Hans Carl Hasselbalch

Philadelphia-Negative Classical Myeloproliferative Neoplasms: Critical Concepts and Management Recommendations From European LeukemiaNet

We present a review of critical concepts and produce recommendations on the management of Philadelphia-negative classical myeloproliferative neoplasms, including monitoring, response definition, first- and second-line therapy, and therapy for special issues. Key questions were selected according the criterion of clinical relevance. Statements were produced using a Delphi process, and two consensus conferences involving a panel of 21 experts appointed by the European LeukemiaNet (ELN) were convened. Patients with polycythemia vera (PV) and essential thrombocythemia (ET) should be defined as high risk if age is greater than 60 years or there is a history of previous thrombosis. Risk stratification in primary myelofibrosis (PMF) should start with the International Prognostic Scoring System (IPSS) for newly diagnosed patients and dynamic IPSS for patients being seen during their disease course, with the addition of cytogenetics evaluation and transfusion status. High-risk patients with PV should be managed with phlebotomy, low-dose aspirin, and cytoreduction, with either hydroxyurea or interferon at any age. High-risk patients with ET should be managed with cytoreduction, using hydroxyurea at any age. Monitoring response in PV and ET should use the ELN clinicohematologic criteria. Corticosteroids, androgens, erythropoiesis-stimulating agents, and immunomodulators are recommended to treat anemia of PMF, whereas hydroxyurea is the first-line treatment of PMF-associated splenomegaly. Indications for splenectomy include symptomatic portal hypertension, drug-refractory painful splenomegaly, and frequent RBC transfusions. The risk of allogeneic stem-cell transplantation-related complications is justified in transplantation-eligible patients whose median survival time is expected to be less than 5 years.

MPL mutations in myeloproliferative disorders: analysis of the PT-1 cohort

Activating mutations of MPL exon 10 have been described in a minority of patients with idiopathic myelofibrosis (IMF) or essential thrombocythemia (ET), but their prevalence and clinical significance are unclear. Here we demonstrate that MPL mutations outside exon 10 are uncommon in platelet cDNA and identify 4 different exon 10 mutations in granulocyte DNA from a retrospective cohort of 200 patients with ET or IMF. Allele-specific polymerase chain reaction was then used to genotype 776 samples from patients with ET entered into the PT-1 studies. MPL mutations were identified in 8.5% of JAK2 V617F(-) patients and a single V617F(+) patient. Patients carrying the W515K allele had a significantly higher allele burden than did those with the W515L allele, suggesting a functional difference between the 2 variants. Compared with V617F(+) ET patients, those with MPL mutations displayed lower hemoglobin and higher platelet levels at diagnosis, higher serum erythropoietin levels, endogenous megakaryocytic but not erythroid colony growth, and reduced bone marrow erythroid and overall cellularity. Compared with V617F(-) patients, those with MPL mutations were older with reduced bone marrow cellularity but could not be identified as a discrete clinicopathologic subgroup. MPL mutations lacked prognostic significance with respect to thrombosis, major hemorrhage, myelofibrotic transformation or survival.

Response criteria for essential thrombocythemia and polycythemia vera: result of a European LeukemiaNet consensus conference

European experts were convened to develop a definition of response to treatment in polycythemia vera (PV) and essential thrombocythemia (ET). Clinicohematologic (CH), molecular, and histologic response categories were selected. In ET, CH complete response (CR) was: platelet count less than or equal to 400 x 10(9)/L, no disease-related symptoms, normal spleen size, and white blood cell count less than or equal to 10 x 10(9)/L. Platelet count less than or equal to 600 x 10(9)/L or a decrease greater than 50% was partial response (PR). In PV, CH-CR was: hematocrit less than 45% without phlebotomy, platelet count less than or equal to 400 x 10(9)/L, white blood cell count less than or equal to 10 x 10(9)/L, and no disease-related symptoms. A hematocrit less than 45% without phlebotomy or response in 3 or more of the other criteria was defined as PR. In both ET and in PV, molecular CR was a reduction of any molecular abnormality to undetectable levels. Molecular PR was defined as a reduction more than or equal to 50% in patients with less than 50% mutant allele burden, or a reduction more than or equal to 25% in patients with more than 50% mutant allele burden. Bone marrow histologic response in ET was judged on megakaryocyte hyperplasia while on cellularity and reticulin fibrosis in PV. The combined use of these response definitions should help standardize the design and reporting of clinical studies.

Incidence, clinical features and outcome of essential thrombocythaemia in a well defined geographical area

Abstract: In an attempt to characterise the clinical features, incidence and outcome of essential thrombocythaemia (ET) we report our experience in a large unselected series of patients from a well defined region. All new cases of ET in the County of Copenhagen were registered during the period 1977–98. We identified 96 cases of ET, yielding an age‐ and sex‐adjusted annual incidence rate of 0.59/100.000 and a point‐prevalence at last follow up of 11/100.000. The overall incidence rate was 0.31 and 1.00 per 100.000 population during the consecutive periods 1977–89 and 1990–98, respectively, corresponding to a 3.2‐fold increase. Median age at diagnosis was 67 yr (females 68 yr, males 66 yr, range 18–87 yr), and the female to male (F/M) ratio was 2.6:1. At diagnosis, 52% of the patients displayed no ET‐related symptoms and were discovered fortuitously by a routine platelet count. Forty‐eight percent presented with thrombohaemorrhagic phenomena, of which microvascular disturbances of the central nervous system (CNS), extremities and skin were most frequently observed (23%). Compared to patients diagnosed after 1989, patients diagnosed before 1990 had a significantly higher mean platelet count, white blood cell (WBC) count, lactate dehydrogenase (LDH) value and alkaline phosphatase value. With a median follow up of 70 months, 5‐yr survival was 76%, significantly lower than the expected survival of an age‐ and sex‐matched control group (p=0.0052). Thirty‐seven patients experienced a total of 55 thrombohaemorrhagic events during follow‐up, corresponding to an incidence of thrombosis and microvascular disturbances or haemorrhage of 8.1% per pt‐yr and 2.5% per pt‐yr, respectively. The number of patients experiencing thrombosis or microvascular disturbances was significantly higher among the 29 patients who never received acetylsalicylic acid (ASA) compared to the 67 patients who received ASA during follow up (45% vs. 21%; p=0.017). This study provides population‐based data suggesting the benefit of treatment with low‐dose ASA in a non‐selected population of patients with ET.

The JAK2 V617F mutation involves B‐ and T‐lymphocyte lineages in a subgroup of patients with Philadelphia‐chromosome negative chronic myeloproliferative disorders

The JAK2 V617F mutation is a frequent genetic event in the three classical Philadelphia‐chromosome negative chronic myeloproliferative disorders (Phneg.‐CMPD), polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). Its occurrence varies in frequency in regards to phenotype. The mutation is found in the majority of patients with PV and about half of the patients with ET and IMF. These diseases are clonal stem cell disorders arising in an early stem cell progenitor. The level in the stem cell hierarchy on which the initiating genetic events and the JAK2 V617F mutation occurs is not known. The mutation has so far been detected in all cells of the myeloid lineage, whereas the potential clonal involvement of the lymphoid lineage is controversial. In this study, we detected the JAK2 V617F mutation by real‐time quantitative PCR (qPCR) in both B‐lymphocytes and T‐lymphocytes in a subgroup of patients with Phneg.‐CMPDs. These results demonstrate the origin of the JAK2 V617F positive disorders in an early stem cell with both lymphoid and myeloid differentiation potential.

Does primary myelofibrosis involve a defective stem cell niche? From concept to evidence

Primary myelofibrosis (PMF) is the rarest and the most severe Philadelphia-negative chronic myeloproliferative syndrome. By associating a clonal proliferation and a mobilization of hematopoietic stem cells from bone marrow to spleen with profound alterations of the stroma, PMF is a remarkable model in which deregulation of the stem cell niche is of utmost importance for the disease development. This paper reviews key data suggesting that an imbalance between endosteal and vascular niches participates in the development of clonal stem cell proliferation. Mechanisms by which bone marrow niches are altered with ensuing mobilization and homing of neoplastic hematopoietic stem cells in new or reinitialized niches in the spleen and liver are examined. Differences between signals delivered by both endosteal and vascular niches in the bone marrow and spleen of patients as well as the responsiveness of PMF stem cells to their specific signals are discussed. A proposal for integrating a potential role for the JAK2 mutation in their altered sensitivity is made. A better understanding of the cross talk between stem cells and their niche should imply new therapeutic strategies targeting not only intrinsic defects in stem cell signaling but also regulatory hematopoietic niche-derived signals and, consequently, stem cell proliferation.

The JAK2 V617F allele burden in essential thrombocythemia, polycythemia vera and primary myelofibrosis – impact on disease phenotype

Background and objectives:  The JAK2 V617F tyrosine kinase mutation is present in the great majority of patients with polycythemia vera (PV), and approximately half of the patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). The three distinct disease entities may be considered as three phenotypic presentations of the same JAK2 V617F positive chronic myleoproliferative disorder. Together with physiological and genetic modifiers the phenotype may be determined by the JAK2 V617F allele burden. In the present study, we aimed to asses the JAK2 mutational load and its impact on phenotype.

Increased circulating platelet–leukocyte aggregates in myeloproliferative disorders is correlated to previous thrombosis, platelet activation and platelet count

Abstract: Platelet–leukocyte adhesion may occur as a consequence of platelet activation and possibly plays a key role in the deposition of activated platelets and fibrin in the thrombotic plug. The aim of the present study was to assess by whole blood flow cytometry the presence of circulating platelet–leukocyte aggregates (PLA) and the platelet–leukocyte response to platelet agonist stimulation (ADP and TRAP) in 50 patients with chronic myeloproliferative disorders (MPD) and 30 controls. PLA were identified as platelet–granulocyte/monocyte aggregates (PGMA), platelet–monocyte aggregates (PMA) and defined as the percentage of leukocytes coexpressing the platelet‐specific marker glycoprotein Ib. Compared to controls the mean percentage of PGMA and PMA was increased in unstimulated whole blood from patients with MPD (7.98 vs. 1.76%; p<0.001 and 12.34 vs. 3.2%; p<0.001, respectively). The percentage of PGMA was correlated to the platelet count (r=0.46; p<0.001), percentage of P‐selectin (r=0.69; p<0.001) and thrombospondin (r=0.58; p<0.001) positive platelets and platelet expression of GPIV (r=0.33; p=0.02). The mean percentage of PGMA and PMA was significantly increased in ADP‐stimulated whole blood of patients (57.14 vs. 47.92%; p=0.009 and 54.91 vs. 45.89%; p<0.001, respectively). Compared to patients without a history of thrombosis, patients having experienced microvascular disturbances or a thrombotic event had a higher mean percentage of PGMA and PMA in non‐stimulated whole blood (10.07 vs. 6.34%; p=0.025 and 14.81 vs. 10.48%; p=0.021, respectively) and a higher percentage of PGMA in ADP stimulated whole blood (64.32 vs. 51.50%; p<0.01). These data document an increased frequency of PLA in non‐stimulated whole blood in MPD associated with a previous history of thrombosis or microvascular disturbances.

Increased platelet activation and abnormal membrane glycoprotein content and redistribution in myeloproliferative disorders

Chronic myeloproliferative disorders (MPDs) are characterized by a high incidence of thrombohaemorrhagic complications, possibly caused by platelet dysfunction. In an attempt to define platelet functional abnormalities, we assessed the expression of activation‐dependent membrane proteins in unstimulated and agonist [ADP and thrombin receptor‐activating peptide (TRAP)]‐stimulated platelets using quantitative whole blood flow cytometry in samples from 50 MPD patients and 30 controls. The receptor densities of activation markers and glycoproteins (GPs) were quantified using standardized fluorescent beads. Compared with controls, the mean percentage of P‐selectin‐positive (15·3% vs. 7·2%; P < 0·001) and thrombospondin (TSP)‐positive (6·6% vs. 3·7%; P = 0·003) platelets was increased in unstimulated platelets from patients. Patients having experienced a thrombotic event had a higher mean percentage of TSP‐positive non‐stimulated platelets than patients without a history of thrombosis (9·0% vs. 4·6%; P = 0·02) and a higher GPIV molecules of equivalent fluorochrome (MEF) value (33113 vs. 24471 MEF; P = 0·02). Mean MEF values of monoclonal antibodies (mAbs) against GPIb (34055 vs. 38945 MEF; P < 0·001) and GPIIb/IIIa (1416 vs. 1648 MEF; P < 0·001) were significantly reduced among patients, whereas surface expression of GPIV was increased in patients (28273 vs. 16258 MEF; P < 0·001). In TRAP (10 µmol/l) stimulated whole blood, the MEF of P‐selectin (9611 vs. 13293 MEF; P = 0·004) and CD63 (2385 vs. 5177 MEF; P < 0·001) and the ratio of PAC‐1/GPIIb/IIIa MEF (0·98 vs. 2·00; P < 0·001) was reduced in patients, indicating either a reduced granule GP content or an intrinsic cellular defect in receptor‐mediated granule secretion and activation of the GPIIb/IIIa complex. Expressed as the relative change of MEF compared with unstimulated platelets, TRAP induced decrease of GPIb (7·8% vs. 45%; P < 0·001) and increase of GPIIb/IIIa (49·1% vs. 95·7%; P < 0·001) and GPIV expression (17·8% vs. 55·2%; P < 0·001) was attenuated in patients.

Chronic myeloproliferative neoplasms and subsequent cancer risk: a Danish population-based cohort study

Patients with chronic myeloproliferative neoplasms, including essential thrombocythemia (ET), polycythemia vera (PV), and chronic myeloid leukemia (CML), are at increased risk of new hematologic malignancies, but their risk of nonhematologic malignancies remains unknown. In the present study, we assessed the risk of both types of malignancies after an ET, PV, or CML diagnosis. We linked 2 population-based nationwide registries, the Danish National Registry of Patients, covering all Danish hospitals and the Danish Cancer Registry, and assessed subsequent cancer risk in a cohort of all 7229 patients diagnosed with a chronic myeloproliferative neoplasm during 1977-2008. We compared the incidence of subsequent cancer in this cohort with that expected on the basis of cancer incidence in the general population (standardized incidence ratio). Overall, ET, PV, and CML patients were at increased risk of developing both new hematologic and nonhematologic cancers. The standardized incidence ratio for developing a nonhematologic cancer was 1.2 (95% confidence interval [95% CI]): 1.0-1.4) for patients with ET, 1.4 (95% CI: 1.3-1.5) for patients with PV, and 1.6 (95% CI: 1.3-2.0) for patients with CML. We conclude that patients with chronic myeloproliferative neoplasms are at increased risk of developing a new malignant disease.