Elevated telomerase activity in essential thrombocythemia with extreme thrombocytosis.

INTRODUCTION: We performed a comparative analysis of telomerase activity (TA) in patients with myeloproliferative neoplasm (MPN) and myelodysplastic syndrome (MDS). The relationships between TA and known prognostic factors were also analyzed. MATERIALS AND METHODS: A telomeric repeat amplification protocol was performed with bone marrow hematopoietic cells from 96 normal controls, 44 MPNs, and 40 MDSs. RESULT: TA (measured as molecules/reaction) showed no correlation with age in the control group (R(2)=0.0057, p=0.464). MPN showed elevated TA compared with controls (15,537.57 vs. 7775.44, p=0.020). Patients with essential thrombocythemia showed markedly elevated TA (22,688.56, p=0.030), particularly in cases with extreme thrombocytosis versus those without extreme thrombocytosis (34,522.19 vs. 9375.71, p=0.041). MDS patients showed a TA value of 7578.50. CONCLUSION: There was no association between age and TA in bone marrow hematopoietic cells. TA was elevated in MPN but borderline in MDS, probably because of differences in the nature of the diseases. Elevated TA in patients with essential thrombocythemia, especially those with extreme thrombocytosis, suggests that an anti-telomerase strategy could be beneficial in the prevention of thrombotic complications.

Refractory anemia with ringed sideroblasts and thrombocytosis without JAK2 V617F mutation: report of three cases.

In the WHO classification, there is a provisional entity called Myelodysplastic/Myeloproliferative Neoplasm, Unclassifiable (MDS/MPN, U). Refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T) was included in this category. Recently published studies report a small percentage of patients with RARS-T. Sixty percent of these have JAK2 V617F mutation, which can suggest the coexistence of two pathological conditions (MDS and MPN). In this paper, we analyzed three patients diagnosed with RARS-T in the Department of Hematology, "Fundeni" Clinical Institute, Bucharest, Romania, during the period 2005-2011. The patients were investigated with cytogenetic exam and molecular biology. In these three cases were identified morphological features of multilineage dysplasia (two-lineage dysplasia in two cases and three-lineage dysplasia in one case). In two cases, thrombocytosis was under 1000×10(3)/µL and clinical evolution was similar to the myelodysplastic syndrome (transfusion dependent anemia with response to administration of erythropoietin). In the third case, the platelets were over 1000×10(3)/µL and with response to the treatment with Hydrea, which improved anemia. JAK2 V617F mutation was not identified in any case. RARS-T remains a provisional entity and requires a complex investigation of patients for the correct diagnosis of these patients. Therapeutic options should be personalized to each case in part because there is not yet a standardized treatment of these patients.

Deficiency of 5-hydroxyisourate hydrolase causes hepatomegaly and hepatocellular carcinoma in mice.

With the notable exception of humans, uric acid is degraded to (S)-allantoin in a biochemical pathway catalyzed by urate oxidase, 5-hydroxyisourate (HIU) hydrolase, and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase in most vertebrate species. A point mutation in the gene encoding mouse HIU hydrolase, Urah, that perturbed uric acid metabolism within the liver was discovered during a mutagenesis screen in mice. The predicted substitution of cysteine for tyrosine in a conserved helical region of the mutant-encoded HIU hydrolase resulted in undetectable protein expression. Mice homozygous for this mutation developed elevated platelet counts secondary to excess thrombopoietin production and hepatomegaly. The majority of homozygous mutant mice also developed hepatocellular carcinoma, and tumor development was accelerated by exposure to radiation. The development of hepatomegaly and liver tumors in mice lacking Urah suggests that uric acid metabolites may be toxic and that urate oxidase activity without HIU hydrolase function may affect liver growth and transformation. The absence of HIU hydrolase in humans predicts slowed metabolism of HIU after clinical administration of exogenous urate oxidase in conditions of uric acid-related pathology. The data suggest that prolonged urate oxidase therapy should be combined with careful assessment of toxicity associated with extrahepatic production of uric acid metabolites.

Class prediction models of thrombocytosis using genetic biomarkers.

Criteria for distinguishing among etiologies of thrombocytosis are limited in their capacity to delineate clonal (essential thrombocythemia [ET]) from nonclonal (reactive thrombocytosis [RT]) etiologies. We studied platelet transcript profiles of 126 subjects (48 controls, 38 RT, 40 ET [24 contained the JAK2V(617)F mutation]) to identify transcript subsets that segregated phenotypes. Cross-platform consistency was validated using quantitative real-time polymerase chain reaction (RT-PCR). Class prediction algorithms were developed to assign phenotypic class between the thrombocytosis cohorts, and by JAK2 genotype. Sex differences were rare in normal and ET cohorts (< 1% of genes) but were male-skewed for approximately 3% of RT genes. An 11-biomarker gene subset using the microarray data discriminated among the 3 cohorts with 86.3% accuracy, with 93.6% accuracy in 2-way class prediction (ET vs RT). Subsequent quantitative RT-PCR analysis established that these biomarkers were 87.1% accurate in prospective classification of a new cohort. A 4-biomarker gene subset predicted JAK2 wild-type ET in more than 85% patient samples using either microarray or RT-PCR profiling, with lower predictive capacity in JAK2V(617)F mutant ET patients. These results establish that distinct genetic biomarker subsets can predict thrombocytosis class using routine phlebotomy.

Superoxide dismutase, copper and zinc concentrations in platelet-rich plasma of pneumonia patients.

BACKGROUND: The aim of this study was to analyse platelet superoxide dismutase (SOD) activities (total SOD, manganese SOD and copper zinc SOD) and copper (Cu) and zinc (Zn) concentrations during the course of community-acquired pneumonia (CAP), and to compare them between patients with normal platelet count and those who have developed reactive thrombocytosis (RT). METHODS: Platelet count, SOD activities and Cu and Zn concentrations in platelet-rich plasma were measured in patients with CAP on admission and at discharge. RESULTS: Post-therapeutic platelet count increased significantly from the value recorded on admission. By the end of treatment, 42% of patients developed RT. All platelet SOD activities as well as Cu concentration were significantly lower in CAP patients than in control subjects. The initial Zn concentration was greater in CAP patients compared with controls and showed a decrease at discharge. On admission, there was no difference in all SOD activities between either subgroup with normal platelet count or subgroup with RT. At discharge all SOD activities were significantly lower in patients with RT. Also, catalytic activities of those enzymes were significantly lower in both subgroups in comparison with the initial values. Post-therapeutic Cu value was lower in patients with RT in comparison with patients having normal platelet count. Zn concentration decreased significantly at discharge when compared with the initial values only in patients with RT. CONCLUSION: The pattern of changes might be indicative of a certain role of platelets in antioxidant response during treatment in CAP patients.

The role of JAK2 mutations in RARS and other MDS.

Acquired sideroblastic anemia with unilineage dysplasia (WHO RARS) is a clonal stem cell disorder characterized by erythroid dysplasia, mitochondrial accumulation of mitochondrial ferritin, defective erythroid maturation and anemia. A fraction of these patients also show elevated platelet counts; since 2001 this has been defined as RARS with marked thrombocytosis (RARS-T). It has recently been described that around half of RARS-T patients, along with a small subset of other MDS and mixed myelodysplastic/ myeloproliferative disorders, carry the JAK2 mutation, and that MPL mutations are found in single patients. Clinically, RARS-T patients show features of both RARS, essential thrombocythmia (ET) and to some extent also myelofibrosis. However, the degree of anemia and overall survival is more similar to RARS than myeloproliferative disorders. The occurrence of JAK2 mutations and features of ET in RARS is too frequent to be the result of chance only, and it is possible that this link may provide a key to an increased understanding of the genetic abnormalities causing ring sideroblast formation.

The JAK2V617F mutation in polycythemia vera and other myeloproliferative disorders: one mutation for three diseases?

The discovery of the JAK2V617F mutation has made the diagnosis of polycythemia vera (PV) much easier, but the pathogenesis of PV is still incompletely understood. In particular, it is not yet elucidated how a single mutation can be found in multiple myeloproliferative disorders (MPD) and myelodysplastic syndromes with ring sideroblasts and whether the sole JAK2V617F is sufficient to induce a MPD in humans. Several hypotheses are under investigation such as differences in the targeted hematopoietic stem cells (HSC), host modifier polymorphisms, intensity of JAK2V617F signaling, presence of other somatic mutations, or the presence of a pre-JAK2 event that may vary according to the MPD phenotype. Multiple studies have provided some evidence for and against each hypothesis, but it now seems possible to reconcile these hypotheses into a model that will need to be tested using newly developed tools. Recent investigations have also led to new treatment modalities that could benefit patients with PV.

Occurrence of the JAK2 V617F mutation in the WHO provisional entity: myelodysplastic/myeloproliferative disease, unclassifiable-refractory anemia with ringed sideroblasts associated with marked thrombocytosis.

The JAK2/V617F mutation has been noted in essential thrombocytemia. We investigated 19 cases with refractory anemia with ringed sideroblasts (RARS), including three RARS with thrombocytosis (RARS-T). Only the RARS-T patients showed this mutation. More cases need to be analyzed to determine the prevalence of the JAK2/V617F mutation in RARS-T.

Increased megakaryocytopoiesis in Lyn-deficient mice.

Previous studies in cell lines have shown Lyn kinase to be a negative regulator of thrombopoietin (TPO)-induced proliferation. To further investigate the role of Lyn during megakaryocytopoiesis, Lyn-deficient mice (lyn(-/-)) were analyzed. We observed that lyn(-/-) mice have more bone marrow-derived GPIIB (CD41) and Mpl(+) cells when compared to their wild-type littermates. In addition, colony-forming unit-megakaryocytes (CFU-MK) are increased and TPO-induced expansion of primary marrow cells yielded a greater number of mature megakaryocytes (MKs) with increased nuclear ploidy. Histopathology of bone marrow and spleens from lyn(-/-) mice showed an increase in the number of MKs. Mechanistic studies revealed that TPO stimulation of MKs from lyn(-/-) mice did not affect phosphorylation of Janus kinase 2 (JAK2), signal transducer and activator of transcription (STAT) 3, STAT5, or MAP kinase kinase (MEK). Lyn-deficient MKs supported greater TPO-mediated phosphorylation and kinase activity of both Erk1/2 (mitogen-activated protein kinase, MAPK) and Akt. In contrast, there was a reduction of tyrosine phosphorylation of the inositol phosphatase, SHIP. This is the first direct evidence using primary MKs from Lyn-deficient mice that confirms our prior data from cell lines that Lyn kinase is a negative regulator of TPO signaling.

The JAK2(V617F) tyrosine kinase mutation in myelofibrosis with myeloid metaplasia: lineage specificity and clinical correlates.

An association between an activating JAK2 mutation (JAK2(V617F)) and BCR/ABL-negative myeloproliferative disorders was recently reported in multiple simultaneous publications. In the current study, mutation analysis for JAK2(V617F) was performed in peripheral blood mononuclear cells (PBMC) from 157 patients with myelofibrosis with myeloid metaplasia (MMM) including 117 with agnogenic (AMM), 22 with postpolycythaemic (PPMM), and 18 with post-thrombocythaemic (PTMM) myeloid metaplasia. The detection rate for JAK2(V617F) was significantly higher in PPMM (91%; homozygous in 18%) compared with either AMM (45.3%; homozygous in 2.6%) or PTMM (38.9%; homozygous in 11.1%). Concomitant analysis in granulocytes (n=57) and CD34(+) cells (n=25) disclosed a higher incidence of homozygous JAK2(V617F) mutation but the overall mutation rate was similar to that obtained from PBMC. JAK2(V617F) was not detected in DNA derived from T cells (n=19). In AMM, the presence of JAK2(V617F) was associated with an older age at diagnosis and a history of thrombosis or pruritus. Multivariate analysis identified only age and the Dupriez prognostic score as independent prognostic factors; JAK2(V617F) had no prognostic significance. In conclusion, JAK2(V617F) is a myeloid lineage-specific event, its incidence in MMM is significantly higher with an antecedent history of polycythaemia vera (PV), and its presence in AMM does not affect prognosis but is associated with PV-characteristic clinical features.

Platelets express steroidogenic 17beta-hydroxysteroid dehydrogenases. Distinct profiles predict the essential thrombocythemic phenotype.

Human blood platelets have important, regulatory functions in diverse hemostatic and pathological disorders, including vascular remodeling, inflammation, and wound repair. Microarray analysis was used to study the molecular basis of essential thrombocythemia, a myeloproliferative disorder with quantitative and qualitative platelet defects associated with cardiovascular and thrombohemorrhagic symptoms, not infrequently neurological. A platelet-expressed gene (HSD17B3) encoding type 3 17beta-hydroxysteroid dehydrogenase (previously characterized as a testis-specific enzyme catalyzing the final step in gonadal synthesis of testosterone) was selectively down-regulated in ET platelets, with reciprocal induction of the type 12 enzyme (HSD17B12). Functional 17beta-HSD3 activity corresponding to approximately 10% of that found in murine testis was demonstrated in normal platelets. The induction of HSD17B12 in ET platelets was unassociated with a concomitant increase in androgen biosynthesis, suggesting distinct functions and/or substrate specificities of the types 3 and 12 enzymes. Application of a molecular assay distinguished ET from normal platelets in 20 consecutive patients (p < 0.0001). These data provide the first evidence that distinct subtypes of steroidogenic 17beta-HSDs are functionally present in human blood platelets, and that the expression patterns of HSD17B3 and HSD17B12 are associated with an uncommon platelet disorder manifest by quantitative and qualitative platelet defects.

Activity of platelet alpha-6-fucosyltransferase is inversely related to blood platelet concentration.

The activity of serum alpha-6-fucosyltransferase, a platelet derived enzyme, determined in sera of 22 normal individuals and 86 patients with various disorders was positively correlated with platelet counts. When the enzyme activity in 1 microliters serum was calculated per 1000 of platelets in blood (coefficient F/P) an inverse correlation became evident in that F/P was proportionally the higher the lower was platelet count in blood. The F/P values were in a good agreement with the results of direct assays of enzyme activities in isolated platelets. Neither granulocytes, lymphocytes nor red cells significantly contributed to serum enzyme activity though granulocytes enhanced the thrombin-induced enzyme release from platelets. In platelets separated by centrifugation in density gradients the enzyme was shown to be present in platelets of intermediate and high density but missing from the light ones. It is suggested that alpha-6-fucosyltransferase of platelets may be a marker of the ploidy level of megakaryocytes.

Platelet glyoxalases in thrombocytosis.

In platelets of patients suffering from thrombocytosis due to myeloproliferative disorders, glyoxalase I activity is significantly higher than in controls (P less than 0.01), while glyoxalase II levels are the same (P less than 0.3). The cellular concentration of glutathione is also increased in patients. Km values for methylglyoxal (glyoxalase I) and S-lactoylglutathione (glyoxalase II) are identical both in normal and pathological subjects, as are the thermostability of the two enzymes. The higher activity observed for glyoxalase I in patients could be related to a specific role of this enzyme in platelets.

Essential thrombocythemia: a clonal disorder of hematopoietic stem cell.

We studied 5 patients with essential thrombocythemia utilizing glucose-6-phosphate dehydrogenase (G-6-PD) enzyme as a cell marker for determining clonality. One of the patients was found to be heterozygous for isoenzymes B and A in the nonhaemopoietic tissues such as fibroblasts, but manifested only isoenzyme type B in the erythrocytes, neutrophils, and platelets. Our studies support the concept that essential thrombocythemia is a clonal disorder arising in a multipotent stem cell.