Philip A. Beer

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.

The Lin28b–let-7–Hmga2 axis determines the higher self-renewal potential of fetal haematopoietic stem cells

Mouse haematopoietic stem cells (HSCs) undergo a postnatal transition in several properties, including a marked reduction in their self-renewal activity. We now show that the developmentally timed change in this key function of HSCs is associated with their decreased expression of Lin28b and an accompanying increase in their let-7 microRNA levels. Lentivirus-mediated overexpression of Lin28 in adult HSCs elevates their self-renewal activity in transplanted irradiated hosts, as does overexpression of Hmga2, a well-established let-7 target that is upregulated in fetal HSCs. Conversely, HSCs from fetal Hmga2−/− mice do not exhibit the heightened self-renewal activity that is characteristic of wild-type fetal HSCs. Interestingly, overexpression of Hmga2 in adult HSCs does not mimic the ability of elevated Lin28 to activate a fetal lymphoid differentiation program. Thus, Lin28b may act as a master regulator of developmentally timed changes in HSC programs with Hmga2 serving as its specific downstream modulator of HSC self-renewal potential.

JAK2 V617F impairs hematopoietic stem cell function in a conditional knock-in mouse model of JAK2 V617F-positive essential thrombocythemia

The JAK2 V617F mutation is found in most patients with a myeloproliferative neoplasm and is sufficient to produce a myeloproliferative phenotype in murine retroviral transplantation or transgenic models. However, several lines of evidence suggest that disease phenotype is influenced by the level of mutant JAK2 signaling, and we have therefore generated a conditional knock-in mouse in which a human JAK2 V617F is expressed under the control of the mouse Jak2 locus. Human and murine Jak2 transcripts are expressed at similar levels, and mice develop modest increases in hemoglobin and platelet levels reminiscent of human JAK2 V617F-positive essential thrombocythemia. The phenotype is transplantable and accompanied by increased terminal erythroid and megakaryocyte differentiation together with increased numbers of clonogenic progenitors, including erythropoietin-independent erythroid colonies. Unexpectedly, JAK2(V617F) mice develop reduced numbers of lineage(-)Sca-1(+)c-Kit(+) cells, which exhibit increased DNA damage, reduced apoptosis, and reduced cell cycling. Moreover, competitive bone marrow transplantation studies demonstrated impaired hematopoietic stem cell function in JAK2(V617F) mice. These results suggest that the chronicity of human myeloproliferative neoplasms may reflect a balance between impaired hematopoietic stem cell function and the accumulation of additional mutations.

Hematopoietic Stem Cell Heterogeneity Takes Center Stage

Over the past 10 years, increasing evidence has accumulated that heterogeneity is a feature of hematopoietic stem cell (HSC) proliferation, self-renewal, and differentiation based on examination of these properties at a clonal level. The heterogeneous behavior of HSCs reflects the operation of a complex interplay of intrinsic and extrinsic variables. In this review, we discuss key findings from the last 5 years that reveal new insights into the mechanisms involved.

Distinct clinical phenotypes associated with JAK2V617F reflect differential STAT1 signaling

The JAK2V617F mutation is associated with distinct myeloproliferative neoplasms, including polycythemia vera (PV) and essential thrombocythemia (ET), but it remains unclear how it generates disparate disorders. By comparing clonally-derived mutant and wild-type cells from individual patients, we demonstrate that the transcriptional consequences of JAK2V617F are subtle, and that JAK2V617F-heterozygous erythroid cells from ET and PV patients exhibit differential interferon signaling and STAT1 phosphorylation. Increased STAT1 activity in normal CD34-positive progenitors produces an ET-like phenotype, whereas downregulation of STAT1 activity in JAK2V617F-heterozygous ET progenitors produces a PV-like phenotype. Our results illustrate the power of clonal analysis, indicate that the consequences of JAK2V617F reflect a balance between STAT5 and STAT1 activation and are relevant for other neoplasms associated with signaling pathway mutations.

Correlation of blood counts with vascular complications in essential thrombocythemia: analysis of the prospective PT1 cohort

Essential thrombocythemia, a myeloproliferative neoplasm, is associated with increased platelet count and risk of thrombosis or hemorrhage. Cytoreductive therapy aims to normalize platelet counts despite there being only a minimal association between platelet count and complication rates. Evidence is increasing for a correlation between WBC count and thrombosis, but prospective data are lacking. In the present study, we investigated the relationship between vascular complications and 21 887 longitudinal blood counts in a prospective, multicenter cohort of 776 essential thrombocythemia patients. After correction for confounding variables, no association was seen between blood counts at diagnosis and future complications. However, platelet count outside of the normal range during follow-up was associated with an immediate risk of major hemorrhage (P = .0005) but not thrombosis (P = .7). Elevated WBC count during follow-up was correlated with thrombosis (P = .05) and major hemorrhage (P = .01). These data imply that the aim of cytoreduction in essential thrombocythemia should be to keep the platelet count, and arguably the WBC count, within the normal range. This study is registered at the International Standard Randomized Controlled Trials Number Registry (www.isrctn.org) as number 72251782.

Novel exon 12 mutations in the HIF2A gene associated with erythrocytosis.

Erythrocytosis can arise from deregulation of the erythropoietin (Epo) axis resulting from defects in the oxygen-sensing pathway. Epo synthesis is controlled by the hypoxia inducible factor (HIF) complex, composed of an alpha and a beta subunit. There are 2 main alpha subunits, HIF-1 alpha and HIF-2 alpha. Recently, a HIF-2 alpha Gly537Trp mutation was identified in a family with erythrocytosis. This raises the possibility of HIF2A mutations being associated with other cases of erythrocytosis. We now report a subsequent analysis of HIF2A in a cohort of 75 erythrocytosis patients and identify 4 additional patients with novel heterozygous Met535Val and Gly537Arg mutations. All patients presented at a young age with elevated serum Epo. Mutations at Gly-537 account for 4 of 5 HIF2A mutations associated with erythrocytosis. These findings support the importance of HIF-2 alpha in human Epo regulation and warrant investigation of HIF2A in patients with unexplained erythrocytosis.

How I treat essential thrombocythemia

In the past 5 years we have witnessed significant advances in both the diagnostic process and optimal therapy for patients with essential thrombocythemia (ET). Insights into the underlying molecular mechanisms have been accompanied by the development of new diagnostic tests and by an improved understanding of the relationship between ET and other related myeloproliferative neoplasms, such as polycythemia vera and primary myelofibrosis. In the first part of this review, we describe how recent molecular and histologic studies can be integrated into a streamlined diagnostic process that is applicable to everyday clinical practice. We also address areas of current diagnostic controversy, including heterogeneity within ET and the phenotypic overlap between ET, polycythemia vera, and primary myelofibrosis. In the second part, we provide an overview of our current approach to the treatment of ET, including risk stratification, choice of cytoreductive agent, and a consideration of special situations such as the pregnant or perioperative patient. Areas of controversy discussed include the identification of those at high risk of complications and therapeutic decisions in the younger patient.

Inhibition of the Bcl-xL Deamidation Pathway in Myeloproliferative Disorders

BACKGROUND The myeloproliferative disorders are clonal disorders with frequent somatic gain-of-function alterations affecting tyrosine kinases. In these diseases, there is an increase in DNA damage and a risk of progression to acute leukemia. The molecular mechanisms in myeloproliferative disorders that prevent apoptosis induced by damaged DNA are obscure. METHODS We searched for abnormalities of the proapoptotic Bcl-x(L) deamidation pathway in primary cells from patients with chronic myeloid leukemia (CML) or polycythemia vera, myeloproliferative disorders associated with the BCR-ABL fusion kinase and the Janus tyrosine kinase 2 (JAK2) V617F mutation, respectively. RESULTS The Bcl-x(L) deamidation pathway was inhibited in myeloid cells, but not T cells, in patients with CML or polycythemia vera. DNA damage did not increase levels of the amiloride-sensitive sodium-hydrogen exchanger isoform 1 (NHE-1), intracellular pH, Bcl-x(L) deamidation, and apoptosis. Inhibition of the pathway was reversed by enforced alkalinization or overexpression of NHE-1, leading to a restoration of apoptosis. In patients with CML, the pathway was blocked in CD34+ progenitor cells and mature myeloid cells. Imatinib or JAK2 inhibitors reversed inhibition of the pathway in cells from patients with CML and polycythemia vera, respectively, but not in cells from a patient with resistance to imatinib because of a mutation in the BCR-ABL kinase domain. CONCLUSIONS BCR-ABL and mutant JAK2 inhibit the Bcl-x(L) deamidation pathway and the apoptotic response to DNA damage in primary cells from patients with CML or polycythemia vera.

Clonal diversity in the myeloproliferative neoplasms: independent origins of genetically distinct clones

This study looked for clonal diversity in patients with a myeloproliferative neoplasm associated with more than one acquired genetic lesion. A tyrosine kinase mutation and a cytogenetic lesion were present in the same clone in six of seven patients. By contrast, the genetic lesions were present in separate clones in all six patients with two tyrosine kinase pathway mutations. Moreover, in two patients the clones were genetically unrelated by X‐chromosome inactivation studies. These data demonstrated clonal diversity in a subset of patients with early stage haematopoietic malignancy and showed, for the first time, that such clones may arise independently.