Sanford B. Krantz

Identification of an Acquired JAK2 Mutation in Polycythemia Vera

Polycythemia vera (PV) is a human clonal hematological disorder. The molecular etiology of the disease has not been identified. PV hematopoietic progenitor cells exhibit hypersensitivity to growth factors and cytokines, suggesting possible abnormalities in protein-tyrosine kinases and phosphatases. By sequencing the entire coding regions of cDNAs of candidate enzymes, we identified a G:C→ T:A point mutation of the JAK2 tyrosine kinase in 20 of 24 PV blood samples but none in 12 normal samples. The mutation has varying degrees of heterozygosity and is apparently acquired. It changes conserved Val617 to Phe in the pseudokinase domain of JAK2 that is known to have an inhibitory role. The mutant JAK2 has enhanced kinase activity, and when overexpressed together with the erythropoietin receptor in cells, it caused hyperactivation of erythropoietin-induced cell signaling. This gain-of-function mutation of JAK may explain the hypersensitivity of PV progenitor cells to growth factors and cytokines. Our study thus defines a molecular defect of PV.

Erythroid response to treatment with G-CSF plus erythropoietin for the anaemia of patients with myelodysplastic syndromes : proposal for a predictive model

Previous studies have shown that approximately 40% of patients with myelodysplastic syndrome (MDS) and anaemia respond to treatment with human recombinant granulocyte‐CSF (G‐CSF) plus erythropoietin (epo). The present study was designed to investigate pre‐treatment variables for their ability to predict erythroid responses to this treatment. 98 patients with MDS (30 RA, 31 RARS, 32 RAEB, five RAEB‐t) were treated with a combination of G‐CSF (0.3–3.0 μg/kg/d, s.c.) and epo (60–300 U/kg/d, s.c.) for at least 10 weeks. Minimum criteria for erythroid response was a 100% reduction of red blood cell (RBC) transfusion need or an increase in haemoglobin level of  1.5 g/dl. 35 patients (36%) showed responses to treatment. Medium duration of response was 11–24 months. In multivariate analysis, serum erythropoietin levels and initial RBC‐transfusion need retained high statistical significance (P < 0.01). Using pre‐treatment serum epo levels as a ternary variable (< 100, 100–500 or > 500 U/l) and RBC transfusion need as a binary variable (< 2 or  2 units per month), the analysis provided a predictive score for erythroid response. This score divided patients into three groups: one group with a high probability of erythroid responses (74%), one intermediate group (23%) and one group with poor responses to treatment (7%). This predictive scoring system could be used in decisions regarding use of these cytokines for treating the anaemia of MDS, both for defining patients who should not be given the treatment and for selecting patients for inclusion in prospective trials.

Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin.

Morphological and biochemical studies of human colony-forming units-erythroid (CFU-E) have been hindered by their extreme rarity. Since burst-forming units-erythroid (BFU-E) develop into CFU-E, we used normal human blood BFU-E to generate large numbers of highly purified CFU-E in vitro. Using density centrifugation, sheep erythrocyte rosetting, surface immunoglobulin-positive cell depletion, adherence to plastic, and negative panning with monoclonal antibodies, human blood BFU-E were purified from 0.017 to 0.368%, a 22-fold purification with a 43% yield. The panned cells were cultured in methylcellulose with recombinant erythropoietin (rEp) and conditioned medium for 9 d. These cells were then collected and CFU-E were further purified using adherence and density centrifugation. This yielded almost 10(7) erythroid colony forming cells with a purity of 70 +/- 18%. Analysis of these cells by light and electron microscopy showed 94% erythroid cells. The prominent cell was a primitive blast with high nuclear/cytoplasmic ratio, dispersed nuclear chromatin and a distinct large nucleolus. The relation between the number of erythroid colonies and the number of day 9 cells plated in plasma clots was a straight line through the origin with a maximum number of erythroid colonies at 1 U/ml of rEp and no erythroid colonies without rEp. Specific binding with 125I-rEp showed that 60% of the binding was inhibited by excess pure erythropoietin (Ep), but not by albumin, fetal calf serum, and a variety of growth factors or glycoproteins. By days 12-13 of cell culture, when the progenitor cells matured to late erythroblasts, specific binding markedly declined. In this study, human CFU-E have been isolated in sufficient purity to characterize the morphology of these rare cells and in sufficient numbers to measure specific binding of Ep.

Human colony-forming units-erythroid do not require accessory cells, but do require direct interaction with insulin-like growth factor I and/or insulin for erythroid development.

The presence of heterogeneous erythroid progenitor cells, contaminant cells, or serum may alter erythroid colony development in vitro. To obtain highly purified colony-forming units-erythroid (CFU-E), we cultured partially purified human blood burst-forming units-erythroid (BFU-E) in methylcellulose with recombinant human erythropoietin (rHuEPO) for 7 d and generated cells that consisted of 30-60% CFU-E, but no BFU-E. A serum-free medium was used that allowed development of the same number of erythroid colonies as serum containing medium, but with a greater percentage of larger colonies. This medium consisted of delipidated crystalline bovine serum albumin, iron saturated transferrin, lipid suspension, fibrinogen, thrombin, Iscoves modified Dulbeccos medium/F-12[HAM], and insulin plus rHuEPO. When CFU-E were cultured in a limiting dilution assay and the percentage of nonresponder wells was plotted against cell concentration, both serum-free cultures and serum-containing cultures yielded overlapping straight lines through the origin indicating that CFU-E development did not depend on accessory cells and that insulin acted directly on the CFU-E. Human recombinant interleukin 3 (IL-3) and/or granulocyte-macrophage colony-stimulating factor had no effect on CFU-E growth, while they markedly enhanced BFU-E growth. Physiological concentrations of recombinant human insulin-like growth factor I (IGF-I) enhanced CFU-E growth in the absence of insulin and, together with rHuEPO in serum-free medium, provided a plating efficiency equal to that of serum-containing medium. Limiting dilution analysis in serum-free medium with IGF-I showed a straight line through the origin indicating that IGF-I also acted directly on the CFU-E and not through an effect on accessory cells. These data demonstrate that CFU-E do not require accessory cells, but do require IGF-I and/or insulin which act directly on the CFU-E.

Distinct roles of erythropoietin, insulin-like growth factor I, and stem cell factor in the development of erythroid progenitor cells.

Erythropoietin (EP), insulin-like growth factor I (IGF-I) and stem cell factor (SCF) each reduce apoptosis of human erythroid progenitor cells. To determine if these growth factors have additional roles in stimulating erythropoiesis, the proliferation, maturation, and survival of highly purified human erythroid colony-forming cells (ECFCs) were studied during the application of different combinations of these growth factors in a serum-free liquid culture. EP maintained cell viability and supported heme synthesis during erythroid maturation, with little increase in viable cell number or stimulation of DNA synthesis. The addition of SCF with EP resulted in a substantial increase in DNA synthesis, which was greater than that seen with the addition of EP and was associated with a large expansion in the number of ECFCs. Thus EP, by itself, produces little increase in cell proliferation, and expansion of the number of erythroid cells depends upon the presence of SCF with EP. The addition of IGF-I with EP led to enhanced heme synthesis and moderate cellular proliferation, but also greatly enhanced nuclear condensation and enucleation in the late erythroblasts. Thus EP, by itself, is not sufficient for complete end-terminal nuclear condensation/enucleation and the presence of IGF-I is necessary for this complete process. While EP greatly reduced apoptosis during 16 h of incubation at 37 degrees C, the addition of SCF and IGF-I with EP had little additional effect, but these additions enhanced DNA synthesis > 3.4-fold. Thus SCF may have an additional role in directly stimulating proliferation through a process that is distinct from apoptosis. Our observations indicate that EP prevents apoptosis and maintains erythroid cell viability and development. IGF-I enhances erythroid maturation and proliferation, but the proliferation of erythroid progenitors is mainly controlled by the addition of SCF with EP, independent of an effect on apoptosis.

The Anemia of Primary Autonomic Failure and its Reversal with Recombinant Erythropoietin

Table. SI Units An association between the sympathetic nervous system and erythropoiesis has been postulated for several years and examined in a few animal studies. Takaku and colleagues [1] showed in 1961 that the reticulocyte response to acute blood-letting was greatly diminished in rats when their kidneys were functionally denervated [2]. Intravenous administration of the -adrenergic receptor agonist salbutamol increased plasma concentrations of erythropoietin-like factor in rabbits, measured with a bioassay in polycythemic rats [3]. Conversely, -blockers were shown to blunt the erythropoietin response to hypoxia [4, 5]. The conclusion derived from these and other studies was that sympathetic stimulation, acting through 2-adrenoreceptors, positively modulates erythropoiesis through increased erythropoietin production. The physiologic significance of these findings and their relevance to humans, however, has not previously been studied. In caring for patients with severe autonomic failure, we realized that anemia was not an uncommon occurrence. In exceptional patients, anemia was severe enough that it was thought to contribute to the patients symptoms and was treated with blood transfusions. No obvious cause for anemia was apparent in these patients. Therefore, we hypothesized that if indeed the sympathetic nervous system modulated erythropoiesis, patients with severe autonomic failure may present with hypoproliferative anemia. We also studied the effect of recombinant erythropoietin therapy in these patients. Preliminary reports of these studies have been presented previously [6, 7]. Methods Patients We analyzed the clinical and laboratory data of 84 consecutive patients with primary autonomic failure. These patients were referred to Vanderbilt Universitys Autonomic Dysfunction Center from 1983 to 1992 because of symptomatic orthostatic hypotension and, therefore, represent a selected sample of patients. Patients were diagnosed as having either multiple system atrophy (the Shy-Drager syndrome) or pure autonomic failure, based on their clinical features. Patients with pure autonomic failure are characterized by isolated involvement of the autonomic nervous system (orthostatic hypotension without compensatory heart rate increase, impaired sinus arrhythmia, lack of blood pressure overshoot during phase IV of the Valsalva maneuver, and impaired pressor response to isometric exercise and the cold pressor test). They also have decreased sweating, impaired gastric emptying, and impotence. Patients with multiple system atrophy, in addition to autonomic impairment, have central neurologic involvement that usually includes, but is not limited to, Parkinson disease. Patients with secondary forms of autonomic failure (for example, diabetes mellitus) were excluded from the study. Patients with known causes of anemia (for example, iron deficiency, megaloblastic anemia) or concurrent illnesses known to cause the anemia of chronic disease (for example, inflammatory processes) were excluded from evaluation on the basis of clinical findings and laboratory results. All protocols were approved by the institutional review board, and patients gave informed consent. Measurements Patients were admitted to Vanderbilt Universitys Clinical Research Center and received a diet containing 150 mEq of sodium and 60 mEq of potassium per day. All medications were withheld. Autonomic evaluation was done as previously described [8]. Routine laboratory studies and hematologic values were determined in our clinical pathology laboratory. Serum erythropoietin levels were measured in the last 28 patients evaluated, using an enzyme immunoassay (Clinigen; R&D Systems, Inc., Minneapolis, Minnesota) [9]. Reticulocyte counts were corrected for the degree of anemia using the following formula: corrected reticulocyte count = reticulocyte count x (hematocrit/40). Erythrocyte volume was measured with Chromium-51-labeled erythrocytes. Plasma volume was measured with Iodine-131-labeled albumin [10]. Total blood volume was calculated by adding erythrocyte volume plus plasma volume. Blood samples for catecholamine levels and plasma renin activities were obtained through an indwelling catheter placed in a peripheral vein. Samples were obtained after patients had been lying down overnight (supine position) and after they had been standing for 30 minutes (upright position). Patients were encouraged to remain standing upright throughout this 30-minute period but were allowed to sit down at intervals if symptoms developed. Plasma norepinephrine levels were determined by high-pressure liquid chromatography [11]. Plasma renin activity was measured by the conversion of angiotensinogen to angiotensin I and was expressed as nanograms of angiotensin I produced per millilitre of plasma per hour [12]. Because there is no consensus about what hemoglobin level to use (cutoff value) for diagnosing anemia in elderly persons, we divided our patients arbitrarily into three groups depending on their hemoglobin level: group I, hemoglobin levels less than 120 g/L; group II, hemoglobin levels 120 to 130 g/L; and group III, hemoglobin levels greater than 130 g/L. Therapeutic Trials Five patients with anemia (hemoglobin levels < 120 g/L) and isolated autonomic failure were treated with human recombinant erythropoietin (epoetin alfa: Procrit; Ortho Pharmaceuticals, Raritan, New Jersey; or Epogen: Amgen Inc., Thousand Oaks, California). Patients received an initial dose of 25 units/kg body weight subcutaneously, three times a week. Doses were increased by 25 units/kg at 3-week intervals until the hematocrit returned to normal. Patients also received oral iron (300 mg of ferrous sulfate one to three times daily as tolerated), as has been used in other erythropoietin trials [13], and were allowed to continue on their regular medication as long as it was kept constant during erythropoietin therapy. Complete blood cell and reticulocyte counts were done weekly for the first 4 weeks and every 3 weeks thereafter. Erythrocyte mass and plasma volume were measured before treatment and at the time the hematocrit returned to normal. Statistical Analysis Group differences were assessed by analysis of variance (ANOVA) using NCSS statistical software (NCSS, Kaysville, Utah). Mean differences were determined by paired or unpaired t-tests as appropriate. All hypotheses were two tailed, and the criterion of significance was P < 0.05. Results Patient Characteristics The clinical characteristics of the patients are presented in Table 1. As shown by our sample of patients, primary autonomic failure typically occurs in the sixth and seventh decade of life. Patients had dramatic decreases in blood pressure while standing upright. The expected compensatory heart rate increase was virtually absent despite the profound orthostatic hypotension, indicating the severity of their autonomic failure. Plasma norepinephrine levels and plasma renin activity measured in patients lying supine or standing upright were lower than those in normal persons, an inappropriate response considering the magnitude of orthostatic hypotension observed in these patients. Table 1. Clinical Characteristics of Patients with Autonomic Failure Grouped by Severity of Anemia* Characterization of Anemia in Autonomic Failure Patients were divided into three groups depending on their hemoglobin level (Table 1). There were more men in group 3 (P = 0.008 by ANOVA), but no sex differences were apparent in the group with the lowest hemoglobin level (group 1). The only other difference found between groups was in plasma norepinephrine concentrations measured in patients standing upright (P < 0.002 by ANOVA). Differences in plasma renin activity in patients standing upright were of borderline significance (P = 0.09 by ANOVA). No age differences were found among the groups characterized by the severity of the anemia. The diagnoses of pure autonomic failure and multiple system atrophy were evenly distributed within groups 1 and 2, but more patients in group 3 had multiple system atrophy. Patients with the greatest degree of anemia (group 1) had a mean blood hemoglobin concentration of 108 g/L (range, 87 to 118 g/L) and a hematocrit of 0.33 (range, 0.26 to 0.37). Average values for mean corpuscular hemoglobin and mean corpuscular volume were 29 pg and 89 fL (89 microns3), respectively. The mean serum iron was 16.5 mol/L (92 g/dL); the mean total iron binding capacity, 43.3 mol/L (242 g/dL); and the mean ferritin level, 184 g/L. Mean serum vitamin B12 and folate levels were 410 pmol/L (556 pg/mL) and 22.7 nmol/L (10 ng/mL), respectively. The mean corrected reticulocyte count was 0.008. These patients had been treated previously with oral iron, folic acid, and parenteral vitamin B12, with no improvement of the anemia. We measured total blood volume in 14 patients to determine if peripheral venous hemoglobin adequately reflected erythrocyte mass in patients with primary autonomic failure. The mean hemoglobin level in this subset of patients was 123 g/L (range, 106 to 156 g/L). The mean total erythrocyte volume, corrected for body weight, was lower in patients with autonomic failure (21.4 mL/kg) than predicted values (27.1 mL/kg; P = 0.009). The average percent difference in mean erythrocyte volume ([measured mean erythrocyte volume 100/predicted mean erythrocyte volume] 100)was 20.5%(range, 49.3% to 2.4%). A relation was found between blood hemoglobin levels and the percent decrease in erythrocyte volume (hemoglobin = percent decrease in mean erythrocyte volume 0.066 + 13.7; n = 14; r = 0.56; P = 0.01). On the other hand, the mean total blood volume in patients with autonomic failure (65.2 mL/kg; 95% CI, 58.7 to 71.7 mL/kg) was not different from values in normal persons (67.0 mL/kg; CI, 64.4 to 69.6 mL/kg) because of a slightly higher plasma volume in patients with autonomic failure (43.8 mL/kg) compared with predicted values (40.2 mL/kg; P = 0.1). Thes

Pathogenesis and Treatment of the Anemia of Chronic Disease

The anemia of chronic disease may be viewed simply as the anemia that accompanies chronic inflammatory, infectious, or neoplastic disorders. Because these conditions are very common, the anemia of chronic disease is one of the most frequent anemias encountered, and is only second in incidence to iron-deficiency anemia. The anemia of chronic disease is primarily an anemia due to underproduction of red cells, with low reticulocyte production, and is most often a normochromic, normocytic anemia. However, in 30% to 50% of patients, the red cells are hypochromic and microcytic and, most often, the serum iron, total iron-binding capacity, and transferrin saturation are reduced in the presence of adequate iron stores. Although the differential diagnosis includes other underproduction anemias, such as those caused by vitamin and mineral deficiencies, renal failure, endocrinopathies, and myelodysplasia, it generally is easily distinguished from these conditions. Nevertheless, an understanding of the pathogenesis of this condition, as well as a means of alleviating the anemia when the chronic disorder persists, has remained elusive. Recently, major advances have occurred toward understanding the pathogenesis of the anemia of chronic disease and its treatment, and these advances are reviewed.

Studies on Red Cell Aplasia. V. PRESENCE OF ERYTHROBLAST CYTOTOXICITY IN γG-GLOBULIN FRACTION OF PLASMA

The marrow cells of a patient with pure red cell aplasia markedly increased their rate of heme synthesis when they were freed from the host environment and were incubated in vitro. When the red cell aplasia was treated with cyclophosphamide and prednisone, marrow cell incorporation of (59)Fe into heme in vitro increased several weeks before a reticulocytosis was apparent, and was the earliest effect noted. The plasma gammaG-globulins of this patient inhibited heme synthesis by normal marrow cells or the patients own marrow cells obtained after remission of the disease. Since the inhibition of heme synthesis could be the result of damage to erythroblasts, the patients posttreatment marrow cells or normal marrow cells were labeled with (59)Fe and were then incubated with the patients pretreatment, treatment, and posttreatment gammaG-globulins as well as normal gammaG-globulins. At the end of this incubation the supernatant and cells were separated and counted. Heme was extracted and also was counted. Treatment of the cells with the patients pretreatment gammaG-globulins resulted in a release of 40% of the radioactive heme from the cells. This represented the loss of radioactive hemoglobin and was an index of erythroblast cytotoxicity. A progressive disappearance of the cytotoxic factor in the gammaG-globulins occurred in the 3 wk period preceding the onset of reticulocytes in the patients blood. Posttreatment and normal gammaG-globulins did not produce this effect and increased injury of red cells and lymphocytes was not produced by the patients pretreatment gammaG-globulins. These studies demonstrate a method for measuring erythroblast cytoxicity and show that red cell aplasia is associated with gammaG-globulins that specifically damage erythroblasts. Whether interference with new erythroblast development also occurs and contributes to the inhibition of heme synthesis has not yet been ascertained.

The pathogenesis of anemia in rheumatoid arthritis: A clinical and laboratory analysis☆

Principal concepts concerning the anemia of RA are summarized in Tables 7 and 8. These concepts have been validated by our analysis of 93 anemic RA patients and by our review of the literature. The fact that anemia in RA may have one or more etiologies, occasionally in the same patient, mandates a reasoned approach to the analysis of anemia in every RA patient in whom it may occur. In particular, iron deficiency is common and determination of bone marrow iron content via an aspirate may be required for a definitive diagnosis. In those RA patients with anemia of chronic disease, the best therapy remains control of the underlying disease, most commonly with second line drugs and/or corticosteroids. The place for recombinant erythropoietin in the therapy of this anemia has not been defined; one specific role for erythropoietin may be in the preparation of RA patients for elective surgery, particularly hip arthroplasty, where correction of the anemia may either obviate the need for transfusion or may allow for donation of blood for purposes of autologous transfusion perioperatively. The pathogenesis of the anemia of chronic disease, as seen in RA anemia, is not completely understood. Inflammatory mediators, particularly the cytokines, appear to be important factors in the impairment of erythropoiesis. The mechanism by which these cytokines impair erythroid progenitor growth and hemoglobin production in developing erythrocytes is an important area for future study.

Polycythemia vera blood burst-forming units-erythroid are hypersensitive to interleukin-3.

Because polycythemia vera (PV) is a clonal hematopoietic stem cell disease with a trilineage hyperplasia, and interleukin-3 (IL-3) stimulates trilineage hematopoiesis, we have studied the response of highly purified PV blood burst-forming units-erythroid (BFU-E) to recombinant human IL-3 (rIL-3). Whereas the growth of normal blood BFU-E in vitro rapidly declined by 40 and 60% after 24 and 48 h of incubation without 50 U/ml of rIL-3, the growth of PV BFU-E declined by only 10 and 30% under the same conditions, demonstrating a reduced dependence on rIL-3. A reduced dependence of PV BFU-E on recombinant human erythropoietin (rEP) was also present. Dose-response experiments showed a 117-fold increase in PV BFU-E sensitivity to rIL-3, and a 6.5-fold increase in sensitivity to rEP, compared to normal BFU-E, whereas blood BFU-E from patients with secondary polycythemia responded like normal BFU-E. Endogenous erythroid colony (EEC) formation, which is independent of the addition of rEP, was reduced by 50% after erythroid colony-forming cells were generated from PV BFU-E in vitro without rIL-3 for 3 d, whereas rEP-stimulated erythroid colonies were unaffected. These studies demonstrate a striking hypersensitivity of PV blood BFU-E to rIL-3, which may be the major factor in the pathogenesis of increased erythropoiesis without increased EP concentrations.