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Subclones homozygous for JAK2V617F are more common in polycythemia vera (PV) than essential thrombocythemia (ET), but their prevalence and significance remain unclear. The JAK2 mutation status of 6495 BFU-E, grown in low erythropoietin conditions, was determined in 77 patients with PV or ET. Homozygous-mutant colonies were common in patients with JAK2V617F-positive PV and were surprisingly prevalent in JAK2V617F-positive ET and JAK2 exon 12-mutated PV. Using microsatellite PCR to map loss-of-heterozygosity breakpoints within individual colonies, we demonstrate that recurrent acquisition of JAK2V617F homozygosity occurs frequently in both PV and ET. PV was distinguished from ET by expansion of a dominant homozygous subclone, the selective advantage of which is likely to reflect additional genetic or epigenetic lesions. Our results suggest a model in which development of a dominant JAK2V617F-homzygous subclone drives erythrocytosis in many PV patients, with alternative mechanisms operating in those with small or undetectable homozygous-mutant clones.

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.

Therapeutic decision-making in primary myelofibrosis (PMF) is becoming more challenging because of the increasing use of allogeneic stem cell transplantation and new investigational drugs. To enhance this process by developing a highly discriminative prognostic system, 1054 patients consecutively diagnosed with PMF at 7 centers were studied. Overall median survival was 69 months (95% confidence interval [CI]: 61-76). Multivariate analysis of parameters obtained at disease diagnosis identified age greater than 65 years, presence of constitutional symptoms, hemoglobin level less than 10 g/dL, leukocyte count greater than 25 x 10(9)/L, and circulating blast cells 1% or greater as predictors of shortened survival. Based on the presence of 0 (low risk), 1 (intermediate risk-1), 2 (intermediate risk-2) or greater than or equal to 3 (high risk) of these variables, 4 risk groups with no overlapping in their survival curves were delineated; respective median survivals were 135, 95, 48, and 27 months (P< .001). Compared with prior prognostic models, the new risk stratification system displayed higher predictive accuracy, replicability, and discriminating power. In 409 patients with assessable metaphases, cytogenetic abnormalities were associated with shorter survival, but their independent contribution to prognosis was restricted to patients in the intermediate-risk groups. JAK2V617F did not cluster with a specific risk group or affect survival.

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.

The Janus kinase 2 mutation, JAK2617V>F, is myeloid neoplasm-specific; its presence excludes secondary polycythemia, thrombocytosis, or bone marrow fibrosis from other causes. Furthermore, JAK2617V>F or a JAK2 exon 12 mutation is present in virtually all patients with polycythemia vera (PV), whereas JAK2617V>F also occurs in approximately half of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF). Therefore, JAK2 mutation screening holds the promise of a decisive diagnostic test in PV while being complementary to histology for the diagnosis of ET and PMF; the combination of molecular testing and histologic review should also facilitate diagnosis of ET associated with borderline thrombocytosis. Accordingly, revision of the current World Health Organization (WHO) diagnostic criteria for PV, ET, and PMF is warranted; JAK2 mutation analysis should be listed as a major criterion for PV diagnosis, and the platelet count threshold for ET diagnosis can be lowered from 600 to 450 x 10(9)/L. The current document was prepared by an international expert panel of pathologists and clinical investigators in myeloproliferative disorders; it was subsequently presented to members of the Clinical Advisory Committee for the revision of the WHO Classification of Myeloid Neoplasms, who endorsed the document and recommended its adoption by the WHO.

The identification of an acquired mutation of JAK2 in patients with myeloproliferative disorders has raised questions about the relationship between mutation-positive and mutation-negative subtypes, timing of the JAK2 mutation, and molecular mechanisms of disease progression. Here we demonstrate that patients with V617F(-) essential thrombocythemia do not commonly progress to become V617F(+). Consistent with the concept of distinct pathogenetic mechanisms, we show that patients with and without the JAK2 mutation have different patterns of cytogenetic abnormality, with virtually all patients carrying the 20q deletion or trisomy 9 being V617F(+). We also investigated the existence of a "pre-JAK2" phase by comparing the proportion of clonally derived granulocytes, estimated from X-chromosome inactivation patterns (XCIPs), with the proportion of V617F(+) granulocytes. Our results demonstrate that inherent XCIP variability between granulocytes and T cells produces a systematically biased pattern of results that may be misinterpreted as evidence for an excess of clonally derived granulocytes, an observation that limits the utility of XCIP analysis in this context. Lastly, we studied 4 patients with V617F(+) myeloproliferative disorders who subsequently developed acute myeloid leukemia. In 3 patients the leukemic cells were V617F(-), suggesting that in these patients the leukemia arose in a V617F(-) cell.

Myelofibrosis with myeloid metaplasia (MMM) is a clinicopathologic entity characterized by stem cell-derived clonal myeloproliferation, ineffective erythropoiesis, extramedullary hematopoiesis, and bone marrow fibrosis and osteosclerosis. Patients with MMM have shortened survival and their quality of life is compromised by progressive anemia, marked hepatosplenomegaly, and severe constitutional symptoms including cachexia. After decades of frustration with ineffective therapy, patients are now being served by promising treatment approaches that include allogeneic hematopoietic stem cell transplantation and immunomodulatory drugs. Recent information regarding disease pathogenesis, including a contribution to the myeloproliferative disorder phenotype by a gain-of-function JAK2 mutation (JAK2(V617F)), has revived the prospect of targeted therapeutics as well as molecular monitoring of treatment response. Such progress calls for standardization of response criteria to accurately assess the value of new treatment modalities, to allow accurate comparison between studies, and to ensure that the definition of response reflects meaningful health outcome. Accordingly, an international panel of experts recently convened and delineated 3 response categories: complete remission (CR), partial remission (PR), and clinical improvement (CI). Bone marrow histologic and hematologic remissions characterize CR and CR/PR, respectively. The panel agreed that the CI response category is applicable only to patients with moderate to severe cytopenia or splenomegaly.

Most patients with polycythemia vera and half with idiopathic myelofibrosis and essential thrombocythemia have an acquired V617F mutation in JAK2. Using sensitive polymerase chain reaction (PCR)-based methods, we genotyped 152 patients with idiopathic myelofibrosis to establish whether there were differences in presentation and outcome between those with and those without the mutation. Patients positive for V617F had higher neutrophil and white cell counts (P = .02) than did patients negative for V617F, but other diagnostic features were comparable between the 2 groups. Patients positive for V617F were less likely to require blood transfusion during follow-up (P = .03). Despite this, patients positive for V617F had poorer overall survival, even after correction for confounding factors (P = .01).

Myelofibrosis with myeloid metaplasia (MMM) is currently classified as a classic (i.e. not yet molecularly defined) myeloproliferative disorder (MPD), along with essential thrombocythemia (ET) and polycythemia vera (PV). All three MPDs represent stem-cell-derived clonal myeloproliferation that, in the case of MMM, is accompanied by an intense bone marrow stromal reaction that includes collagen fibrosis, osteosclerosis, and angiogenesis. To date, both the molecular basis of the primary clonal process and the pathogenetic mechanisms that underlie the secondary histological changes remain elusive. Clinically, MMM is characterized by anemia, multi-organ extramedullary hematopoiesis that often involves the spleen and liver, constitutional symptoms, and premature death from either leukemic transformation or other disease complications. Current diagnosis is based on characteristic but not diagnostic bone marrow histological features. Modern therapy remains palliative but allogeneic stem cell transplantation might be curative to a selected group of patients. This chapter reviews both the old and the new therapy with regard to non-transplant treatment options for MMM.

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 Council's 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.

The European Myelofibrosis Network (EUMNET), a European research network on myelofibrosis with myeloid metaplasia (MMM), has developed a definition of response for the disease by using clinicohematologic, histologic, and cytogenetic criteria. A core set of 5 clinicohematologic criteria was selected out of 9 candidates on the basis of their sensitivity to change measured in 196 patients treated either during clinical trials or routine clinical practice. A consensus panel of 16 international experts was convened and asked to score the level of response in 104 patient profiles as major, moderate, minor, or no response according to changes of the clinicohematologic criteria. Using the experts' consensus as the gold standard, the performance of 100 possible definitions of response was evaluated. Criteria for major or moderate clinicohematologic response were determined to be changes in hemoglobin (Hb) and spleen size and the presence of constitutional symptoms, while changes in platelet count and white blood cell (WBC) count served as complementary criteria and were of value for defining minor responses. A histologic response was defined by changes in bone marrow fibrosis and cellularity grades. The combined use of these response definitions should help standardize the design and reporting of future clinical studies in MMM.

In this report, we have evaluated the effects of a TransFix-based stabilisation technique on leukocyte scatter characteristics, immunophenotyping, membrane permeability, absolute cell counting and morphology to extend previously reported flow cytometric data focused on the lymphocyte population. We show that scatter characteristics, immunophenotyping and absolute cell counting are well preserved, particularly in the lymphocyte population. Nevertheless, a general increase in membrane permeability, evaluated by propidium iodide (PI) uptake, was observed in TransFix-treated leukocyte subsets. Ultrastructural observations show selective morphological preservation (up to 10 days of storage) of lymphocytes and, to a lesser extent, of monocytes. In contrast, granulocytes have necrosis-like features, although the plasma membrane seems well preserved. Therefore, electron microscopy observations reflect modifications induced in different cell populations as evidenced by flow cytometry (FC). The data indicate that this short-term stabilisation method is particularly suitable for the analysis of human lymphocytes and it is a good procedure for quality control programmes for inter- and intra-laboratory performance evaluation; good results are obtained with respect to antigen definition and absolute cell counting procedures. Any apoptotic pathways in leukocyte subsets are blocked for at least 10 days.

Haematopoiesis is controlled by a number of growth factors and cytokines, a number of which act through binding to high-affinity receptor tyrosine kinases (RTKs). Approximately 20 different RTK classes have been identified, all of which share a similar structure that includes a ligand binding extracellular domain, a single transmembrane domain and an intracellular tyrosine kinase domain. Recent studies have linked an increasing number of mutations in the RTKs to the pathogenesis of both acute and chronic leukaemia. For example, the FLT3 receptor, a RTK class III, is the most commonly mutated gene in acute myeloid leukaemia, while c-kit mutations are strongly linked to the development of mast cell malignancy. This review summarizes the RTK classes that are known to be expressed on normal haematopoietic tissue and highlights the many 'gain-of-function' mutations involved in leukaemogenesis. It is to be hoped that this knowledge will provide important new insights for targeted therapy in leukaemia.

We tested the feasibility and precision of affordable CD4+ T cell counting in resource-poor settings using a recently standardised fixative, TransFix in whole blood (WB) by flow cytometry (FCM). The precision of the assays was established under optimal conditions for single-platform FCM such as the volumetric CytoronAbsolute and the bead-based FACSCan. Fresh WB samples from HIV-seropositive and seronegative patients were tested in Tanzania and South Africa, fixed and sent to the UK for reanalysis 7 days later. Correlation, bias and limits of agreements were analysed by linear regression and the Bland-Altman test. Absolute CD4+ T cell counts remained stable for at least 10 days when TransFix was added to WB in 1:10 dilution at 20-25 degrees C, and for 7 days when added in 1:10 or 1:5 dilution to samples stored to mimic 'tropical' conditions at 37 degrees C. Higher temperatures such as 42 degrees C were tolerated for only short periods since the recovery had decreased to 63% by day 3. The reproducibility of lymphocyte subset analysis remained unchanged by TransFix with coefficient of variations <6% for all T cell subsets. Absolute CD4+ T cell counts and CD4+ T cell % values on fixed samples in the UK showed a high correlation with the results using fresh samples in Tanzania (r=0.993 and 0.969, respectively) and with the samples handled in Johannesburg (r=0.991 and 0.981) with minimal bias. Primary CD4 gating using only a single CD4 antibody also remained accurate in TransFixed samples (r=0.999). Thus, TransFix permits optimal fixation and transport of WB samples in the developing world for FCM to local regional laboratories and for quality assurance in international centres. When used together with inexpensive primary CD4 gating, TransFix will allow reliable and affordable CD4+ T cell counting by FCM in resource-poor settings.

Idiopathic myelofibrosis is a chronic myeloproliferative disorder characterized by bone marrow fibrosis, extramedullary haematopoiesis and a leucoerythroblastic blood picture. The marrow fibrosis results from an increased deposition of various interstitial and basement membrane glycoproteins, including collagen types I, III, IV, V and VI, fibronectin, vitronectin, laminin and tenascin. In addition, a marked neovascularization is present, even in the early proliferative phase of the disease. In contrast to the clonal haematopoiesis, the increased bone marrow stromal tissue is thought to be a reactive phenomenon, resulting from the inappropriate release of megakaryocyte/platelet-derived growth factors, including PDGF, TGF-beta, EGF, bFGF and calmodulin. Recent cytogenetic studies have highlighted three defects, namely del(13q), del(20q) and partial trisomy 1q, that account for nearly 70% of all abnormalities at diagnosis, and suggests that in many patients gene loss and/or inactivation may be an important pathogenetic mechanism. The median survival is approximately 4 years, although individual survival varies greatly. Prognostic schema enable the identification of patients with a limited life expectancy, for whom bone marrow transplantation should be considered. However, for the majority of patients therapy is supportive and consists of blood transfusions, androgens to sustain erythropoiesis, cytoreductive agents to prevent thrombocythaemia and, in carefully selected cases, splenectomy. The role for a number of experimental therapies, such as vitamin D3 analogues, alpha and gamma interferons and erythropoietin has yet to be defined.