive 100 mg of aspirin 1, 2, or 3 times daily for 2 weeks. Serum thromboxane B2 (sTXB2), a validated biomarker of platelet COX-1 activity, and urinary prostacyclin metabolite (PGIM) excretion were measured at randomization and after 2 weeks, as primary surrogate end points of efficacy and safety, respectively. Urinary TX metabolite (TXM) excretion, gastrointestinal tolerance, and ET-related symptoms were also investigated. Evaluable patients assigned to the twice-daily and thrice-daily regimens showed substantially reduced interindividual variability and lower median (interquartile range) values for sTXB2 (ng/mL) compared with the once-daily arm: 4 (2.1-6.7; n = 79), 2.5 (1.4-5.65, n = 79), and 19.3 (9.7-40; n = 85), respectively. Urinary PGIM was comparable in the 3 arms. Urinary TXM was reduced by 35% in both experimental arms. Patients in the thrice-daily arm reported a higher abdominal discomfort score. In conclusion, the currently recommended aspirin regimen of 75 to 100 once daily for cardiovascular prophylaxis appears to be largely inadequate in reducing platelet activation in the vast majority of patients with ET. The antiplatelet response to low-dose aspirin can be markedly improved by shortening the dosing interval to 12 hours, with no improvement with further reductions (EudraCT 2016-002885-30).

Identification of a new VHL exon and complex splicing alterations in familial erythrocytosis or von Hippel-Lindau disease.

Chuvash polycythemia is an autosomal recessive form of erythrocytosis associated with a homozygous p.Arg200Trp mutation in the von Hippel-Lindau (VHL) gene. Since this discovery, additional VHL mutations have been identified in patients with congenital erythrocytosis, in a homozygous or compound-heterozygous state. VHL is a major tumor suppressor gene, mutations in which were first described in patients presenting with VHL disease, which is characterized by the development of highly vascularized tumors. Here, we identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. More importantly, we identify mutations in E1' in 7 families with erythrocytosis (1 homozygous case and 6 compound-heterozygous cases with a mutation in E1' in addition to a mutation in VHL coding sequences) and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, we show that the mutations induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. In addition, we demonstrate a pathogenic role for synonymous mutations in VHL exon 2 that altered splicing through E2-skipping in 5 families with erythrocytosis or VHL disease. In all the studied cases, the mutations differentially affected splicing, correlating with phenotype severity. This study demonstrates that cryptic exon retention and exon skipping are new VHL alterations and reveals a novel complex splicing regulation of the VHL gene. These findings open new avenues for diagnosis and research regarding the VHL-related hypoxia-signaling pathway.

Splanchnic vein thromboses associated with myeloproliferative neoplasms: An international, retrospective study on 518 cases.

Myeloproliferative Neoplasms (MPN) course can be complicated by thrombosis involving unusual sites as the splanchnic veins (SVT). Their management is challenging, given their composite vascular risk. We performed a retrospective, cohort study in the framework of the International Working Group for MPN Research and Treatment (IWG-MRT), and AIRC-Gruppo Italiano Malattie Mieloproliferative (AGIMM). A total of 518 MPN-SVT cases were collected and compared with 1628 unselected, control MPN population, matched for disease subtype. Those with MPN-SVT were younger (median 44 years) and enriched in females compared to controls; PV (37.1%) and ET (34.4%) were the most frequent diagnoses. JAK2V617F mutation was highly prevalent (90.2%), and 38.6% of cases had an additional hypercoagulable disorder. SVT recurrence rate was 1.6 per 100 patient-years. Vitamin K-antagonists (VKA) halved the incidence of recurrence (OR 0.48), unlike cytoreduction (OR 0.96), and were not associated with overall or gastrointestinal bleeding in multivariable analysis. Esophageal varices were the only independent predictor for major bleeding (OR 17.4). Among MPN-SVT, risk of subsequent vascular events was skewed towards venous thromboses compared to controls. However, MPN-SVT clinical course was overall benign: SVT were enriched in PMF with lower IPSS, resulting in significantly longer survival than controls; survival was not affected in PV and slightly reduced in ET. MPN-U with SVT (n = 55) showed a particularly indolent phenotype, with no signs of disease evolution. In the to-date largest, contemporary cohort of MPN-SVT, VKA were confirmed effective in preventing recurrence, unlike cytoreduction, and safe; the major risk factor for bleeding was esophageal varices that therefore represent a major therapeutic target.

Heterozygous FXII deficiency is not associated with an increased incidence of thrombotic events: Results of a long term study.

OBJECTIVE: To investigate the incidence of thrombotic events in patients heterozygous for FXII deficiency during a long observation period. PATIENTS AND METHODS: 103 heterozygotes for FXII deficiency, 49 female and 54 male were followed for 19.6 years (range 5-32 years). As controls 103 unaffected family members of same sex and similar age (±5 years) were enrolled. The thrombotic end points were: myocardial infarction, deep vein thrombosis and ischemic stroke. The mean Factor XII level in the heterozygotes was 48.5%: range (35-60%) that of control was 96.5% (range 70-155%). The heterozygotes showed one myocardial infarction, two deep vein thromboses and no ischemic stroke. The unaffected family members observed 2 myocardial infarctions, one deep vein thrombosis and one ischemic stroke. There were seven deliveries (five women) among the heterozygotes and six (five women) among the controls. Furthermore, four and five surgical procedures were carried out in the patient and in the control group, respectively. Immobilization times for surgical procedures or pregnancies were 50 days and 57 days for the heterozygotes and the unaffected family members, respectively. Heterozygotes for FXII deficiency did not show an increased incidence of thrombotic events as compared with unaffected family members during a long follow up.

Thrombotic events in severe FXII deficiency in comparison with unaffected family members during a long observation period.

To investigate the occurrence of thrombotic events (myocardial infarction, deep vein thrombosis or ischemic stroke) in a group of 39 cases of severe FXII deficiency during a mean 22.5 years follow-up. All patients seen in Padua during the years 1968-2006 will the object of this investigation. FXII was less than or 1% of normal in all cases. Factor FXII activity in unaffected family members was 98% (range 90-140%). No patient or control had a thrombotic event in the past and none were on anticoagulant therapy. FV Leiden was present in one patient and in two controls whereas the G to A20210 prothrombin polymorphism was absent in both groups. There was one death among the patients (breast cancer) and one among the control (car accident). There were two thrombotic events (myocardial infarction and deep vein thrombosis) in the patient group and three (myocardial infarction and two deep vein thrombosis) in the control group. Heterozygous FV Leiden was present in the patient who had venous thrombosis, One of the two control subjects who developed venous thrombosis had heterozygous FV Leiden and was on oral contraception. The second control subject who developed venous thrombosis was on oral contraception and had varicose veins. No ischemic stroke was observed in the patients or controls. Periods of immobilization were 42 days and 38 days, respectively for FXII deficient patients and for the controls. Patients with severe FXII deficiency may present thrombotic events but these are similar to these presented by unaffected family members. As a consequence it may be stated that severe FXII deficiency does not appear to effect thrombotic events.

Systemic Mastocytosis and Essential Thrombocythemia: Case Report and Literature Overview.

Mastocytosis is a rare disease in which heightened amounts of mast cells accumulate in the skin, bone marrow, and other visceral organs. Upon activation, mast cells release a wide variety of preformed or newly synthesized mediators which can induce allergic symptoms and inflammatory reactions. Mastocytosis is diagnosed by biopsy and can be divided into cutaneous and systemic mastocytosis (SM). The first one affects the skin and is relatively benign, whilst SM, which involves bone marrow and other organs, may be aggressive and associate with both myelodisplastic and myeloproliferative diseases. Here we present a case of SM associated with essential thrombocythemia and complicated by severe osteoporosis, successfully treated with hydroxyurea, low-dose aspirin and zolendronic acid.

Thromboses and hemorrhages are common in MPN patients with high JAK2V617F allele burden.

The most common causes of morbidity and mortality in myeloproliferative neoplasms (MPN) are thrombotic and hemorrhagic complications. The JAK2V617F mutation, commonly found in MPN, correlates with several clinical and laboratory characteristics even if the relevance of JAK2V617F allele burden in the natural history of these diseases is unclear. In this study we searched, a relation between thrombotic and hemorrhagic complications and JAK2V617F allele burden level in MPN patients. We evaluated 253 consecutive MPN [121 essential thrombocythemia (ET), 124 polycythemia vera (PV), and 8 primary myelofibrosis (PMF)] patients in whom the JAK2V617F allele burden was available, all studied and followed (median 8.8 years) in our department. Patients were stratified accordingly to their JAK2V617F allele burden, into four quartiles (1st <25%, 2nd 26-50%, 3rd 51-75%, and 4th >75%). Significantly higher incidence of thromboses (p = 0.001) and hemorrhages (p < 0.001) during follow-up has been observed in higher quartiles when compared to lower ones. Thrombosis- and hemorrhage-free survivals were poorer in patients belonging to the highest quartile. Our data suggest that MPN patients with JAK2V617F allele burden higher than 75% have to be considered as high risk patients, being prone to develop thrombo-hemorrhagic complications during the disease course.

Cardiovascular events and intensity of treatment in polycythemia vera.

BACKGROUND: Current treatment recommendations for patients with polycythemia vera call for maintaining a hematocrit of less than 45%, but this therapeutic strategy has not been tested in a randomized clinical trial. METHODS: We randomly assigned 365 adults with JAK2-positive polycythemia vera who were being treated with phlebotomy, hydroxyurea, or both to receive either more intensive treatment (target hematocrit, <45%) (low-hematocrit group) or less intensive treatment (target hematocrit, 45 to 50%) (high-hematocrit group). The primary composite end point was the time until death from cardiovascular causes or major thrombotic events. The secondary end points were cardiovascular events, cardiovascular hospitalizations, incidence of cancer, progression to myelofibrosis, myelodysplasia or leukemic transformation, and hemorrhage. An intention-to-treat analysis was performed. RESULTS: After a median follow-up of 31 months, the primary end point was recorded in 5 of 182 patients in the low-hematocrit group (2.7%) and 18 of 183 patients in the high-hematocrit group (9.8%) (hazard ratio in the high-hematocrit group, 3.91; 95% confidence interval [CI], 1.45 to 10.53; P=0.007). The primary end point plus superficial-vein thrombosis occurred in 4.4% of patients in the low-hematocrit group, as compared with 10.9% in the high-hematocrit group (hazard ratio, 2.69; 95% CI, 1.19 to 6.12; P=0.02). Progression to myelofibrosis, myelodysplasia or leukemic transformation, and bleeding were observed in 6, 2, and 2 patients, respectively, in the low-hematocrit group, as compared with 2, 1, and 5 patients, respectively, in the high-hematocrit group. There was no significant between-group difference in the rate of adverse events. CONCLUSIONS: In patients with polycythemia vera, those with a hematocrit target of less than 45% had a significantly lower rate of cardiovascular death and major thrombosis than did those with a hematocrit target of 45 to 50%. (Funded by the Italian Medicines Agency and others; ClinicalTrials.gov number, NCT01645124, and EudraCT number, 2007-006694-91.).

Idiopathic erythrocytosis: a study of a large cohort with a long follow-up.

Idiopathic erythrocytosis (IE) is an absolute erythrocytosis with no known cause, diagnosed by exclusion of primary and secondary erythrocytosis. Familial erythrocytosis (FE) is a rare disease and as the rare patients with JAK2-wild-type polycythemia vera (PV) may be misdiagnosed as IE. We compared 78 patients with IE, 21 with FE and 136 with PV in the effort to identify simple features capable of discriminating between them. FE patients were younger at diagnosis either than IE and PV (p < 0.001); IE and FE had lower WBC, platelet counts and higher serum EPO levels, and had splenomegaly and thrombotic events less frequently than PV patients. Phlebotomies to obtain a haematocrit lower than 45 % induce platelet count increase in 70 % of PV but not in IE. Mainly in men, normal spleen, normal platelet counts and no history of thrombosis at diagnosis argue against PV; diagnosis of IE could be supported by means of a cycle of venesection to see how it affects their platelet count. No simple data capable of distinguishing between IE and FE were identified; therefore, a case of sporadic erythrocytosis in a young patient should be investigated as a possible genetic cause.

Complex history of the discovery and characterization of congenital factor X deficiency.

Factor X (FX) plays a pivotal role in blood coagulation. FX represents the point where all coagulation systems converge and, once activated, it converts prothrombin into thrombin. The discovery and definition of FX are based on the description between 1956 and 1957 about three patients and their families with a peculiar defect later demonstrated to be almost identical. These patients were an American (Mr. Stuart), a British (Ms. Prower), and a Swiss with Italian background (infant Delia B). We stated "almost identical" because immunological and molecular biology studies subsequently revealed that even though the basic clotting defect was identical, the FX protein level and the mutation were different in each case. Mr. Stuart had no FX protein in his plasma and the mutation was Val298Met (homozygote). Ms. Prower instead had a normal level of FX protein and the mutation was Arg287Trp + Asp282Asn (compound heterozygote). Unfortunately, the status of the Swiss patient in this regard is not known. Subsequent studies described a few major variants (FX Friuli, FX Melbourne, FX Padua, and other similar patients), which showed peculiar activation patterns (FX Friuli had a normal Russell viper venom clotting time; FX Melbourne was defective only in the intrinsic coagulation system; FX Padua, on the contrary, was defective only in the extrinsic coagulation system). All these studies have informed on the great heterogeneity and complexity of the FX defect. The story of the discovery and classification of FX deficiency has contributed considerably to our understanding of blood coagulation. The three original families and the families of the major variants, together with the researchers that discovered them, should be remembered with deep respect and gratitude.

Patterns of presentation and thrombosis outcome in patients with polycythemia vera strictly defined by WHO-criteria and stratified by calendar period of diagnosis.

Most studies in polycythemia vera (PV) include patients with both remote and most recent diagnostic periods and are therefore vulnerable to inaccurate interpretation of time-dependent data. We addressed the particular issue by analyzing presenting characteristics and outcome data among 1,545 patients with WHO-defined PV stratified by a diagnosis period of before or after 2005, which coincides with the first description of JAK2V617F as the molecular marker of PV. Patients diagnosed after 2005 displayed lower hemoglobin values (P < 0.0001) and older age (P = 0.007) at diagnosis; we suggest ease of diagnosis offered by a molecular marker enabled earlier diagnosis and broader application across older age groups that is further enhanced by recent trends in increased attention and health monitoring for the elderly. Post-2005 diagnosed patients were also more or less likely to receive aspirin and cytoreductive therapy, respectively, and, despite their older age distribution, displayed significantly lower risk of thrombosis in high risk disease. Regardless of the contributing factors to the latter phenomenon, our observations underscore the need to reassess current demographics and frequencies of thrombosis in clinical trial designs including thrombosis prevention in PV.

Genetic basis of congenital erythrocytosis: mutation update and online databases.

Congenital erythrocytosis (CE), or congenital polycythemia, represents a rare and heterogeneous clinical entity. It is caused by deregulated red blood cell production where erythrocyte overproduction results in elevated hemoglobin and hematocrit levels. Primary congenital familial erythrocytosis is associated with low erythropoietin (Epo) levels and results from mutations in the Epo receptor gene (EPOR). Secondary CE arises from conditions causing tissue hypoxia and results in increased Epo production. These include hemoglobin variants with increased affinity for oxygen (HBB, HBA mutations), decreased production of 2,3-bisphosphoglycerate due to BPGM mutations, or mutations in the genes involved in the hypoxia sensing pathway (VHL, EPAS1, and EGLN1). Depending on the affected gene, CE can be inherited either in an autosomal dominant or recessive mode, with sporadic cases arising de novo. Despite recent important discoveries in the molecular pathogenesis of CE, the molecular causes remain to be identified in about 70% of the patients. With the objective of collecting all the published and unpublished cases of CE the COST action MPN&MPNr-Euronet developed a comprehensive Internet-based database focusing on the registration of clinical history, hematological, biochemical, and molecular data (http://www.erythrocytosis.org/). In addition, unreported mutations are also curated in the corresponding Leiden Open Variation Database.

Development and validation of an International Prognostic Score of thrombosis in World Health Organization-essential thrombocythemia (IPSET-thrombosis).

Accurate prediction of thrombosis in essential thrombocythemia (ET) provides the platform for prospective studies exploring preventive measures. Current risk stratification for thrombosis in ET is 2-tiered and considers low- and high-risk categories based on the respective absence or presence of either age > 60 years or history of thrombosis. In an international study of 891 patients with World Health Organization (WHO)-defined ET, we identified additional independent risk factors including cardiovascular risk factors and JAK2V617F. Accordingly, we assigned risk scores based on multivariable analysis-derived hazard ratios (HRs) to age > 60 years (HR = 1.5; 1 point), thrombosis history (HR = 1.9; 2 points), cardiovascular risk factors (HR = 1.6; 1 point), and JAK2V617F (HR = 2.0; 2 points) and subsequently devised a 3-tiered prognostic model (low-risk = < 2 points; intermediate-risk = 2 points; and high-risk = > 2 points) using a training set of 535 patients and validated the results in the remaining cohort (n = 356; internal validation set) and in an external validation set (n = 329). Considering all 3 cohorts (n = 1220), the 3-tiered new prognostic model (low-risk n = 474 vs intermediate-risk n = 471 vs high-risk n = 275), with a respective thrombosis risk of 1.03% of patients/y versus 2.35% of patients/y versus 3.56% of patients/y, outperformed the 2-tiered (low-risk 0.95% of patients/y vs high-risk 2.86% of patients/y) conventional risk stratification in predicting future vascular events.

Disease characteristics and clinical outcome in young adults with essential thrombocythemia versus early/prefibrotic primary myelofibrosis.

In the present study, we investigated disease characteristics and clinical outcome in young patients (< 40 years) with World Health Organization (WHO)-defined essential thrombocythemia (ET) compared with early/prefibrotic primary myelofibrosis (PMF) with presenting thrombocythemia. We recruited 213 young patients (median age, 33.6 years), including 178 patients (84%) with WHO-defined ET and 35 patients (16%) showing early PMF. Median follow-up time was 7.5 years. A trend for more overall thrombotic complications, particularly arterial, was seen in early PMF compared with ET. Progression to overt myelofibrosis was 3% in ET and 9% in early PMF, but no transformation into acute leukemia was observed. Combining all adverse events (thrombosis, bleeding, and myelofibrosis), the rate was significantly different (1.29% vs 3.43% of patients/year, P = .01) in WHO-ET and early PMF, respectively. In multivariate analysis, early PMF and the JAK2V617F mutation emerged as independent factors predicting cumulative adverse events.

Initial bone marrow reticulin fibrosis in polycythemia vera exerts an impact on clinical outcome.

We examined the prevalence and prognostic relevance of bone marrow reticulin fibrosis in 526 patients with World Health Organization-defined polycythemia vera evaluated at the time of initial diagnosis. Seventy-four patients (14%) displayed mostly grade 1 reticulin fibrosis, with only 2 cases showing higher-grade fibrosis. Presenting clinical and laboratory characteristics, including JAK2V617F allele burden, between patients with and without fibrosis were similar for the most part, with the exception of a higher prevalence of palpable splenomegaly in patients with fibrosis (P < .01). Patients with fibrosis were less prone to experience thrombosis during their clinical course (1.1 vs 2.7 per 100 patient-years; P = .03) and more prone to develop post-polycythemia vera myelofibrosis (2.2 vs 0.8 per 100 patient-years; P = .01). There was no significant difference between the 2 groups in terms of overall or leukemia-free survival. The present study clarifies the incidence, degree, and prognostic relevance of bone marrow fibrosis obtained at time of initial diagnosis of polycythemia vera.