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.     

Haptoglobin Metabolism in Polycythemia Vera

1. Turnover studies with ¹²⁵I-labeled haptoglobin (Hp) were performed in7 patients with polycythemia vera, 2 patients with erythrocytosis of unknownetiology, and 2 control subjects.

2. The T of plasma radioactivity was shortened in 6 of the 7 patients withpolycythemia vera; 3 of these had diminished plasma Hp levels but lackedother evidence of hemolysis.

3. The fractional catabolic rate exceeded 40 per cent/day in all subjectswith a shortened half-time of plasma radioactivity.

4. Increases in the fractional catabolic rate were not accompanied by increases in Hp turnover (mg./kg./day), suggesting that accelerated Hp catabolism per se does not provide a stimulus to Hp production.

5. It is concluded that patients with polycythemia vera catabolize Hp morerapidly than nonpolycythemic subjects, possibly because of increased formation and removal of the haptoglobin-hemoglobin complex.

Submitted on November 5, 1968 Accepted on January 21, 1969     

Granulocyte Kinetic Studies in Patients with Proliferative Disorders of the Bone Marrow

Granulocyte kinetic studies with DFP³² were done in four patients withchronic myelocytic leukemia, three patients with polycythemia vera, one patient with essential thrombocythemia, and one patient with persistent, unexplained granulocytosis. The increased blood granulocyte concentrationfound in the patients with polycythemia vera, essential thrombocythemia andunexplained granulocytosis was at least in part the result of increased granulocyte production. Precise calculations of granulocyte pool sizes and turnoverrates in the patients with chronic myelocytic leukemia were not possiblebecause of unresolved problems related to the non-uniform population ofmyeloid cells in the blood of these patients. However, within the limitationsof the method, a greater number of myeloid cells were turned over per daythrough blood than in normal subjects. The findings support the conceptthat a widespread disorder of marrow proliferation exists in chronic myelocytic leukemia, polycythemia vera, and essential thrombocythemia.

Submitted on December 20, 1962 Accepted on March 23, 1963     

Chronic Myeloid Leukaemia: Evidence for Basophil Differentiation and Histamine Synthesis from Cultured Peripheral Blood Cells

S ummary . We have attempted to assess basophil differentiation in vitro in 15 patients with chronic myeloid leukaemia (CML). Using a sensitive radioassay, whole blood histamine values were found to be elevated in 10 of 14 patients tested, and correlated well with peripheral blood basophil counts (r=+0.70). In seven of nine CML patients, but not in controls, total histamine content of separated peripheral blood cells suspended in a modified Marbrook system was shown to rise after 7 d in vitro. Further study showed that both histamine (mean, five-fold increase) and basophils (mean, three-fold increase) were significantly elevated over control values at 1,2 and 3 weeks in vitro. Total cell-associated histamine content (in pg per 100 viable cells) was greater in CML cultures than in controls at 3 weeks (P<0.01). Serial cultures of cells from one patient revealed substantial in vitro rises in basophils and histamine at an accelerated, but not at a chronic, phase of disease. Cells from this patient and two others studied at the time of blast crisis demonstrated higher indices of basophil and histamine increases when compared to the group of CML patients in chronic phases of the disease (P<0.05). We conclude that basophil precursors exist in increased numbers in the peripheral blood of CML patients. Assays for basophil differentiation may prove useful in following disease activity in this and other myeloproliferative disorders.     

Aquagenic pruritus in polycythemia vera: Characteristics and influence on quality of life in 441 patients

Aquagenic pruritus (AP) is a symptom typical for polycythemia vera, but very little is known about its exact frequency, characteristics, influence on quality of life, and proper treatment. Therefore, we investigated these aspects in a large cohort of German patients with polycythemia vera using a patient directed questionnaire. Our analysis revealed that 301 of 441 analyzed patients suffered from AP. In 64.8%, AP occurred on average 2.9 years prior to diagnosis of polycythemia vera. Only in 15.4% did this lead to a hematological investigation. AP occurs primarily on the trunk and proximal parts of the extremities. Most patients complain about itching (71.8%), the remainder about tickling, stinging, or burning sensations. Forty‐four patients (14.6%) classified the pruritus as “unbearable.” Patients with AP reported reduced global health status and higher fatigue, pain, and dyspnea. Only 24% of patients received pruritus specific treatment for pruritus consisting mostly of histamine antagonists, which ameliorated symptoms in about half of the patients. In 5.6% of patients, polycythemia vera directed therapy (phlebotomy/cytoreduction) resolved the symptoms. In summary, AP is a serious symptom in patients with polycythemia vera, which until recently was difficult to treat. The advent of the novel JAK2 inhibitors, however, may open new ways for therapy. Am. J. Hematol. 88:665–669, 2013. © 2013 Wiley Periodicals, Inc.     

Polycythemia vera masked due to severe iron deficiency anemia

Polycythemia vera is one of the chronic myeloproliferative diseases and very few patients present with its actual clinical manifestations. The most common findings are increased red cell mass and an increased leukocyte count with decreased erythropoietin. We present a case where there was a delay in the diagnosis of polycythemia because of menorrhagia in the past. On admission, the patient presented with elevated red and white blood cell counts, erythropoietin was low, and polycythemia was then suspected. A bcr-abl test was performed to rule out chronic myelogenous leukemia. JAK2 mutation was positive, and the patient was diagnosed with polycythemia vera.     

STUDIES ON THE PHYSIOLOGY OF THE WHITE BLOOD CELL: THE GLYCOGEN CONTENT OF LEUKOCYTES IN LEUKEMIA AND POLYCYTHEMIA

The technic of determining glycogen in isolated white blood cells was appliedto the study of the different types of leukemia and of polycythemia, in order toobtain information on the physiology of the white blood cell. From this study itis concluded that the granulated leukocyte is the only carrier of glycogen in wholeblood. The "reducing substances" in lymphocytes and blast cells are not consideredas true glycogen.

The glycogen content of wet white blood cells in the rabbit amounts to about1 per cent. In the human being a range of from 0.17 to 0.67 per cent was calculated.In disease higher percentages occur, in polycythemia up to 1.64 per cent and inglycogen storage disease up to 3.05 per cent.

The glycogen concentration of normal white blood cells is within the same rangeas that of the striated muscle.

Note: I acknowledge with gratitude my indebtedness to Dr. William Dameshek for giving me the opportunity of analyzing the blood of some of the patients studied. Miss M. H. Campbell, Miss H. A. Clark,and Miss L. M. Garofalo have aided in carrying out many of the blood counts.

     

Leukocyte Histidine Decarboxylase: Properties and Activity in Myeloproliferative Disorders

1. Histidine decarboxylase was assayed in extracts from human leukocytesand the properties of the enzyme studied.

2. Leukocyte histidine decarboxylase was found to be substrate-specific, torequire pyridoxal phosphate as co-enzyme, and to be inhibited by alpha-hydrazino analog of histidine (MK 785), a selective inhibitor of the specifichistidine decarboxylase occuring in rat tissue. A non-specific L-aromatic aminoacid decarboxylase was also demonstrated in leukocyte extracts, which possessed little activity toward histidine.

3. Cellular localization studies revealed that mature neutrophils and basophils possessed most of the histidine decarboxylase activity exhibited by mixedleukocyte preparations. Mature eosinophils, small lymphocytes, and immaturemyeloid cells (myeloblasts and promyelocytes) showed little histidine decarboxylase activity.

4. In the clinical studies, patients with uncontrolled polycythemia vera,"spent" polycythemia, myelofibrosis with myeloid metaplasia, and chronic myelocytic leukemia, showed increased leukocyte enzyme activity when comparedto a control group composed of normal subjects and patients with relativepolycythemia. This increased activity appears to represent a true increase inenzyme activity per granulocyte, and is believed to account for the elevatedleukocyte histamine content demonstrated in patients with myeloproliferativedisorders.

Submitted on August 30, 1967 Accepted on November 7, 1967     

Inflammation and thrombosis in essential thrombocythemia and polycythemia vera: different role of C-reactive protein and pentraxin 3

We tested the hypothesis that levels of pentraxin high sensitivity C-reactive protein and pentraxin 3 might be correlated with cardiovascular complications in patients with essential thrombocythemia and polycythemia vera. High sensitivity C-reactive protein and pentraxin 3 were measured in 244 consecutive essential thrombocythemia and polycythemia vera patients in whom, after a median follow up of 5.3 years (range 0-24), 68 cardiovascular events were diagnosed. The highest C-reactive protein tertile was compared with the lowest (>3 vs. <1 mg/L) and correlated with age (P=0.001), phenotype (polycythemia vera vs. essential thrombocythemia, P=0.006), cardiovascular risk factors (P=0.012) and JAK2V617F allele burden greater than 50% (P=0.003). Major thrombosis rate was higher in the highest C-reactive protein tertile (P=0.01) and lower at the highest pentraxin 3 levels (P=0.045). These associations remained significant in multivariate analyses and indicate that blood levels of high sensitivity C-reactive protein and petraxin 3 independently and in opposite ways modulate the intrinsic risk of cardiovascular events in patients with myeloproliferative disorders.     

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.     

Gastric pepsin secretion and ABO blood groups in polycythemia vera

Summary A study of the ABO blood group distribution among 332 patients with polycythemia vera revealed 3.5 per cent excess of group O. Although this difference was in the predicted direction, it was not significant. In 73 individuals with polycythemia vera and 38 with chronic granulocytic leukemia, measurement of plasma pepsinogen concentration was made by the indirect method of plasma pepsinogen determination. The mean value for polycythemic patients was slightly higher and for leukemic subjects slightly lower than among controls. Neither difference was statistically significant. The increased occurrence of peptic ulcer claimed for these 2 diseases, if indeed real, may be due to factors other than elevated acid-pepsin activity.The authors wish to express their gratitude to Dr. Edward H. Reinhard, Washington University, and to Dr. Ovid O. Meyer, University of Wisconsin, for permitting them to study their patients.     

The Determination of Iron Absorption and Loss by Whole Body Counting

A technic for the study of radioiron absorption and loss is described employing an NaI (T1) crystal-detector whole body counter and 1-10 µc. Fe⁵⁹in 250 µg. elemental iron. Changes in whole body Fe⁵⁹ activity during thefirst few hours and the next 90-100 days after oral ingestion are describedand their significance discussed. Normal absorption with this technic rangesfrom 5.7-24.7 per cent of the administered tracer. In 14 patients with polycythemia vera, 12 previously phlebotomized and 2 with a recent history ofgastrointestinal hemorrhage, iron deficiency as evidenced by increased ironabsorption (20.6 per cent-96.9 per cent) correlates well with the extent ofpreceding phlebotomy, and relatively well with the plasma iron at the timeof study. Although other parameters reflect iron deficiency, none correlatewell with the absorption of radioiron. Next to increased iron absorption, depletion of iron stores in the marrow seems to be the earliest evidence of irondeficiency.

Iron absorption and erythrocyte incorporation of radioiron was also studiedin several other hematologic disorders, including four heavily menstruatingwomen, three cases of aplastic anemia, and a small number of other conditions.The findings are described and discussed.

Radioiron loss in three normal patients was 0.110 per cent, 0.110 per cent,and 0.182 per cent daily, and in two patients with aplastic anemia 0.103 percent and 0.173 per cent daily, defining the normal range of tracer loss overdays 20-100. Radioiron loss in the polycythemics ranged from 0-0.044 per centdaily. An unusual case of pyridoxine-responsive anemia with increased absorption of radioiron (69.1 per cent), but no red cell incorporation, lost only0.026 per cent/day. Some problems in the interpretation of such data arediscussed.

The results demonstrate the effectiveness of the technic of whole bodycounting in the study of various aspects of iron metabolism.

Submitted on December 26, 1961 Accepted on July 21, 1962     

Reticulated platelets are increased in chronic myeloproliferative disorders, pure erythrocytosis, reactive thrombocytosis and prior to hematopoietic reconstitution after intensive chemotherapy

Reticulated platelets with a high RNA content represent the most recently released platelets and are regarded to reflect thrombopoiesis. In the present study we used flow cytometric analysis to determine the percentage of reticulated platelets in peripheral blood for patients with chronic myeloproliferative disorders (polycythemia vera, essential thrombocytosis) and acute myelogenous leukemia (AML) patients with severe chemotherapy-induced thrombocytopenia. Patients with essential thrombocytosis and polycythemia vera showed increased levels of reticulated platelets compared with healthy controls, and these levels persisted after normalization of the platelet count by hydroxyurea or interferon-alpha treatment. Patients with reactive thrombocytosis or thrombocytopenia with increased platelet turnover often had higher levels of circulating reticulated platelets than patients with myeloproliferative disorders. Furthermore, AML patients with severe chemotherapy-induced cytopenia showed low levels that started to increase 1-9 days prior to hematopoietic reconstitution. To summarize and conclude: (i) circulating reticulated platelets are increased in patients with chronic myeloproliferative disorders, reactive thrombocytosis and thrombocytopenia due to increased platelet turnover; (ii) patients with pure erythrocytosis often have additional abnormalities in the thrombopoiesis; and (iii) the levels of reticulated platelets seem to predict hematopoietic reconstitution for patients receiving intensive AML therapy.     

Increased serum procollagen III aminoterminal peptide in myelofibrosis

Myelofibrosis has been shown to involve an increase in type III collagen in the marrow. The aminoterminal procollagen III (PC III) peptide fragment is released during the production of PC III by fibroblasts and its serum level is therefore a marker for type III collagen synthesis. Using a recently developed sensitive radioimmunoassay, serum levels of PC III peptide were measured in 30 patients with myeloproliferative disease and 23 normal volunteers. Levels were found to be elevated above normal values in patients with polycythemia vera, even more elevated in patients with polycythemia and evidence of secondary myelofibrosis with myeloid metaplasia, and most strikingly elevated in patients with agnogenic myeloid metaplasia and severe marrow fibrosis. There was a significant association between serum levels of PC III peptide and the extent of reticulin fibrosis in bone marrow biopsies. Serum PC III level appears to be a quantitative marker for myelofibrosis.     

Elongation index of erythrocytes, study of activity of chosen erythrocyte enzymes, and the levels of glutathione, malonyldialdehyde in polycythemia vera (PV)

The principal aim of the study was to investigate rheological properties of erythrocytes obtained from patients admitted to the clinic, and diagnosed with polycythemia vera. The polycythemia vera diagnosis was based on the WHO criteria for polycythemia vera. Using a laser rheometer SSD Rheometer-Rheodyn, the elongation index of erythrocytes was determined, indicating an increased rigidity of the erythrocytes in this disease compared with the erythrocytes in healthy people. In order to explain (albeit partially) the reason for reduced elasticity, the erythrocytes of patients with polycythemia were studied for the activity of enzymes - glucose-6-phosphate dehydrogenase and acetylcholinesterase membrane enzyme, as well as the levels of glutathione and malonyldialdehyde. The elevated activities of these enzymes, the glutathione level, and elevated ‰ of reticulocytes, indicated an increased pool of juvenile erythrocyte forms; furthermore, the elevated value of malonyldialdehyde may suggest a lipid peroxidative damage in certain pool of the erythrocyte membrane in blood circulation.     

Relation of mastocytoma to mast cell leukemia,and of heparin,histamine and serotonin to mast cells

A highly functional, transplantable neoplasm of mast cells is described.It causes solitary slowly growing tumors localized at the site of graft in muscles and subcutaneous tissues.

Mast cell leukemia results when isolated cells are injected intravenously.The blood of mice with mast cell leukemia produces solid mastocytomas wheninjected intramuscularly.

These and other observations suggest the essential identity of blood andtissue mast cells and suggest that mast cells are an independent cell typewith primary residence in tissues outside the hemopoietic organs.

In mastocytomas, heparin, histamine and serotonin are present in greatquantities. Some histamine is released and partly excreted in the urine.

In the mast cell leukemias, histamine plasma levels are slightly raised andurine levels are highly elevated.

Plasma heparin values may be slightly raised in mice with mast cell tumors and are greatly increased in mast cell leukemias without a hemorrhagicstate.

The liver histamine and heparin values appear to be related to the number of infiltrating mast cells.

Submitted on August 18, 1958 Accepted on October 23, 1958     

Therapy of polycythemia vera with myleran

Myleran was used in the therapy of nine relapses of polycythemia verain five patients. Clinical examinations, blood studies, and, in three instances,radioisotope tracer tests before and after treatment demonstrated the effectiveness, safety and simplicity of the treatment. Further trial of Mylerantherapy in polycythemia vera seems warranted.

Submitted on September 6, 1957 Accepted on December 16, 1957     

The Jak2V617F mutation, PRV-1 overexpression, and EEC formation define a similar cohort of MPD patients

Recently, a Jak2V617F mutation has been described in the vast majority of patients with polycythemia vera (PV) as well as in subsets of patients with essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). The question arises whether this mutation is observed in those patients with ET and IMF who have also displayed previously described molecular markers, notably the ability to form endogenous erythroid colonies (EECs), overexpression of polycythemia rubra vera 1 (PRV-1), and decreased c-Mpl expression. We therefore analyzed the Janus kinase 2 (Jak2) DNA sequence, EEC growth, PRV-1 expression, and c-Mpl (myeloproliferative) levels in a cohort of 78 myeloproliferative disorder (MPD) patients (42 ET, 22 PV, and 14 IMF). Presence of the Jak2V617F mutation was very highly correlated with PRV-1 overexpression and the ability to form EECs in all 3 subtypes of MPDs (P < .001). (     

The relationship of the basophil to blood histamine in man

Data are presented correlating 22 determinations of the histamine content of blood in patients with chronic granulocytic leukemia with (1) the basophils (2) the other myeloid granulocytes. Expressing the histamine in each caseon the basis of the amount in 10⁸ granulocytes, there is a readily evident positivecorrelation with the basophil percentage and a negative correlation with thepercentage of other myeloid elements. This is strikingly apparent in instanceswhere blood with a very high percentage of basophils was analyzed. The dataindicate that in chronic granulocytic leukemia, the basophil is predominantlyand, perhaps exclusively, responsible for the marked elevations in blood histamine. Inferentially, it appears probable that this cell type, though small innumbers, may be the principal carrier of histamine in non-leukemic blood.

Submitted on April 26, 1954 Accepted on May 24, 1954