Lorenza Torti

Markers of myeloproliferative diseases in childhood polycythemia vera and essential thrombocythemia

PURPOSE Polycythemia vera (PV) and essential thrombocythemia (ET) can present in pediatric age as sporadic or familial diseases. To define the biologic profile of childhood PV and ET, we evaluated specific markers in a cohort of pediatric patients affected by PV and ET, including cases with familial occurrence. PATIENTS AND METHODS Thirty-eight children with PV and ET were investigated. The control group included 58 adults with PV and ET. Endogenous erythroid colonies, qualitative reverse transcriptase polymerase chain reaction for polycythemia rubra vera-1 (PRV-1) RNA expression, human androgen receptor assay and allele specific polymerase chain reaction for JAK2 V617F mutation were undertaken in all patients. Thrombopoietin, thrombopoietin receptor (c-mpl), and erythropoietin receptor mutation analysis was performed by direct sequencing in familial cases. RESULTS The JAK2 V617F mutation in children with PV was significantly less frequent than in adult PV. The most common myeloproliferative marker found in these patients was PRV-1 RNA overexpression. Children and adults with sporadic ET showed a similar proportion of patients with PRV-1 RNA overexpression, JAK2 V617F mutation, and clonality, while none of the familial ET showed JAK2 V617F mutation and clonality. Also, PRV-1 RNA overexpression was significantly less common. Furthermore, most patients with familial ET exhibited the dominant-positive activating mutation of c-mpl. Finally, children with PV and ET had a significant lower incidence of thrombosis than adults. CONCLUSION This study demonstrates that familial and sporadic ET recognize different pathogenetic mechanisms. Myeloproliferative markers are specific tests for the diagnosis of ET in children with sporadic forms, while a significant proportion of children with PV can prove negative.

A novel heterozygous HIF2AM535I mutation reinforces the role of oxygen sensing pathway disturbances in the pathogenesis of familial erythrocytosis

This report shows that two members of a family with idiopathic erythrocytosis carried a mutation in the hypoxia-inducible factor-2A (HIF2A) gene. See related perspective article on page 963. HIF2A transcription factor plays a central role in the regulation of the hypoxia responding pathway in mammalian cells, by modulating erythropoiesis and angiogenesis. Molecular alterations of oxygen sensing pathway constituents are implicated in hereditary erythrocytosis. Here we show that 2 members of a family with idiopathic erythrocytosis exhibited a new heterozygous G to A mutation at base 1605 of the exon 12 of hypoxia-inducible factor-2A (HIF2A) gene. This mutation determines the replacement of methionine by isoleucine at the position 535, very close to the position 531, where the hydroxyl acceptor prolyne is located. In addition, we found that mRNA expression of erythropoietin receptor, vascular endothelial growth factor, transferrin receptor, adrenomedullin and N-myc downstream regulated gene 1, up-regulated by HIF2A or hypoxia, were significantly higher in patients carrying the mutation than in normal controls. These results suggest that the HIF2AM535I gene mutation could induce hereditary erythrocytosis at a young age.

Epigenetic alteration of SOCS family members is a possible pathogenetic mechanism in JAK2 wild type myeloproliferative diseases

In polycythemia vera (PV) and essential thrombocythemia (ET) specific JAK2 mutations constitutively activate the JAK‐STAT pathway, explaining biologic findings such as endogenous erythroid colony (EECs) growth or PRV‐1 RNA overexpression. Since these markers are detected also in JAK2 wild type patients, we hypothesized that, in these cases, the activation of the JAK‐STAT pathway could be produced by a deregulation of the suppressor of cytokine signaling (SOCS) protein system. Eighty‐one patients with PV and ET (53 adults and 28 children) were investigated for the methylation status of the SOCS‐1, SOCS‐2 and SOCS‐3 CpG islands and for several myeloproliferative markers (including JAK2 and MPL mutations and clonality of hematopoiesis). SOCS‐1 or SOCS‐3 hypermethylation was identified in 23 patients and was associated with a significant decrease of SOCS‐1 or SOCS‐3 RNA and protein levels. The gene expression was restored by exposing cells to the demethylating agent 2‐deoxyazacytidin. Interestingly, SOCS‐1 or SOCS‐3 hypermethylation was detected in 6 female patients, proved negative for JAK2 or MPL mutations and exhibiting monoclonal hematopoiesis. In conclusion, SOCS‐1 or SOCS‐3 hypermethylation can activate the JAK‐STAT signaling pathway in alternative or together with JAK2 mutations. These alterations might represent a potential therapeutic target.

Hereditary thrombocytosis caused by MPLSer505Asn is associated with a high thrombotic risk, splenomegaly and progression to bone marrow fibrosis

Background The MPLSer505Asn mutation has been reported to be a cause of hereditary thrombocythemia. Recently, we detected this mutation in a large proportion of children with familial thrombocythemia, suggesting that in Italy the incidence of MPLSer505Asn mutation could be underestimated. Design and Methods We extended the search for this mutation to all patients with essential thrombocythemia who had a positive family history for thrombocytosis or essential thrombocythemia. We identified eight Italian families positive for the MPLSer505Asn mutation. Clinical and hematologic data were available for members of seven families, including 21 patients with a proven mutation and 20 relatives with thrombocytosis. Results Fifteen major thrombotic episodes, nine of which were fatal, were recorded among 41 patients. The thrombotic manifestation was stroke in four cases, myocardial infarction in seven cases, fetal loss in two cases, deep vein thrombosis of the leg in one case and Budd Chiari syndrome in one case. Almost all patients over 20 years old had splenomegaly and bone marrow fibrosis, while these were rarely observed in patients under 20 years old, suggesting that these manifestations are associated with aging. Finally, the life expectancy of family members with thrombocytosis was significantly shorter than that of members without thrombocytosis (P=0.003). Conclusions Patients with familial thrombocytosis caused by a MPLSer505Asn mutation have a high risk of thrombosis and, with aging, develop splenomegaly and bone marrow fibrosis, significantly affecting their life expectancy.

Endothelial progenitor cells are clonal and exhibit the JAK2 V617F mutation in a subset of thrombotic patients with Ph-negative myeloproliferative neoplasms

In this study we investigated whether neoplastic transformation occurring in Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs) could involve also the endothelial cell compartment. We evaluated the level of endothelial colony-forming cells (E-CFCs) in 42 patients (15 with polycythemia vera, 12 with essential thrombocythemia, and 15 with primary myelofibrosis). All patients had 1 molecular abnormality (JAK2(V617F) or MPL(W515K) mutations, SOCS gene hypermethylation, clonal pattern of growth) detectable in their granulocytes. The growth of colonies was obtained in 22 patients and, among them, patients with primary myelofibrosis exhibited the highest level of E-CFCs. We found that E-CFCs exhibited no molecular abnormalities in12 patients, had SOCS gene hypermethylation, were polyclonal at human androgen receptor analysis in 5 patients, and resulted in JAK2(V617F) mutated and clonal in 5 additional patients, all experiencing thrombotic complications. On the whole, patients with altered E-CFCs required antiproliferative therapy more frequently than patients with normal E-CFCs. Moreover JAK2(V617F)-positive E-CFCs showed signal transducer and activator of transcription 5 and 3 phosphorylation rates higher than E-CFCs isolated from healthy persons and patients with MPN without molecular abnormalities. Finally, JAK2(V617F)-positive E-CFCs exhibited a high proficiency to adhere to normal mononuclear cells. This study highlights a novel mechanism underlying the thrombophilia observed in MPN.

Endothelial progenitor cell trafficking in human immunodeficiency virus-infected persons.

Objective:Human immunodeficiency virus (HIV)-infected people exhibit a high incidence of vascular diseases. Since in the general population the high cardiovascular risk has been associated with an impaired endothelial cell function, we investigated circulating endothelial progenitor cells in HIV-positive patients. Design:We evaluated circulating colony-forming unit–endothelial cell (CFU-EC) and endothelial colony-forming cell (ECFC) progenitors in 14 antiviral therapy-naive HIV-positive patients, in comparison with 15 normal controls. Methods:CFU-EC and ECFC derived from peripheral blood mononuclear cells from HIV-infected and HIV-uninfected individuals were recovered and evaluated for HIV genome presence by PCR. Vascular endothelial growth factor (VEGF) and apolipoprotein B mRNA-editing enzyme catalytic polypeptide like (APOBEC) subunits expression were evaluated in infected colonies by real-time PCR. Results:We found that circulating CFU-EC but not ECFC were significantly reduced in HIV-positive patients and that proviral HIV DNA was detectable only in CFU-EC but not in ECFC. Furthermore, the expression of APOBEC subunits was significantly lower in CFU-EC than in circulating monocytes. Accordingly, the CFU-EC displayed a high content of proviral DNA copies, suggesting that these cells have a high sensitivity to the HIV infection. Conclusions:Although HIV does not affect the ‘true endothelial progenitor’ compartment, it infects and strongly depletes circulating endothelial progenitors with hematopoietic signature. We unravel a novel pathogenetic mechanism by which HIV infection might cause vascular diseases.

Detrimental clinical interaction between ritonavir-boosted protease inhibitors and vinblastine in HIV-infected patients with Hodgkin's lymphoma

In order to analyze the clinical relevance of the pharmacokinetic interactions between vinblastine and antiretrovirals described in literature, we evaluated all HIV-infected patients with Hodgkins lymphoma treated with vinblastine-containing regimens and combination antiretroviral therapy, in a single clinical center. The use of protease inhibitors was independently associated with WHO grade III–IV neutropenia. Moreover, an inverse correlation between dosage of ritonavir and mean nadir neutrophil count was found. The concomitant administration of vinblastine-containing chemotherapy regimens with protease inhibitors can lead to higher levels of neutropenia than those of different classes of drugs such as nonnucleoside reverse transcriptase inhibitors or integrase inhibitors.

Hypoxia-inducible factor-1α(Pro-582-Ser) polymorphism prevents iron deprivation in healthy blood donors.

BACKGROUND Frequent blood loss induces progressive depletion of iron stores, leading to iron deficiency and, ultimately, to overt iron-deficient anaemia. The erythropoietin-mediated bone marrow response to anaemia is under the control of hypoxia-inducible factors (HIF), the master regulators of oxygen and iron homeostasis. Since the HIF-1α(Pro-582-Ser) variant is associated with elevated trans-activation capacity of hypoxia responsive elements of target genes, we investigated whether the HIF-1α(Pro-582-Ser) polymorphism might influence the response to repeated blood withdrawals. MATERIALS AND METHODS Using polymerase chain reaction analysis and DNA sequencing, we retrospectively investigated the presence of HIF-1α(Pro-582-Ser) in a series of 163 blood donors. Haematological findings, serum ferritin levels and frequency of donations were compared according to the mutational status of the HIF-1α gene. RESULTS We found that male carriers of the HIF-1α(Pro-582-Ser) polymorphism had higher haemoglobin and ferritin levels than individuals homozygous for the wild-type allele. Moreover, the HIF-1α(Pro-582-Ser) polymorphism protected regular blood donors from developing iron deficiency and anaemia and predicted uninterrupted donation activity. DISCUSSION These findings show for the first time that the HIF-1α(Pro-582-Ser) polymorphism significantly affects red blood cell and iron homeostasis after blood loss, conferring to male carriers a resistance to anaemia. Regarding the female gender, large series of individuals should be investigated to establish whether there is an effect of the HIF-1α(Pro-582-Ser) polymorphism in this population. Although these data need to be confirmed in prospective studies, they could have important implications in blood donor selection and donation procedures.

Thrombopoietin Receptor Activation, Thrombopoietin Mimetic Drugs, and Hereditary Thrombocytosis: Remarks on Bone Marrow Fibrosis

Thus,romiplostim administration mimics the increased stimulation byTpo, while chronic eltrombopag administration reminds the consti-tutive activation of MPL. Given that these drugs have to be lifelongadministered, concern emerged when it was noticed that some pa-tients with immune thrombocytopenia (ITP) receiving romiplostindeveloped bone marrow fibrosis.

Detrimental clinical interaction between ritonavir-boosted protease inhibitors and vinblastin in HIV-infected patients with Hodgkin lymphoma

7‐11 November 2010, Tenth International Congress on Drug Therapy in HIV Infection, Glasgow, UK