To study the role of the JAK2-V617F mutation in leukemic transformation, we examined 27 patients with myeloproliferative disorders (MPDs) who transformed to acute myeloid leukemia (AML). At MPD diagnosis, JAK2-V617F was detectable in 17 of 27 patients. Surprisingly, only 5 of 17 patients developed JAK2-V617F-positive AML, whereas 9 of 17 patients transformed to JAK2-V617F-negative AML. Microsatellite analysis in a female patient showed that mitotic recombination was not responsible for the transition from JAK2-V617F-positive MPD to JAK2-V617F-negative AML, and clonality determined by the MPP1 polymorphism demonstrated that the granulocytes and leukemic blasts inactivated the same parental X chromosome. In a second patient positive for JAK2-V617F at transformation, but with JAK2-V617F-negative leukemic blasts, we found del(11q) in all cells examined, suggesting a common clonal origin of MPD and AML. We conclude that JAK2-V617F-positive MPD frequently yields JAK2-V617F-negative AML, and transformation of a common JAK2-V617F-negative ancestor represents a possible mechanism. (Blood.

We determined the allelic frequency of the JAK2-V617F mutation in DNA and assessed the expression levels of the mutant and wild-type JAK2 mRNA in granulocytes from 60 patients with essential thrombocythemia (ET) and 62 patients with polycythemia vera (PV) at the time of diagnosis. Using allele-specific quantitative polymerase chain reaction (qPCR), we detected JAK2-V617F in 75% of ET and 97% of PV at diagnosis. The total JAK2 mRNA levels were elevated in ET, PV, and secondary and idiopathic erythrocytosis, suggesting that hyperactive hematopoiesis alters JAK2 expression. The expression levels of JAK2-V617F mRNA were variable but strongly correlated with the allelic ratio of JAK2-V617F determined in DNA. Thus, differences in JAK2-V617F expression, markedly lower in ET than in PV, reflected different percentages of granulocytes carrying the mutation. Moreover, allelic ratios higher than 50% JAK2-V617F, indicating the presence of granulocytes homozygous for JAK2-V617F, were found in 70% of PV at diagnosis but never in

We previously showed that G i2 proteins interfere with the transduction of CSF-1 receptor (CSF-1R) proliferation signals (Corre and Hermouet, 1995). To identify CSF-1R pathways controlled by G i2 , we transfected v-fms, the oncogenic equivalent of CSF-1R, in NIH3T3 cells in which G i2 proteins were inactivated by stably expressing a dominant negative mutant form of the a subunit of G i2 (a i2 -G204A). Expression of a i2 -G204A resulted in decreased Src-kinase activity, delayed activation of p42 ERK-MAPK, decreased cyclin D1 expression and reduced proliferation in response to serum. In a i2 -G204A cells transfected with v-fms, Src-kinase activity remained de®cient but p42 MAPK activity and cyclin D1 expression were similar to those of vector/v-fms cells, suggesting that v-fms bypasses Src to activate the ERK-MAPK cascade. However, DNA synthesis and focus formation were inhibited by up to 80% in a i2 -G204A/vfms cells compared to vector/v-fms cells. We found that tyrosine phosphorylation of STAT3, also activated by CSF-1R/v-fms, was inhibited in a i2 -G204A/v-fms cells; in addition, expression of an 85 kDa, C-terminal truncated form of STAT3 (STAT3D) was constitutively increased. Both the inhibition of v-fms-induced STAT3 tyrosine phosphorylation and the increased expression of STAT3D were reproduced by transfecting a dominant negative mutant of Src. Last, we show that expression of STAT3D55C, a mutant form of STAT3 lacking the last 55 C-terminal amino acids, is su cient to inhibit DNA synthesis and v-fms-induced transformation in NIH3T3 cells. In summary, adequate regulation by G i2 proteins of the activity of both Src-kinase and STAT3 is required for optimal cell proliferation in response to CSF-1R/vfms.

Congenital Erythrocytosis (CE), also called congenital polycythemia, represents a rare and heterogeneous clinical entity. It is caused by deregulated red blood cell production where erythrocyte overproduction results in elevated hemoglobin and hematocrit levels. 3Primary congenital familial erythrocytosis is associated with low erythropoietin (Epo) levels and generally results from mutations in the erythropoietin-receptor gene (EPOR).Secondary congenital erythrocytosis arises from conditions which cause tissue hypoxia thus resulting in increased Epo production. These include hemoglobin variants with increased affinity for oxygen (genes HBB, HBA1 and HBA2), decreased production of 2,3-biphosphoglycerate due to mutations in the BPGM gene, or mutations in the genes involved in the hypoxia sensing pathway (VHL, EPAS1 and EGLN1). Depending on the affected gene CE can be inherited either in an autosomal dominant or recessive mode, with sporadic cases arising de novo.Despite recent important discoveries in the molecular pathogenesis of CE, the molecular causes remain to be identified in about 70% of the patients.With the objective of collecting all the published and unpublished cases of CE the COST action MPN&MPNr-Euronet developed a comprehensive internet-based database focusing on the registration of clinical history, hematological, biochemical and molecular data (http://www.erythrocytosis.org/). In addition, unreported mutations are also curated in the corresponding Leiden Open Variation Database (LOVD).

The discovery of mutated, GTPase-deficient a subunits of G. or

No abstract

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of clonal diseases characterized by the excessive and chronic production of mature cells from one or several of the myeloid lineages. Recent advances in the biology of MPNs have greatly facilitated their molecular diagnosis since most patients present with mutation(s) in the JAK2, MPL, or CALR genes. Yet the roles played by these mutations in the pathogenesis and main complications of the different subtypes of MPNs are not fully elucidated. Importantly, chronic inflammation has long been associated with MPN disease and some of the symptoms and complications can be linked to inflammation. Moreover, the JAK inhibitor clinical trials showed that the reduction of symptoms linked to inflammation was beneficial to patients even in the absence of significant decrease in the JAK2-V617F mutant load. These observations suggested that part of the inflammation observed in patients with JAK2-mutated MPNs may not be the consequence of JAK2 mutation. The aim of this paper is to review the different aspects of inflammation in MPNs, the molecular mechanisms involved, the role of specific genetic defects, and the evidence that increased production of certain cytokines depends or not on MPN-associated mutations, and to discuss possible nongenetic causes of inflammation.

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21 500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6–85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.