David Bareford

Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study

BACKGROUND An acquired V617F mutation in JAK2 occurs in most patients with polycythaemia vera, but is seen in only half those with essential thrombocythaemia and idiopathic myelofibrosis. We aimed to assess whether patients with the mutation are biologically distinct from those without, and why the same mutation is associated with different disease phenotypes. METHODS Two sensitive PCR-based methods were used to assess the JAK2 mutation status of 806 patients with essential thrombocythaemia, including 776 from the Medical Research Councils Primary Thrombocythaemia trial (MRC PT-1) and two other prospective studies. Laboratory and clinical features, response to treatment, and clinical events were compared for V617F-positive and V617F-negative patients with essential thrombocythaemia. FINDINGS Mutation-positive patients had multiple features resembling polycythaemia vera, with significantly increased haemoglobin (mean increase 9.6 g/L, 95% CI 7.6-11.6 g/L; p<0.0001), neutrophil counts (1.1x10(9)/L, 0.7-1.5x10(9)/L; p<0.0001), bone marrow erythropoiesis and granulopoiesis, more venous thromboses, and a higher rate of polycythaemic transformation than those without the mutation. Mutation-positive patients had lower serum erythropoietin (mean decrease 13.8 U/L; 95% CI, 10.8-16.9 U/L; p<0.0001) and ferritin (n=182; median 58 vs 91 mug/L; p=0.01) concentrations than did mutation-negative patients. Mutation-negative patients did, nonetheless, show many clinical and laboratory features that were characteristic of a myeloproliferative disorder. V617F-positive individuals were more sensitive to therapy with hydroxyurea, but not anagrelide, than those without the JAK2 mutation. INTERPRETATION Our results suggest that JAK2 V617F-positive essential thrombocythaemia and polycythaemia vera form a biological continuum, with the degree of erythrocytosis determined by physiological or genetic modifiers.

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.

Guidelines for the diagnosis, investigation and management of polycythaemia/erythrocytosis.

Traditionally, polycythaemia has been used to identify a group of varied disorders with an increase in circulating red cells that are typified by a persistently raised haematocrit (Hct). Since only the red cell lineage is involved, the term erythrocytosis has more validity and will be used throughout this article. Polycythaemia will be retained in relation to the clonal disorder, polycythaemia vera (PV), in which three cell lineages are involved.

Guideline for investigation and management of adults and children presenting with a thrombocytosis.

Guy’s and St Thomas’ NHS Foundation Trust, London, Russells Hall Hospital, Dudley, West Midlands, Arrowe Park Hospital Arrowe Park Road Upton Wirral, Wellcome Trust Sanger Institute, Hinxton, Cambridge, St. James Hospital, James Street, Dublin, Gartnavel General Hospital 21 Shelley Road Glasgow, Addenbrooke’s Hospital, Cambridge, Salisbury Healthcare NHS Trust, Salisbury, Wiltshire, Cambridge Institute for Medical Research, Hills Road, Cambridge, John Radcliffe Hospital, Headley Way, Headington, Oxford, Royal Infirmary, Little France Crescent, Edinburgh, Sandwell and West Birmingham Hospitals, Dudley Road, Birmingham, Royal Hallamshire Hospital, Glossop Road, Sheffield, and Belfast City Hospital, Lisburn Road Belfast, UK

Reticulin Accumulation in Essential Thrombocythemia: Prognostic Significance and Relationship to Therapy

PURPOSE Essential thrombocythemia (ET) manifests substantial interpatient heterogeneity in rates of thrombosis, hemorrhage, and disease transformation. Bone marrow histology reflects underlying disease activity in ET but many morphological features show poor reproducibility. PATIENTS AND METHODS We evaluated the clinical significance of bone marrow reticulin, a measure previously shown to have relatively high interobserver reliability, in a large, prospectively-studied cohort of ET patients. RESULTS Reticulin grade positively correlated with white blood cell (P = .05) and platelet counts (P = .0001) at diagnosis. Elevated reticulin levels at presentation predicted higher rates of arterial thrombosis (hazard ratio [HR], 1.8; 95% CI, 1.1 to 2.9; P = .01), major hemorrhage (HR, 2.0; 95% CI, 1.0 to 3.9; P = .05), and myelofibrotic transformation (HR, 5.5; 95% CI, 1.7 to 18.4; P = .0007) independently of known risk factors. Higher reticulin levels at diagnosis were associated with greater subsequent falls in hemoglobin levels in patients treated with anagrelide (P < .0001), but not in those receiving hydroxyurea (P = .9). Moreover, serial trephine specimens in patients randomly assigned to anagrelide showed significantly greater increases in reticulin grade compared with those allocated to hydroxyurea (P = .0003), and four patients who developed increased bone marrow reticulin on anagrelide showed regression of fibrosis when switched to hydroxyurea. These data suggest that patients receiving anagrelide therapy should undergo surveillance bone marrow biopsy every 2 to 3 years and that those who show substantially increasing reticulin levels are at risk of myelofibrotic transformation and may benefit from changing therapy before adverse clinical features develop. CONCLUSION Our results demonstrate that bone marrow reticulin grade at diagnosis represents an independent prognostic marker in ET, reflecting activity and/or duration of disease, with implications for the monitoring of patients receiving anagrelide.

Amendment to the guideline for diagnosis and investigation of polycythaemia/erythrocytosis

historical review. Leukemia Research, 31, 439–444. Tober, J., Koniski, A., McGrath, K.E., Vemishetti, R., Emerson, R., de Mesy-Bentley, K.K.L., Waugh, R. & Palis, J. (2007) The megakaryocyte lineage originates from hemangioblast precursors and is an integral component both of primitive and of definitive hematopoiesis. Blood, 109, 1433–1441.

Increased soluble P-selectin in patients with haematological and breast cancer: a comparison with fibrinogen, plasminogen activator inhibitor and von Willebrand factor.

Abnormal platelet activation and an increased risk of thrombosis are frequent findings in cancer. As soluble adhesion molecule P-selectin is being increasingly recognized as reflecting increased platelet activation, we hypothesized raised levels in patients with cancer, obtaining plasma from 24 patients with a cross-section of haematological cancers, 41 with breast cancer, and from an equal number of healthy controls for each patient group. Levels of soluble P-selectin were compared with those of von Willebrand factor (vWf), plasminogen activator inhibitor-1 (PAI-1) activity and fibrinogen (markers of endothelial integrity, fibrinolysis and coagulation, respectively). We found raised soluble P-selectin, fibrinogen and vWf in both patient groups compared with their controls (P< 0.01). vWf and soluble P-selectin were higher in the haematological cancers than in breast cancer patients (by 30 and 74%, respectively; bothP< 0.01). There was no significant difference in levels of PAI-1 between any group. There were no differences in soluble P-selectin or vWf when the data from the women with breast cancer were classified according to tumour size, lymph node involvement or presence of vascular invasion. We conclude that the platelet marker soluble P-selectin is raised in both haematological and breast cancer, and is higher in the former, but is unrelated to the type or stage of breast cancer.

Clinical utility of routine MPL exon 10 analysis in the diagnosis of essential thrombocythaemia and primary myelofibrosis.

Approximately 50% of essential thrombocythaemia and primary myelo‐fibrosis patients do not have a JAK2 V617F mutation. Up to 5% of these are reported to have a MPL exon 10 mutation but testing for MPL is not routine as there are multiple mutation types. The ability to routinely assess both JAK2 and MPL mutations would be beneficial in the differential diagnosis of unexplained thrombocytosis or myelofibrosis. We developed and applied a high resolution melt (HRM) assay, capable of detecting all known MPL mutations in a single analysis, for the detection of MPL exon 10 mutations. We assessed 175 ET and PMF patients, including 67 that were JAK2 V617F‐negative by real time polymerase chain reaction (PCR). Overall, 19/175 (11%) patients had a MPL exon 10 mutation, of whom 16 were JAK2 V617F‐negative (16/67; 24%). MPL mutation types were W515L (11), W515K (4), W515R (2) and W515A (1). One patient had both W515L and S505N MPL mutations and these were present in the same haemopoietic colonies. Real time PCR for JAK2 V617F analysis and HRM for MPL exon 10 status identified one or more clonal marker in 71% of patients. This combined genetic approach increases the sensitivity of meeting the World Health Organization diagnostic criteria for these myeloproliferative neoplasms.

Racial background is a determinant of average warfarin dose required to maintain the INR between 2.0 and 3.0

Warfarin is a commonly used prophylactic agent for the prevention of thromboembolic disease. We hypothesized that racial background influenced warfarin dosage, and tested this by recording the international normalized ratio (INR) in 867 patients aged 40–90 routinely passing through our Anticoagulation Service whose target INR was 2–3. Mean (95% confidence interval) dose was 4.1 (4.0–4.2) mg/d in 737 Caucasians, 5.5 (4.9–6.1) mg/d in 72 Asians, and 6.7 (5.8–7.6) mg/d in 58 Afro‐Caribbeans (P < 0.05) between each group). In a subgroup of 302 (41 Asians, 22 Afro‐Caribbeans, 239 Caucasians), body mass index did not influence warfarin use. Despite small numbers, we conclude that racial background, but not body mass index, is a determinant of warfarin dosage. The reasons for this could be genetic, cultural (diet related), or both.

Methylation of the suppressor of cytokine signaling 3 gene (SOCS3) in myeloproliferative disorders

Suppressor of cytokine signaling 3 (SOCS3) is negative regulator of the JAK/STAT pathway. Methylation of this gene might, therefore, contribute to the pathogenesis of myeloproliferative disorders. In this study, SOCS3 promoter methylation was detected in about one third of patients with idiopathic myelofibrosis suggesting a possible role for SOCS3 methylation in this disorder. Background The JAK2 V617F mutation can be found in patients with polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis. Mutation or methylation of other components of JAK/STAT signaling, such as the negative regulators suppressor of cytokine signaling 1 (SOCS1) and SOCS3, may contribute to the pathogenesis of both JAK2 V617F positive and negative myeloproliferative disorders. Design and Methods A cohort of patients with myeloproliferative disorders was assessed for acquired mutations, aberrant expression and/or CpG island hypermethylation of SOCS1 and SOCS3. Results No mutations were identified within the coding region of either gene in 73 patients with myeloproliferative disorders. No disease-specific CpG island methylation of SOCS1 was observed. SOCS1 expression was raised in myeloproliferative disorder granulocytes but the level was independent of JAK2 V617F status. Hypermethylation of the SOCS3 promoter was identified in 16 of 50 (32%) patients with idiopathic myelofibrosis but not in patients with essential thrombocythemia, polycythemia vera or myelofibrosis preceded by another myeloproliferative disorder. Confirmation of methylation status was validated by nested polymerase chain reaction and/or bisulphite sequencing. SOCS3 transcript levels were highest in patients with polycythemia vera and other JAK2 V617F positive myeloproliferative disorders, consistent with SOCS3 being a target gene of JAK2/STAT5 signaling. There was a trend towards an association between SOCS3 methylation and lower SOCS3 expression in JAK2 V617F negative patients with idiopathic myelofibrosis but not in JAK2 V617F positive ones. Finally, SOCS3 methylation was not significantly correlated with survival or other clinical variables. Conclusions SOCS3 promoter methylation was detected in 32% of patients with idiopathic myelofibrosis suggesting a possible role for SOCS3 methylation in this disorder. The pathogenetic consequences of SOCS3 methylation in idiopathic myelofibrosis remain to be fully elucidated.