Carole Tonetti

The impact of JAK2 and MPL mutations on diagnosis and prognosis of splanchnic vein thrombosis: a report on 241 cases

Myeloproliferative diseases (MPDs) represent the commonest cause of splanchnic vein thrombosis (SVT), including Budd-Chiari syndrome (BCS) and portal vein thrombosis (PVT), but their diagnosis is hampered by changes secondary to portal hypertension, while their influence in the outcome of SVT remains unclear. We assessed the diagnostic and prognostic value of JAK2 and MPL515 mutations in 241 SVT patients (104 BCS, 137 PVT). JAK2V617F was found in 45% of BCS and 34% of PVT, while JAK2 exon 12 and MPL515 mutations were not detected. JAK2V617F was found in 96.5% of patients with bone marrow (BM) changes specific for MPD and endogenous erythoid colonies, but also in 58% of those with only one feature and in 7% of those with neither feature. Stratifying MPD diagnosis first on JAK2V617F detection would have avoided BM investigations in 40% of the patients. In BCS, presence of MPD carried significantly poorer baseline prognostic features, required hepatic decompression procedures earlier, but had no impact on 5-year survival. Our results suggest that JAK2V617F testing should replace BM investigations as initial test for MPD in patients with SVT. Underlying MPD is associated with severe forms of BCS, but current therapy appears to offset deleterious effects of MPD on the medium-term outcome.

Role for the Nuclear Factor κB Pathway in Transforming Growth Factor-β1 Production in Idiopathic Myelofibrosis: Possible Relationship with FK506 Binding Protein 51 Overexpression

The release of transforming growth factor-beta1 (TGF-beta1) in the bone marrow microenvironment is one of the main mechanisms leading to myelofibrosis in murine models and probably in the human idiopathic myelofibrosis (IMF). The regulation of TGF-beta1 synthesis is poorly known but seems regulated by nuclear factor kappaB (NF-kappaB). We previously described the overexpression of an immunophilin, FK506 binding protein 51 (FKBP51), in IMF megakaryocytes. Gel shift and gene assays show that FKBP51s overexpression in a factor-dependent hematopoietic cell line, induces a sustained NF-kappaB activation after cytokine deprivation. This activation correlates with a low level of IkappaBalpha. A spontaneous activation of NF-kappaB was also detected in proliferating megakaryocytes and in circulating CD34(+) patient cells. In normal cells, NF-kappaB activation was only detected after cytokine treatment. The expression of an NF-kappaB superrepressor in FKBP51 overexpressing cells and in derived megakaryocytes from CD34(+) of IMF patients revealed that NF-kappaB activation was not involved in the resistance to apoptosis after cytokine deprivation of these cells but in TGF-beta1 secretion. These results highlight the importance of NF-kappaBs activation in the fibrosis development of this disease. They also suggest that FKBP51s overexpression in IMF cells could play an important role in the pathogenesis of this myeloproliferative disorder.

A Senescence-Like Cell-Cycle Arrest Occurs During Megakaryocytic Maturation: Implications for Physiological and Pathological Megakaryocytic Proliferation

During normal megakaryocyte development, in response to thrombopoetin, mature cells enter a senescence-like state in which they shed platelets; this state, characterized by cell cycle arrest, is defective in malignant megakaryocytes.

Expression level and differential JAK2-V617F–binding of the adaptor protein Lnk regulates JAK2-mediated signals in myeloproliferative neoplasms

Activating mutations in signaling molecules, such as JAK2-V617F, have been associated with myeloproliferative neoplasms (MPNs). Mice lacking the inhibitory adaptor protein Lnk display deregulation of thrombopoietin/thrombopoietin receptor signaling pathways and exhibit similar myeloproliferative characteristics to those found in MPN patients, suggesting a role for Lnk in the molecular pathogenesis of these diseases. Here, we showed that LNK levels are up-regulated and correlate with an increase in the JAK2-V617F mutant allele burden in MPN patients. Using megakaryocytic cells, we demonstrated that Lnk expression is regulated by the TPO-signaling pathway, thus indicating an important negative control loop in these cells. Analysis of platelets derived from MPN patients and megakaryocytic cell lines showed that Lnk can interact with JAK2-WT and V617F through its SH2 domain, but also through an unrevealed JAK2-binding site within its N-terminal region. In addition, the presence of the V617F mutation causes a tighter association with Lnk. Finally, we found that the expression level of the Lnk protein can modulate JAK2-V617F-dependent cell proliferation and that its different domains contribute to the inhibition of multilineage and megakaryocytic progenitor cell growth in vitro. Together, our results indicate that changes in Lnk expression and JAK2-V617F-binding regulate JAK2-mediated signals in MPNs.

Plasma Homocysteine Levels Related to Interactions Between Folate Status and Methylenetetrahydrofolate Reductase: A Study in 52 Healthy Subjects

Hyperhomocysteinemia, a risk factor for vascular disease, is related to vitamin B12, vitamin B6, and especially folate deficiency, or to genetic factors such as mutations in methylenetetrahydrofolate reductase (MTHFR), an enzyme involved in the remethylation pathway of homocysteine to methionine. Recently, a C677 --> T mutation identified in the MTHFR gene was found to be frequently associated with decreased MTHFR activity and an elevated plasma homocysteine concentration. Since hyperhomocysteinemia seems to be determined by both genetic and environmental factors, we studied the interactions between MTHFR (phenotype and genotype) and folate status, including methyltetrahydrofolate (methylTHF), the product of MTHFR, on the homocysteine concentration in 52 healthy subjects, (28 women and 24 men; mean age, 32.7 years). MTHFR activity seems to be dependent on folate status, as shown by a lower activity in folate-deficient subjects and a return to normal values after supplementation with folic acid, and also by a decreased enzymatic activity on phytohemagglutinin (PHA)-stimulated lymphocytes grown in a folic acid-deficient medium. Conversely, the C677 --> T mutation seems to influence folate metabolism. Subjects who were homozygous for this mutation (+/+) had significantly higher plasma homocysteine and lower plasma folate and total and methylfolate levels in red blood cells (RBCs) than heterozygous (+/-) and normal (-/-) subjects. The ratio of RBC methylfolate to RBC total folate was, respectively, 0.27 in +/+, 0.66 in +/-, and 0.71 in -/-. This mutation seems to have an impact on methylTHF generation. These data illustrate the interactions between nutritional and genetic factors.

FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Primary myelofibrosis (PMF) is characterized by increased number of hematopoietic progenitors and a dysmegakaryopoiesis which supports the stromal reaction defining this disease. We showed that increased ligand (FL) levels in plasma, hematopoietic progenitors, and stromal cells from PMF patients were associated with upregulation of the cognate Flt3 receptor on megakaryocytic (MK) cells. This connection prompted us to study a functional role for the FL/Flt3 couple in PMF dysmegakaryopoiesis, as a route to reveal insights into pathobiology and therapy in this disease. Analysis of PMF CD34(+) and MK cell transcriptomes revealed deregulation of the mitogen-activated protein kinase (MAPK) pathway along with Flt3 expression. In PMF patients, a higher proportion of circulating Flt3(+)CD34(+)CD41(+) cells exhibited an increased MAPK effector phosphorylation independently of Jak2(V617F) mutation. Activation of FL/Flt3 axis in PMF MK cell cultures, in response to FL, induced activation of the p38-MAPK cascade, which is known to be involved in inflammation, also increasing expression of its target genes (NFATC4, p53, AP-1, IL-8). Inhibiting Flt3 or MAPK or especially p38 by chemical, antibody, or silencing strategies restored megakaryopoiesis and reduced phosphorylation of Flt3 and p38 pathway effectors, confirming the involvement of Flt3 in PMF dysmegakaryopoiesis via p38 activation. In addition, in contrast to healthy donors, MK cells derived from PMF CD34(+) cells exhibited an FL-induced migration that could be reversed by p38 inhibition. Taken together, our results implicate the FL/Flt3 ligand-receptor complex in PMF dysmegakaryopoiesis through persistent p38-MAPK activation, with implications for therapeutic prospects to correct altered megakaryopoiesis in an inflammatory context.

Interlaboratory Development and Validation of a HRM Method Applied to the Detection of JAK2 Exon 12 Mutations in Polycythemia Vera Patients

BACKGROUND Myeloproliferative disorders are characterized by clonal expansion of normal mature blood cells. Acquired mutations giving rise to constitutive activation of the JAK2 tyrosine kinase has been shown to be present in the majority of patients. Since the demonstration that the V617F mutation in the exon 14 of the JAK2 gene is present in about 90% of patients with Polycythemia Vera (PV), the detection of this mutation has become a key tool for the diagnosis of these patients. More recently, additional mutations in the exon 12 of the JAK2 gene have been described in 5 to 10% of the patients with erythrocytosis. According to the updated WHO criteria the presence of these mutations should be looked for in PV patients with no JAK2 V617F mutation. Reliable and accurate methods dedicated to the detection of these highly variable mutations are therefore necessary. METHODS/FINDINGS For these reasons we have defined the conditions of a High Resolution DNA Melting curve analysis (HRM) method able to detect JAK2 exon 12 mutations. After having validated that the method was able to detect mutated patients, we have verified that it gave reproducible results in repeated experiments, on DNA extracted from either total blood or purified granulocytes. This HRM assay was further validated using 8 samples bearing different mutant sequences in 4 different laboratories, on 3 different instruments. CONCLUSION The assay we have developed is thus a valid method, adapted to routine detection of JAK2 exon 12 mutations with highly reproducible results.

Relations between molecular and biological abnormalities in 11 families from siblings affected with methylenetetrahydrofolate reductase deficiency

Methylenetetrahydrofolate reductase (MTHFR) deficiency is an autosomal recessive disorder resulting in elevated homocysteine levels in plasma and urine. MTHFR catalyses the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, a cofactor for homocysteine remethylation to methionine. MTHFR deficiency may be diagnosed from infancy to adulthood with a broad spectrum of clinical symptoms. A molecular analysis of the MTHFR gene combined with an assessment of MTHFR activity, plasma homocysteine and folate in plasma and red blood cells (RBC), especially methylfolate, was assessed in the members of 11 families from children affected with this disorder. This study was performed to try to define the impact of the mutations found in the MTHFR gene on symptoms and biological abnormalities. A total of 14 mutations were found and 10 of them were identified for the first time. Two were found in two families, two more in two other families and one in three families. The position of the mutation spread all over the gene does not predict the degree of biological abnormalities found in parents or healthy siblings bearing the mutation. Two different mutations located not far apart on the same exon may cause mild or severe abnormalities. The thermolabile variant C677T when expressed in an homozygote state in some parents was associated with lower MTHFR activity, higher homocysteine levels, lower folate levels, mainly methylfolate in RBC than in parents without the mutation; conversely, two or more mutations on the same allele had mild effects when the other allele was normal. Conclusion: given the heterogeneity of mutations, no one seems preponderant to predict neurological and/or vascular symptoms.

Cytotoxic synergism of methioninase in combination with 5-fluorouracil and folinic acid.

Potentiation of the cytotoxic activity of 5-fluorouracil (FUra) by folinic acid (5-HCO-H4folate) is due to elevation of the methylene tetrahydrofolate (CH2-H4folate) level, which increases the stability of the ternary complex of thymidylate synthase (TS), fluorodeoxyuridine monophosphate, and CH2-H4folate that inactivates the TS. Methionine deprivation results in the production of tetrahydrofolate (H4folate) and, subsequently, CH2-H4folate from methyl tetrahydrofolate, as a consequence of the induction of methionine synthesis. We hypothesized that the efficacy of FUra could be augmented by the combination of high-concentration 5-HCO-H4folate and recombinant methioninase (rMETase), a methionine-cleaving enzyme. Studies in vitro were performed with the cell line CCRF-CEM. Cytotoxic synergism of FUra + rMETase and FUra + 5-HCO-H4folate + rMETase was demonstrated with the combination index throughout a broad concentration range of FUra and rMETase. A subcytotoxic concentration of rMETase reduced the IC50 of FUra by a factor of 3.6, and by a factor of 7.5, in the absence and in the presence of 5-HCO-H4folate, respectively. 5-HCO-H4folate increased the intracellular concentrations of CH2-H4folate and H4folate from their baseline levels. Concentrations of folates were not changed by exposure to rMETase. Levels of free TS in cells treated with FUra + 5-HCO-H4folate and with FUra + rMETase were lower than those in cells exposed to FUra alone. The decrease of TS was still more pronounced in cells treated with FUra + 5-HCO-H4folate + rMETase. The synergism described in this study will be a basis for further exploration of combinations of fluoropyrimidines, folates, and rMETase.

SOCS3 inhibits TPO-stimulated, but not spontaneous, megakaryocytic growth in primary myelofibrosis

SOCS3 inhibits TPO-stimulated, but not spontaneous, megakaryocytic growth in primary myelofibrosis