Abstract: To elucidate the role of thrombocytosis, alone or in combination with standard (age, previous cardiovascular events) and novel (leukocytosis, JAK2(V617F) mutational status) risk factors, in the cardiovascular events of essential thrombocythemia (ET), we analyzed a cohort of 1063 patients. We found that a platelet count at diagnosis greater than 1000 x 10(9)/L was associated with significantly lower rate of thrombosis in multivariable analysis and, if combined with leukocytes less than 11 x 10(9)/L, pointed to a "low-risk" category with a rate of thrombosis of 1.59% of patients/year. On the contrary, the highest risk category (thrombosis rate, 2.95% of patients/year) was constituted of patients with leukocytosis, lower platelet count, and a JAK2(V617F) mutated genotype in most cases (77% vs 26% in the low-risk group), independently from standard risk factors. These data challenge the theory that elevated platelet count increases thrombosis risk in ET and suggest prospective clinical trials to support this hypothesis.
Abstract: Acquired mutations in the juxtamembrane region of MPL (W515K or W515L), the receptor for thrombopoietin, have been described in patients with primary myelofibrosis or essential thrombocythemia, which are chronic myeloproliferative disorders. We have developed a real-time polymerase chain reaction assay for the detection and quantification of MPL mutations that is based on locked nucleic acid fluorescent probes. Mutational analysis was performed using DNA from granulocytes. Reference curves were obtained using cloned fragments of MPL containing either the wild-type or mutated sequence; the predicted sensitivity level was at least 0.1% mutant allele in a wild-type background. None of the 60 control subjects presented with a MPLW515L/K mutation. Of 217 patients with myelofibrosis, 19 (8.7%) harbored the MPLW515 mutation, 10 (52.6%) with the W515L allele. In one case, both the W515L and W515K alleles were detected by real-time polymerase chain reaction. By comparing results obtained with conventional sequencing, no erroneous genotype attribution using real-time polymerase chain reaction was found, whereas one patient considered wild type according to sequence analysis actually harbored a low W515L allele burden. This is a simple, sensitive, and cost-effective procedure for large-scale screening of the MPLW515L/K mutation in patients suspected to have a myeloproliferative disorder. It can also provide a quantitative estimate of mutant allele burden that might be useful for both patient prognosis and monitoring response to therapy.
Abstract: Among 994 patients with essential thrombocythemia (ET) who were genotyped for the MPLW515L/K mutation, 30 patients carrying the mutation were identified (3.0%), 8 of whom also displayed the JAK2V671F mutation. MPLW515L/K patients presented lower hemoglobin levels and higher platelet counts than did wild type (wt) MPL; these differences were highly significant compared with MPLwt/JAK2V617F-positive patients. Reduced hemoglobin and increased platelet levels were preferentially associated with the W515L and W515K alleles, respectively. MPL mutation was a significant risk factor for microvessel disturbances, suggesting platelet hyperreactivity associated with constitutively active MPL; arterial thromboses were increased only in comparison to MPLwt/JAK2wt patients. MPLW515L/K patients presented reduced total and erythroid bone marrow cellularity, whereas the numbers of megakaryocytes, megakaryocytic clusters, and small-sized megakaryocytes were all significantly increased. These data indicate that MPLW515L/K mutations do not define a distinct phenotype in ET, although some differences depended on the JAK2V617F mutational status of the counterpart.
Abstract: Constitutive mobilization of CD34(+) cells in patients with primary myelofibrosis (PMF) has been attributed to proteolytic disruption of the CXCR4/SDF-1 axis and reduced CXCR4 expression. We document here that the number of circulating CD34(+)/CXCR4(+) cells in PMF patients, as well as the cellular CXCR4 expression, was directly related to CXCR4 mRNA level and that reduced CXCR4 mRNA level was not due to SDF-1-induced downregulation. To address whether epigenetic regulation contributes to defective CXCR4 expression, we studied the methylation status of the CXCR4 promoter using methylation-specific polymerase chain reaction and methylation-specific sequencing in the JAK2V617F-positive HEL cell line and in CD34(+) cells. We found that CD34(+) cells from PMF patients, unlike those from normal subjects, presented hypermethylation of CXCR4 promoter CpG island 1. Following incubation with the demethylating agent 5-Aza-2'-deoxycytidine (5-AzaD), the percentage of PMF CD34(+) cells expressing CXCR4 increased 3-10 times, whereas CXCR4 mRNA level increased approximately 4 times. 5-AzaD-treated PMF CD34(+) cells displayed almost complete reversal of CpG1 island 1 hypermethylation and showed enhanced migration in vitro in response to SDF-1. These data point to abnormal methylation of the CXCR4 promoter as a mechanism contributing to constitutive migration of CD34(+) cells in PMF. Disclosure of potential conflicts of interest is found at the end of this article.
Abstract: BACKGROUND: Fifty to sixty percent of patients with essential thrombocythemia harbor the JAK2(V617F) mutation. The impact of this mutation on clinical phenotype is still debated. The aim of this study was to evaluate possible correlations between JAK2(V617F) mutant allele burden and both clinical presentation and hematologic abnormalities in essential thrombocythemia patients. DESIGN AND METHODS: In this single-center retrospective study, JAK2(V617F) allele load was measured by sensitive quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in the granulocytes of 260 patients diagnosed as having essential thrombocythemia according to WHO criteria. RESULTS: Median V617F allele burden in patients with the mutation (n=165, 63.4%) was 24%, ranging from 1% to 87%; an allele burden greater than 51% was found in 5% of the patients. Older patients presented progressively higher percentages of the V617F allele. Signs of stimulated erythropoiesis and myelopoiesis, as well as higher PRV-1 levels, were found in patients with the mutation, but no linear correlation with load of mutant allele could be ascertained; on the other hand, the frequency of patients with erythropoietin-independent erythroid colonies progressively increased depending on mutant allele load. Splenomegaly and microvessel symptoms were significantly more represented among patients with greater than 50% and 25% JAK2(V617F) allele burden, respectively. Increasing mutant allele load correlated with higher frequency of arterial thrombosis at diagnosis, as confirmed also in multivariate analysis; the relative risk was 3.0 (95% CI 1.3-6.8; p=0.01) in patients having a greater than 25% mutant allele burden. CONCLUSIONS: The JAK2(V617F) mutant allele burden contributes to determining the clinical phenotype in patients with essential thrombocythemia.
Abstract: BACKGROUND: Prior thrombosis is a well-established risk factor for re-thrombosis in polycythemia vera and essential thrombocythemia but scarce data are available on the rate of re-thrombosis and the optimal strategy for prevention of recurrence. DESIGN AND METHODS: We retrospectively estimated the rate of recurrence in a multicenter cohort of 494 patients (poly-cythemia vera/essential thrombocythemia 235/259) with previous arterial (67.6%) or venous thrombosis (31%) or both (1.4%). First thrombosis was cerebrovascular disease in 191 cases, acute coronary syndrome in 106, peripheral arterial thrombosis in 44, and venous thromboembolism in 160. Microcirculatory events were not computed. RESULTS: Thrombosis recurred in 166 patients (33.6%), with an incidence of 7.6% patient-years. Sex, diagnosis (polycythemia vera or essential thrombocythemia), and presence of vascular risk factors did not predict recurrence, whereas age >60 years did (multivariable hazard ratio [HR], 1.67; 95% confidence interval [CI] 1.19-2.32). Increased leukocyte count at the time of the first thrombosis was a risk factor for recurrence in patients <60 years old (HR 3.55; 95% CI 1.02-12.25). Cytoreduction halved the risk in the overall cohort (HR 0.53; 95% CI 0.38-0.73) and the combination with antiplatelet agents or oral anticoagulants was more effective than administration of single drugs. Significant prevention of rethrombosis was independently achieved in patients with venous thromboembolism by both oral anticoagulants (HR 0.32; 95% CI 0.15-0.64) and antiplatelet agents (HR 0.42; 95% CI 0.22-0.77), in those with acute coronary syndrome by cytoreduction (HR 0.30; 95% CI 0.13-0.68), and in those with cerebrovascular disease by antiplatelet agents (HR 0.33; 95% CI 0.16-0.66). The overall incidence of major bleeding was 0.9% patient-years and rose to 2.8% in patients receiving both antiplatelet and anti-vitamin K agents. CONCLUSIONS: In patients with polycythemia vera and essential thrombocythemia, cytoreduction protects against recurrent thrombosis, particularly after acute coronary syndrome. The contemporary use of oral anticoagulants (after venous thromboembolism) or antiplatelet agents (after cerebrovascular disease or venous thromboembolism) further improves the protective effect. Such findings call for prospective studies aimed at investigating whether strategies tailored according to the type of first thrombosis could improve prevention of recurrences.
Abstract: PURPOSE: Established risk factors for thrombosis in essential thrombocythemia (ET) include age and previous vascular events. We aimed to refine this risk stratification by adding baseline leukocytosis. PATIENTS AND METHODS: We enrolled 657 patients with ET followed for a median of 4.5 years who developed 72 major thrombosis. Cox proportional hazard model was performed to analyze the thrombotic risk and to discriminate ET patients with or without thrombosis, multivariable C statistic index was used. We searched for leukocytes cutoff with the best sensitivity and specificity by a receiver operating characteristic curve. RESULTS: Results confirmed that age and prior events are independent risk factors for thrombosis and showed a gradient between baseline leukocytosis and thrombosis. On the contrary, no significant association was found either for JAK2(V617F) allele burden and for other laboratory parameters, including platelet number. In the model with conventional risk factors alone, C statistic ratio for total thrombosis was 0.63 and when leukocytosis was added, the change was small (C = 0.67). In contrast, in younger and asymptomatic patients (low-risk category), C statistic value indicated an high risk for thrombosis in patients with leukocytosis, similar to that calculated in conventionally defined high-risk group (C = 0.65). The best leukocyte cutoff values for predicting the events was found to be 9.4 (x 10(9)/L). CONCLUSION: We suggest to include baseline leukocytosis in the risk stratification of ET patients enrolled in clinical trials.
Abstract: JAK2 and MPL mutations are recurrent in myeloproliferative neoplasms (MPNs). A JAK2 mutation, primarily JAK2V617F, is almost invariably associated with polycythemia vera (PV). However, JAK2V617F also occurs in the majority of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF) as well as in a much smaller percentage of those with other MPNs. The mechanism(s) behind this one allele-multiple phenotypes phenomenon has not been fully elucidated. The issue is further confounded by the presence of marked variation in JAK2V617F allele burden among mutation-positive patients. In the current communication, we discuss potential mechanisms for phenotypic diversity among JAK2V617F-positive MPNs as well as review the current literature in regard to genotype-phenotype correlations (that is clinical correlates and prognostic significance) in the context of both the presence or absence of the mutation (ET and PMF) and its allele burden (PV, ET and PMF).
Abstract: This study was aimed at the characterization of a gene expression signature of the pluripotent hematopoietic CD34(+) stem cell in idiopathic myelofibrosis (IM), which would eventually provide novel pathogenetic insights and/or diagnostic/prognostic information. Aberrantly regulated genes were revealed by transcriptome comparative microarray analysis of normal and IM CD34(+) cells; selected genes were also assayed in granulocytes. One-hundred seventy four differentially expressed genes were identified and in part validated by quantitative polymerase chain reaction. Altered gene expression was corroborated by the detection of abnormally high CD9 or CD164, and low CXCR4, membrane protein expression in IM CD34(+) cells. According to class prediction analysis, a set of eight genes (CD9, GAS2, DLK1, CDH1, WT1, NFE2, HMGA2, and CXCR4) properly recognized IM from normal CD34(+) cells. These genes were aberrantly regulated also in IM granulocytes that could be reliably differentiated from control polycythemia vera and essential thrombocythemia granulocytes in 100% and 81% of cases, respectively. Abnormal expression of HMGA2 and CXCR4 in IM granulocytes was dependent on the presence and the mutational status of JAK2(V617F) mutation. The expression levels of both CD9 and DLK1 were associated with the platelet count, whereas higher WT1 expression levels identified IM patients with more active disease, as revealed by elevated CD34(+) cell count and higher severity score. In conclusion, molecular profiling of IM CD34(+) cells uncovered a limited number of genes with altered expression that, beyond their putative role in disease pathogenesis, are associated with patients' clinical characteristics and may have potential prognostic application.
Abstract: JAK2 617V>F mutation occurs in a homozygous state in 25% to 30% of patients with polycythemia vera (PV) and 2% to 4% with essential thrombocythemia (ET). Whether homozygosity associates with distinct clinical phenotypes is still under debate. This retrospective multicenter study considered 118 JAK2 617V>F homozygous patients (104 PV, 14 ET) whose clinical characteristics were compared with those of 587 heterozygous and 257 wild-type patients. Irrespective of their clinical diagnosis, homozygous patients were older, displayed a higher leukocyte count and hematocrit value at diagnosis, and presented larger spleen volume. Aquagenic pruritus was significantly more common among homozygous PV patients. JAK2 617V>F homozygosity associated with more frequent evolution into secondary myelofibrosis in both PV and ET. After adjustment for sex, age, leukocyte count, and previous thrombosis in a multivariate analysis, homozygous ET patients displayed a significantly higher risk of cardiovascular events (hazard ratio [HR] 3.97, 95% confidence interval [CI] 1.34-11.7; P = .013) than wild-type (HR = 1.0) or heterozygous patients (HR = 1.49). No significant association of JAK2 617V>F homozygosity with thrombosis risk was observed in PV. Finally, JAK2 617V>F homozygous patients were more likely to receive chemotherapy for control of disease. We conclude that JAK2 617V>F homozygosity identifies PV or ET patients with a more symptomatic myeloproliferative disorder and is associated with a higher risk of major cardiovascular events in patients with ET.
Abstract: OBJECTIVE: Expression profiling of microRNA (miRNA) was performed in granulocytes isolated from patients with primary myelofibrosis (PMF), with the aim of identifying abnormally expressed miRNAs in comparison with normal subjects or patients with polycythemia vera (PV) or essential thrombocythemia (ET). PATIENTS AND METHODS: Using stem loop-primed reverse transcription and TaqMan quantitative real-time polymerase chain reaction, the expression of 156 mature miRNAs was evaluated using pooled granulocytes from PMF patients, either wild-type or JAK2(617V>F) mutant with >51% allele burden, and control subjects. Differentially expressed miRNAs were then validated on additional control and PMF samples, and also on PV or ET granulocytes. RESULTS: There was a global downregulation of miRNA expression in PMF granulocytes; 60 miRNAs, of 128 called present, displayed differential expression compared to normal samples. Twelve miRNAs, which had been selected based on statistically different expression level, were finally validated. In PMF granulocytes, levels of miR-31, -150, and -95 were significantly lower, while those of miR-190 significantly greater, than control and PV or ET samples; on the other hand, miR-34a, -342, -326, -105, -149, and -147 were similarly reduced in patients with PMF, PV, or ET compared to controls. Increased expression of miR-182 and -183 correlated with JAK2(617V>F) allele burden. Three in silico-predicted putative target genes (DTR, HMGA2, and MYB), showed deregulated expression in PMF granulocytes that correlated with expression level of regulatory miRNA. CONCLUSIONS: A defined miRNA profile distinguishes PMF granulocytes from those of normal subjects and, partially, also from PV or ET patients.
Abstract: An acquired JAK2 (V617F)mutation has been found in myeloid cells from most patients with chronic idiopathic myelofibrosis (IM), but whether it occurs in a common myelo-lymphoid, rather than a myeloid-restricted, progenitor cell is still debated. Using a sensitive ARMS assay for the quantitative assessment of JAK2 (V617F)cDNA, we detected the mutation in purified B-, T- and NK-cells from about half of 12 patients studied. These results indicate that involvement of lymphoid lineage in IM may be more frequent than previously supposed.
Abstract: The aim of this study was to determine whether the burden of JAK2(V617F) allele correlated with major clinical outcomes in patients with polycythemia vera (PV). To this end, we determined JAK2 mutant allele levels in granulocytes of 173 PV patients at diagnosis. The mean (+/-s.d.) mutant allele burden was 52% (+/-29); 32 patients (18%) had greater than 75% mutant allele. The burden of JAK2(V617F) allele correlated with measurements of stimulated erythropoiesis (higher hematocrit, lower mean cell volume, serum ferritin and erythropoietin levels) and myelopoiesis (higher white cell count, neutrophil count and serum lactate dehydrogenase) and with markers of neutrophil activation (elevated leukocyte alkaline phosphatase and PRV-1 expression). As compared to those with less than 25% mutant allele, patients harboring greater than 75% JAK2(V617F) allele were at higher relative risk (RR) of presenting larger spleen (RR 4.7; P<0.001) or suffering from pruritus (RR 3.1; P<0.001). In these patients, the risk of requiring chemotherapy (RR 1.8; P=0.001) or developing major cardiovascular events (RR 7.1; P=0.003) during follow up were significantly increased. We conclude that a burden of JAK2(V617F) allele greater than 75% at diagnosis points to PV patients with high-risk disease.
Abstract: The clinical and haematological phenotype of patients with myelofibrosis harbouring MPL(W515L/K) mutation has not been thoroughly investigated. Of 217 myelofibrosis subjects, 18 (8.2%) had an MPL mutation, four of which (22%) co-existed with JAK2(V617F) mutation. When compared with MPL wild-type patients, irrespective of JAK2(V617F) status, those with MPL(W515L/K), were more frequently female, were older (61 years vs. 57 years; P = 0.02), presented with more severe anaemia (haemoglobin, 101 g/l vs. 121 g/l; P = 0.002) and were more likely to require regular transfusional support (P = 0.012). These data indicate that MPL mutation in myelofibrosis characterises patients with more severe anaemic phenotype.
Abstract: PURPOSE: We studied the expression of the chemokine receptor CXCR4 on circulating CD34+ cells of patients with myelofibrosis with myeloid metaplasia (MMM), and examined its relationship to the severity of disease. PATIENTS AND METHODS: Surface and intracellular CXCR4 expression were measured flow cytometrically in 84 consecutive MMM patients, 16 patients with polycythemia vera (PV), and 20 healthy subjects. In 23 MMM patients, CXCR4 gene expression level was also quantitated by real time-RT-PCR in CD34+ cells. RESULTS: The expression of CXCR4 on circulating CD34+ cells was significantly reduced in patients with MMM (P<0.001) as compared to normal controls and patients with PV (P=0.01). The levels of CXCR4 mRNA in CD34+ cells were lower in patients with MMM as compared with normal subjects, and were directly correlated with the degree of CXCR4 surface expression, demonstrating that transcriptional defects were the major cause for receptor down-regulation. No statistical association was found between JAK2V617F mutational status and the extent of CXCR4 down-regulation. CXCR4 expression on CD34+ cells inversely correlated with the number of circulating CD34+ cells (R=-0.55; P<0.001), and was severely down-regulated in high risk patients and patients with a high "myelodepletion severity index". CXCR4 down-regulation was associated with advanced patient age, the presence of severe anemia, thrombocytopenia, and degree of bone marrow fibrosis. CONCLUSIONS: Reduced expression of CXCR4 by CD34+ cells is a characteristic of MMM which is associated with the constitutive mobilization of CD34+ cells and occurs in patients with advanced forms of the disease.
Abstract: Few investigators have evaluated the usefulness of the JAK2 V617F mutation for explaining the phenotypic variations and for predicting the risk of major clinical events in primary myelofibrosis (PMF). In a transversal survey we assayed by allele-specific polymerase chain reaction (PCR) the JAK2 V617F mutational status in 304 patients with PMF. Multiple DNA samples were collected prospectively from 64 patients, and a highly sensitive quantitative PCR was used as a confirmatory test. In a longitudinal prospective study we determined the progression rate to clinically relevant outcomes in 174 patients who had JAK2 mutation determined at diagnosis. JAK2 V617F was identified in 63.4% of patients. None of the V617F-negative patients who were sequentially genotyped progressed to become V617F positive, whereas progression rate from heterozygous to homozygous mutation was 10 per 100 patient-years. JAK2 V617F mutation contributed to hemoglobin, aquagenic pruritus, and platelet count variability, whereas homozygous mutation was independently associated with higher white blood cell count, larger spleen size, and greater need for cytoreductive therapies. Adjusting for conventional risk factors, V617F mutation independently predicted the evolution toward large splenomegaly, need of splenectomy, and leukemic transformation. We conclude that JAK2 V617F genotype should be considered in any future risk stratification of patients with PMF.
Abstract: A point mutation in the Janus tyrosine kinase 2 (JAK2) gene has been described in patients with chronic myeloproliferative disorders (MPD), but the clinical significance of JAK2(V617F), which may be harbored in either the heterozygote or homozyote status, is still largely undefined. There are indirect suggestions that clinical phenotype and also some biological characteristics are dependent on the mutated allele levels. We have designed and validated in 179 MPD patients an amplification-refractory mutation sequencing PCR assay that allows the relative quantitation of mutated and normal JAK2 mRNAs using dye-labelled mutation-specific primers and capillary electrophoresis. Direct sequencing confirmed the specificity of the assay, which has a detection limit congruent with1% and allowed to identify 9% more JAK2-mutated patients as compared to conventional allele-specific PCR. The mutated mRNA ratio ranged from 5 to 51% in the JAK2(V617F) heterozygote and from 45 to 100% in the homozygote patients. Expression levels of both PRV-1 and NF-E2 gene, previously found to be overexpressed in MPD patients, were significantly correlated to the amount of mutated JAK2 mRNA. We propose that this method might complement current technologies based on genomic DNA analysis, and lead prospectively to a better clinically oriented assessment of the impact of JAK2(V617F) mutation in MPD.
Abstract: Two murine bipotent erythroid/megakaryocytic cells, the progenitor (MEP) and precursor (PEM) cells, recently have been identified on the basis of the phenotypes of linnegc-kitposSca-1neg CD16/CD32lowCD34low and TER119pos4A5pos or 2D5pos, respectively. However, the functional relationship between these two subpopulations and their placement in the hemopoietic hierarchy is incompletely understood. We compared the biological properties of these subpopulations in marrow and spleen of mice with and without acute or chronic erythroid stress. MEP cells, but not PEM cells, express c-kit, respond to stem cell factor in vitro, and form spleen colonies in vivo. PEM cells comprise up to 50%-70% of the cells in BFU-E-derived colonies but are not present among the progeny of purified MEP cells cultured under erythroid and megakaryocytic permissive conditions. PEM cells increase 10- to 20-fold under acute and chronic stress, whereas MEP cell increases (21%-84%) are observed only in acutely stressed animals. These data suggest that MEP and PEM cells represent distinct cell populations that may exist in an upstream-downstream differentiation relationship under conditions of stress. Whereas the dynamics of both populations are altered by stress induction, the differential response to acute and chronic stress suggests different regulatory mechanisms. A model describing the relationship between MEP, PEM, and common myeloid progenitor cells is presented.
Abstract: Recent studies suggest that extracorporeal photochemotherapy (ECP) may be beneficial in patients with steroid-refractory chronic graft-versus-host disease (cGvHD). However, it is not yet clear whether certain conditions, such as age, mode of onset of cGvHD etc., influence clinical response and whether certain affected organs are more sensitive to ECP than others. We analysed the main clinical and laboratory parameters related to evolution of the disease in 32 steroid-refractory cGvHD patients, to identify any useful response predictors to ECP. ECP affected the course of the disease positively in 78% (25/32) of our cases.
Abstract: The abnormal megakaryocytopoiesis associated with idiopathic myelofibrosis (IM) plays a role in its pathogenesis. Because mice with defective expression of transcription factor GATA-1 (GATA-1(low) mutants) eventually develop myelofibrosis, we investigated the occurrence of GATA-1 abnormalities in IM patients. CD 34(+) cells were purified from 12 IM patients and 8 controls; erythroblasts and megakaryocytes were then obtained from unilineage cultures of CD 34(+) cells. Purified CD 61(+), GPA(+), and CD 34(+) cells from IM patients contained levels of GATA-1, GATA-2, and FOG-1 mRNA, as well as of GATA-2 protein, that were similar to controls. In contrast, CD 61(+) cells from IM patients contained significantly reduced GATA-1 protein. Furthermore, 45% of megakaryocytes in biopsies from IM patients did not stain with anti-GATA-1 antibody, as compared to controls (2%), essential thrombocythemia (4%), or polycythemia vera (11%) patients. Abnormalities in immunoreactivity for FOG-1 were not found, and no mutations in GATA-1 coding sequences were found. The presence of GATA-1(neg) megakaryocytes in bone marrow biopsies was independent of the Val 617 Phe JAK 2 mutation, making it unlikely that a downstream functional relationship exists. We conclude that megakaryocytes from IM patients have reduced GATA-1 content, possibly contributing to disease pathogenesis as in the GATA-1(low) mice and also representing a novel IM-associated marker.
Abstract: Females with the monoclonal type of essential thrombocythaemia (ET), based on the X-chromosome inactivation pattern (XCIP), have previously been shown to present a higher incidence of thrombosis than polyclonal ones. We aimed to assess correlations between XCIP, thrombosis, and three epigenetic markers of ET, namely PRV-1 overexpression, endogenous erythroid colony (EEC) formation, and reduced platelet Mpl content. Fifty-three (60%) of 88 subjects studied had monoclonal myelopoiesis and presented a 32% incidence of major thrombosis compared with 6% of polyclonal subjects (P = 0.009). The frequency of abnormalities of PRV-1, EEC, or Mpl was similar in monoclonal and polyclonal subjects (respectively, 28%, 48%, 75%, and 37%, 27%, 63%), and none of them correlated with thrombosis. We conclude that the exploited epigenetic markers constitute independent phenotypic variations and are not clustered according to monoclonality of myelopoiesis in ET; none of them could serve as a surrogate marker of thrombotic risk in male subjects with ET.
