Extent of hematopoietic involvement by TET2 mutations in JAK2V⁶¹⁷F polycythemia vera

TET2 mutations are found in polycythemia vera and it was initially reported that there is a greater TET2 mutational burden than JAK2(V617F) in polycythemia vera stem cells and that TET2 mutations precede JAK2(V617F). We quantified the proportion of TET2, JAK2(V617F) mutations and X-chromosome allelic usage in polycythemia vera cells, BFU-Es and in vitro expanded erythroid progenitors and found clonal reticulocytes, granulocytes, platelets and CD34(+) cells. We found that TET2 mutations may also follow rather than precede JAK2(V617F) as recently reported by others. Only a fraction of clonal early hematopoietic precursors and largely polyclonal T cells carry the TET2 mutation. We showed that in vitro the concomitant presence of JAK2(V617F) and TET2 mutations favors clonal polycythemia vera erythroid progenitors in contrast with non-TET2 mutated progenitors. We conclude that loss-of-function TET2 mutations are not the polycythemia vera initiating events and that the acquisition of TET2 somatic mutations may increase the aggressivity of the polycythemia vera clone.     

Intracellular growth factors in polycythemia vera and other myeloproliferative disorders.

In polycythemia vera, idiopathic myelofibrosis, and essential thrombocytosis, hematopoietic cell proliferation is increased in the absence of a recognizable stimulus, suggesting the autonomous production of growth factors in these disorders. Sonicates of peripheral blood mononuclear cells (PBMNC) from patients with polycythemia vera, idiopathic myelofibrosis, and essential thrombocytosis contained soluble factors that stimulated the proliferation of quiescent-confluent 3T3 cells. PBMNC sonicates from normal individuals; from patients with secondary erythrocytosis, chronic myelogenous leukemia, B-cell chronic lymphocytic leukemia, and acute myelogenous leukemia; and from K-562 and HL-60 cells did not stimulate proliferation. Polycythemia vera PBMNC sonicates also induced anchorage-independent colony formation in soft agar by normal rat kidney fibroblasts. Both the mitogenic and transforming activities of the polycythemia vera PBMNC sonicates resided in the T-lymphocyte-depleted mononuclear fraction of the PBMNC and were not secreted. By gel filtration, reversed-phase HPLC and NaDodSO4/PAGE, the mitogenic and transforming activities in the polycythemia vera PBMNC were localized to three proteins with molecular masses of 13-, 17-, and 65-kDa. The 13-kDa protein was only mitogenic, and the 17-kDa protein was only transforming, whereas the 65-kDa protein had both mitogenic and transforming activity. These proteins may be involved in the autonomous hematopoiesis that characterizes polycythemia vera, idiopathic myelofibrosis, and essential thrombocytosis.     

Endogenous erythroid colony formation by peripheral blood mononuclear cells from patients with myelofibrosis and polycythemia vera.

Peripheral blood mononuclear cells from patients with polycythemia vera or myelofibrosis with myeloid metaplasia were studied for their erythroid colony growth characteristics in plasma clot cultures. In both diseases, erythroid colonies formed early in culture in the absence of added erythropoietin (endogenous colonies). In no instance did early, endogenous colony formation occur with peripheral blood cells from normals or patients with secondary polycythemia. A normal response to erythropoietin was observed with both control and patients' peripheral blood cells. Spleen mononuclear cells obtained from one patient with myelofibrosis also produced endogenous colonies and showed a response to erythropoietin. This study suggests that culture of peripheral blood mononuclear cells might serve as a useful tool in discriminating polycythemia vera from secondary polycythemia.     

Polycythemia vera and other primary polycythemias

PURPOSE OF REVIEW: Diagnosis and therapy of polycythemia vera are controversial since the molecular basis of polycythemia vera remains unknown. Distinguishing between polycythemia vera and other polycythemic disorders can be very challenging. The purpose of this review is to discuss the recent progress in this area and critically review the published data in context of our knowledge of other polycythemic disorders. RECENT FINDINGS: Erythropoietin is the principal regulator of regulator of erythropoiesis; its production is regulated by the degree of hypoxia. Our knowledge of cellular responses to hypoxia has recently exploded and led to the elucidation of the molecular basis of a polycythemia caused by augmentation of hypoxic sensing, Chuvash polycythemia. Similar progress in understanding the molecular basis of polycythemia vera has been elusive. A simple, readily available laboratory test to establish a diagnosis of polycythemia vera would be highly desirable; however, none exists. The value of quantization of neutrophil PRV-1 mRNA, platelet c-mpl expression, in vitro assays of erythroid progenitor cells, serum erythropoietin levels, establishing clonality in female subjects using assays employing X-chromosome-based polymorphism assays, and the progress in the chromosomal location of the gene is discussed. Integration of this information underlies the complexity of the molecular biology of polycythemia vera and indicates likely interaction of multiple genetic events in the genesis of polycythemia vera. SUMMARY: The existence of family clustering of PV may facilitate the search for PV molecular basis. Only collaborative interaction of clinical researchers and laboratory scientists will lead to meaningful progress in determining the molecular basis of PV.     

Possible selective effects of Interferon α-2b on a malignant clone in a case of polycythemia vera

Summary A patient with polycythemia vera associated with chromosomal translocation (6; 8) (q27; p11) and IgA monoclonal gammopathy was treated with interferon -2b. Interferon -2b induced good hematological control and reduced only IgA levels, but not IgG and IgM levels. In addition, cytogenetic improvement was obtained, the abnormal cells with chromosomal translocation declining from 100% to 50%. Interferon a may have selective effects on a malignant clone in polycythemia vera.     

Aspirin-responsive painful red,blue, black toe,or finger syndrome in polycythemia vera associated with thrombocythemia

Five patients with red, purple blue, or black toes or fingers due to thrombocythemia associated with polycythemia vera (polycythemia and thrombocythemia vera) in four and essential thrombocythemia (thrombocythemia vera) in one are described. The microvascular erythromelalgic syndrome of thrombocythemia was overlooked and progressed to cold blue swollen and painful fingers or black toes in three patients with polycythemia and thrombocythemia vera due to arteriographically documented occlusions of digital or large peripheral arteries with no evidence of preexistent atherosclerotic vascular disease. Concomitant erythromelalgia of the hand palm could be confirmed by the histopathological findings of arteriolar thrombotic lesions in the reticular dermis in two patients with polycythemia and thrombocythemia vera. The increased hematocrit in the presented patients with polycythemia and thrombocythemia vera contributed to the progression of the microvascular syndrome of thrombocythemia to major occlusive ischemic events of the extremities. Standard therapy with oral anticoagulants and reduction of the hematocrit to normal by bloodletting did not affect the platelet-mediated microvascular erythromelalgic, ischemic symptoms in the patients with polycythemia vera because thrombocythemia vera persisted. Complete relief of pain and restoration of the ischemic acral circulation disturbances in patients with thrombocythemia vera or thrombocythemia associated with polycythemia vera in maintained remission by bloodletting could be obtained by long-term treatment with low-dose aspirin.     

Apoptosis and polycythemia vera

Polycythemia vera is an acquired clonal myeloproliferative disorder characterized by increased numbers of erythroid cells, often with a concomitant rise in neutrophils and/or megakaryocytes. Normally, erythropoietin is essential for the survival and proliferation of erythroid progenitors; however in polycythemia vera the erythroid progenitor cells can survive and develop in the absence of erythropoietin. Members of the Bcl-2 family of apoptosis regulators have been shown to mediate the erythropoietin-dependent survival of erythroid cells. In this article, recent advances in understanding the mechanisms used by erythroid progenitors from patients with polycythemia vera to control apoptosis, are discussed.     

Tissue plasminogen activator levels in different types of polycythemia

The plasma level of tissue plasminogen activator antigen (t-PA-Ag) was examined in 86 patients with polycythemia (29 polycythemia vera, 11 secondary polycythemia and 46 with spurious polycythemia) and 24 healthy volunteers. Tissue plasminogen activator antigen was significantly decreased in patients with polycythemia vera in comparison with healthy controls. On the other hand, in patients with spurious polycythemia and secondary polycythemia t-PA-Ag concentration was significantly increased. There was no significant difference in t-PA-Ag levels in polycythemic patients with or without thromboembolic disease. A significant correlation was detected between t-PA-Ag level and hemoglobin or hematocrit concentration in patients with polycythemia vera (p = 0.02, r = 0.43). However, in patients with secondary polycythemia and spurious polycythemia, no significant correlation between t-PA-Ag and hemoglobin level was found. Plasminogen activator inhibitor (PAI) levels in patients with polycythemia vera and healthy volunteers did not differ significantly.     

Treatment of the myeloproliferative disorders with 32P

Abstract: The use of radioactive phosphorus (³²P) to treat the myeloproliferative disorders (chronic leukemia, polycythemia vera and essential thrombocythemia) began in 1939 when John H. Lawrence treated the first patient on the basis of work done in the laboratory anima that found localization of the radioisotope in the spleen, liver, bone and in leukemic cells sufficient to indicate a therapeutic potential. After World War II when ³²P became widely available, it was used extensively to treat the chronic leukemias and polycythemia vera. Its use in the treatment of essential thrombocythemia began later in 1950. Today it is not widely used in the treatment of the chronic leukemia, if at all, its use in polycythemia vera appears to have decreased substantially and replaced by hydroxyurea, and its use in the management of essential thrombocythemia is not widespread. In each instance it has been replaced by a drug developed for use in cancer chemotherapy, and in some instances by interferon. It probably has wider use in polycythemia vera in the rest of Western Europe than in the UK, and there are cogent reasons to suggest that it may be the best tool for the treatment of polycythemia vera. Thus have we discarded a treatment modality that in polycythemia vera may be the best?     

Familial polycythemia vera with Budd-Chiari syndrome in childhood

Polycythemia vera is a myeloproliferative disorder that, in most cases, occurs sporadically with a median age at presentation of 60 years. Familial cases are very rare and usually manifest in elderly family members. The Budd-Chiari syndrome, characterized by the obstruction and occlusion of the suprahepatic veins, is a rare typical complication in polycythemia vera patients. To date, only two children or adolescents with polycythemia vera and Budd-Chiari syndrome have been described. Here, we report an 11-year-old girl with Budd-Chiari syndrome as the initial symptom of familial polycythemia vera, which was also found in the girl's grandmother. Details of the diagnostic procedures used and the clinical course are reported. The patient underwent orthotopic liver transplantation and is being treated with hydroxyurea. The available literature on familial polycythemia vera and polycythemia vera in childhood with and without Budd-Chiari syndrome is reviewed.     

Erythropoietin-dependent primary pure erythrocytosis.

We investigated the pathogenesis of isolated erythrocytosis of 14 yr duration in a 28-yr-old man. The increase in red cell mass was attributed to increased erythropoietin production. An extensive search for recognized causes of secondary erythrocytosis was unrevealing. Family members were found to be hematologically normal. After reduction of the circulating red cell mass by 20%, erythropoietin activity nearly quadrupled, thus suggesting a normal erythropoietin response to phlebotomy. When bone marrow cells of the patient were cultured in plasma clots in the absence of added erythropoietin, endogenous erythroid colony formation was observed, a pattern previously believed to be specific for polycythemia vera bone marrow cells. Our observations suggest that the erythrocytosis in this individual is best explained by an abnormal "servoregulatory" mechanism of erythropoietin production. In addition, this is the first instance in which the rule that endogenous erythroid colony formation is correlated with the diagnosis of polycythemia vera has not held.     

The JAK2V617 mutation induces constitutive activation and agonist hypersensitivity in basophils from patients with polycythemia vera

Background

The JAK2V617F mutation has been associated with constitutive and enhanced activation of neutrophils, while no information is available concerning other leukocyte subtypes.

Design and Methods

We evaluated correlations between JAK2V617F mutation and the count of circulating basophils, the number of activated CD63⁺ basophils, their response in vitro to agonists as well as the effects of a JAK2 inhibitor.

Results

We found that basophil count was increased in patients with JAK2V617F -positive myeloproliferative neoplasms, particularly in those with polycythemia vera, and was correlated with the V617F burden. The burden of V617F allele was similar in neutrophils and basophils from patients with polycythemia vera, while total JAK2 mRNA content was remarkably greater in the basophils; however, the content of JAK2 protein in basophils was not increased. The number of CD63⁺ basophils was higher in patients with polycythemia vera than in healthy subjects or patients with essential thrombocythemia or primary myelofibrosis and was correlated with the V617F burden. Ultrastructurally, basophils from patients with polycythemia vera contained an increased number of granules, most of which were empty suggesting cell degranulation in vivo. Ex vivo experiments revealed that basophils from patients with polycythemia vera were hypersensitive to the priming effect of interleukin-3 and to f-MLP-induced activation; pre-treatment with a JAK2 inhibitor reduced polycythemia vera basophil activation. Finally, we found that the number of circulating CD63⁺ basophils was significantly greater in patients suffering from aquagenic pruritus, who also showed a higher V617F allele burden.

Conclusions

These data indicate that the number of constitutively activated and hypersensitive circulating basophils is increased in polycythemia vera, underscoring a role of JAK2V617F in these cells’ abnormal function and, putatively, in the pathogenesis of pruritus.     

Isolated cerebellar infarction as a presenting symptom of polycythemia vera.

Although polycythemia vera is one of the reported causes for cerebral infarction, isolated cerebellar infarction, a rare disorder, was never reported in combination with polycythemia vera. This is a report of a 72-year-old woman in whom isolated cerebellar infarction was the presenting manifestation of polycythemia vera. The patient was treated with recurrent phlebotomies until the hematocrit decreased to < 45%. This treatment was followed by marked neurological improvement. A better awareness of the possibility of cerebellar infarction in polycythemia vera may disclose additional cases.     

Cytogenetic studies in polycythemia vera

A group of 54 patients with the original diagnosis of polycythemia vera were subjected to cytogenetic examination. Six (17.6%) of the 34 cases examined in the period of the advanced phase of the polycythemia vera had a chromosomal change. Thirteen (65%) of the 20 patients undergoing the cytogenetic examination in the period when the polycythemia vera turned into another myeloproliferative disease showed chromosomal aberration. This suggests a relationship between the number of chromosomal changes and the transformation of the disease. No connection between the cytogenetic changes and myelosuppressive cures could be confirmed in our material. The chromosomal change 20q- considered to be the most frequent kind in the polycythemia vera was not discovered until in patients with the polycythemia vera transformed into a different myeloproliferative disease.     

Burst-stimulating activity in sera of patients with polycythemia vera

Summary Sera from patients with polycythemia vera without prior cytostatic therapy stimulate the growth of bursts during in vitro methylcellulose cultures of peripheral mononuclear cells from normal donors. Sera from patients with polycythemia vera in remission after treatment with ³²P or cyclophosphamid, did not significantly stimulate the growth of bursts during incubation with normal peripheral mononuclear cells. The stimulating factor was demonstrable again in patients during relapse.Supported by grants of Landesamt für Forschung NW     

Serum erythropoietin (ESF) titers in polycythemia

Serum ESF titers were measured in 42 polycythemic patients using the fetal mouse liver cell bioassay. ESF titers in patients with secondary polycythemia differed significantly from those in patients with polycythemia vera (p less than 0.0001). Among the 21 patients with secondary polycythemia, 1 patient had an ESF titer less than 10 mU/ml (the lower limit of sensitivity) and 20 had ESF titers that ranged between 11 and 112 mU/ml, with a mean titer of 56 mU/ml. Among the 21 patients with polycythemia vera, 13 patients had ESF titers less than 10 mU/ml and 8 had ESF titers ranging between 12 and 55 mU/ml, with a mean titer of 26 mU/ml. The mean hemoglobin concentration in the 8 patients with ESF titers greater than 10 mU/ml was significantly below that in the 13 polycythemia vera patients with ESF titers less than 10 mU/ml (p less than 0.03). If ESF titers less than 10 mU/ml had been indicative of polycythemia vera and ESF titers greater than 10 mU/ml had been indicative of secondary polycythemia in patients with hemoglobin concentrations greater than 17.7 g/dl, but not indicative of either condition in patients with hemoglobin concentrations less than 17.7 g/dl, 71.5% of the polycythemic patients in this study would have been diagnosed correctly, 9.5% incorrectly, and in the 19% the diagnosis would have remained uncertain. It was concluded that measurement of serum ESF titers using this in vitro bioassay can be of clinical importance in differentiating between polycythemia vera and secondary polycythemia.     

Overexpression of microRNA-16-2 contributes to the abnormal erythropoiesis in polycythemia vera

Deregulated expression of microRNAs is associated with neoplasia. Here, we show that mature miR-16 levels are abnormally increased in CD34(+) cells of patients with polycythemia vera as a consequence of preferential expression of miR-16-2 on chromosome 3 rather than of miR-16-1 on chromosome 13. Forced expression of miRNA-16 in normal CD34(+) cells stimulated erythroid cell proliferation and maturation. Conversely, exposure of polycythemia vera CD34(+) cells to small interfering RNA against pre-miR-16-2 reduced erythroid colonies and largely prevented formation of erythropoietin-independent colonies; myeloid progenitors remained unaffected. Experiments with knock down of JAK2 indicated that overexpression of miR-16 was independent of JAK/STAT pathway activation. Mice injected with an miR-16 antagomir showed a blunted erythroid response to exogenous erythropoietin, which indicates a role of miR-16 in normal erythropoiesis. These data suggest that deregulation of miR-16-2 contributes to abnormal expansion of erythroid lineage in polycythemia vera. However, the mechanisms for miR-16-2 overexpression remain to be elucidated, because no genetic abnormalities at the miR-16-2 locus were discovered.