Vanessa Ponziani

Prospective identification of high-risk polycythemia vera patients based on JAK2(V617F) allele burden.

The aim of this study was to determine whether the burden of JAK2V617F allele correlated with major clinical outcomes in patients with polycythemia vera (PV). To this end, we determined JAK2 mutant allele levels in granulocytes of 173 PV patients at diagnosis. The mean (±s.d.) mutant allele burden was 52% (±29); 32 patients (18%) had greater than 75% mutant allele. The burden of JAK2V617F allele correlated with measurements of stimulated erythropoiesis (higher hematocrit, lower mean cell volume, serum ferritin and erythropoietin levels) and myelopoiesis (higher white cell count, neutrophil count and serum lactate dehydrogenase) and with markers of neutrophil activation (elevated leukocyte alkaline phosphatase and PRV-1 expression). As compared to those with less than 25% mutant allele, patients harboring greater than 75% JAK2V617F allele were at higher relative risk (RR) of presenting larger spleen (RR 4.7; P<0.001) or suffering from pruritus (RR 3.1; P<0.001). In these patients, the risk of requiring chemotherapy (RR 1.8; P=0.001) or developing major cardiovascular events (RR 7.1; P=0.003) during follow up were significantly increased. We conclude that a burden of JAK2V617F allele greater than 75% at diagnosis points to PV patients with high-risk disease.

Clinical implications of the JAK2 V617F mutation in essential thrombocythemia.

istic upmodulation of CD20 and downmodulation of CD10, comparable to the immunophenotypic modulation observed in vivo. In contrast, the viable 7-AAD-negative cells retained their original immunophenotype (Figure 2e, f). For each antibody, the MFI differed between the viable and dead cells, but within these two populations the MFI was not affected by the type and dose of the cytotoxic agents to which the cells were exposed (data not shown). Further analysis of dead and viable cells showed that dead cells had a higher MFI for each isotype control antibody than viable cells. This increase in background staining was comparable to the upmodulation of CD20 on CD20-negative leukemic cells observed in day 15 and 28 samples. Altogether, these data indicate that the immunophenotypic modulation observed in vitro is due to the induction of cell kill, resulting in a higher aspecific staining and a decreased expression of membrane antigens. Our data suggest that in ALL patients undergoing treatment, immunophenotypic modulation is caused by drug-induced cell death, resulting in loss of membrane antigens and higher aspecific staining. Our data are in agreement with a recent study, which showed that the loss of expression of lineage antigens (such as CD19) is a common feature of lymphocytes undergoing apoptosis and that the MFI for different antigens might drop to undetectable levels during different stages of apoptosis. Antigens coexpressed on the same cells showed different degrees of loss in the different stages of apoptosis, suggesting that it is a specific, active process, rather than a general degradation of cell components. Our observation that immunophenotypic modulation is due to apoptotic cells also explains why ALL blast cells in resistant patients show no modulation. In conclusion, our data indicate that immunophenotypic modulation in ALL patients undergoing treatment can (at least in part) be explained by the induction of cell death. As the presence of these dying cells may still be clinically relevant, usage of strict gating procedures, based on the exact immunophenotype of the blast cells at diagnosis, should be avoided for the analysis of MRD.

Identification of patients with poorer survival in primary myelofibrosis based on the burden of JAK2V617F mutated allele.

A total of 186 patients with primary myelofibrosis (PMF) were genotyped for JAK2V617F at diagnosis aimed at analyzing the correlation of mutational status and mutated allele burden with outcome variables, including time to anemia, leukocytosis, leukopenia, thrombocytopenia, massive splenomegaly, leukemia, and with overall survival. A total of 127 JAK2V617F-mutated patients (68% of whole series) were divided in quartiles of V617F allele burden. After a median follow-up of 17.2 months, 23 patients died, 15 because of leukemia. A JAK2V617F mutated status did not impact on the rate of leukemia transformation or overall survival. Patients in the lower quartile had shorter time to anemia and leukopenia and did not progress to large splenomegaly. Furthermore, survival was significantly reduced in the lower quartile compared with upper quartiles and JAK2 wild-type patients. In multivariate analysis, factors associated with reduced survival were age, a blast count more than 1%, and a JAK2V617F burden within first quartile. Causes of death in the lower quartile were represented mainly by systemic infections. We conclude that a low JAK2V617F allele burden at diagnosis is preferentially associated with a myelodepletive rather than myeloproliferative phenotype and represents an independent factor associated with shortened survival in patients with PMF.

Influence of JAK2V617F allele burden on phenotype in essential thrombocythemia.

Variable proportions of mutant alleles are found in patients with JAK2 (V617F)-positive myeloproliferative disorders. This study shows that this variable mutant allele burden influences the clinical phenotype of JAK2 (V617F)-positive essential thrombocythemia. Background Fifty to sixty percent of patients with essential thrombocythemia harbor the JAK2V617F mutation. The impact of this mutation on clinical phenotype is still debated. The aim of this study was to evaluate possible correlations between JAK2V617F mutant allele burden and both clinical presentation and hematologic abnormalities in essential thrombocythemia patients. Design and Methods In this single-center retrospective study, JAK2V617F allele load was measured by sensitive quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in the granulocytes of 260 patients diagnosed as having essential thrombocythemia according to WHO criteria. Results Median V617F allele burden in patients with the mutation (n=165, 63.4%) was 24%, ranging from 1% to 87%; an allele burden greater than 51% was found in 5% of the patients. Older patients presented progressively higher percentages of the V617F allele. Signs of stimulated erythropoiesis and myelopoiesis, as well as higher PRV-1 levels, were found in patients with the mutation, but no linear correlation with load of mutant allele could be ascertained; on the other hand, the frequency of patients with erythropoietin-independent erythroid colonies progressively increased depending on mutant allele load. Splenomegaly and microvessel symptoms were significantly more represented among patients with greater than 50% and 25% JAK2V617F allele burden, respectively. Increasing mutant allele load correlated with higher frequency of arterial thrombosis at diagnosis, as confirmed also in multivariate analysis; the relative risk was 3.0 (95% CI 1.3–6.8; p=0.01) in patients having a greater than 25% mutant allele burden. Conclusions The JAK2V617F mutant allele burden contributes to determining the clinical phenotype in patients with essential thrombocythemia.

Hypermethylation of CXCR4 Promoter in CD34+ Cells from Patients with Primary Myelofibrosis

Constitutive mobilization of CD34+ cells in patients with primary myelofibrosis (PMF) has been attributed to proteolytic disruption of the CXCR4/SDF‐1 axis and reduced CXCR4 expression. We document here that the number of circulating CD34+/CXCR4+ cells in PMF patients, as well as the cellular CXCR4 expression, was directly related to CXCR4 mRNA level and that reduced CXCR4 mRNA level was not due to SDF‐1‐induced downregulation. To address whether epigenetic regulation contributes to defective CXCR4 expression, we studied the methylation status of the CXCR4 promoter using methylation‐specific polymerase chain reaction and methylation‐specific sequencing in the JAK2V617F‐positive HEL cell line and in CD34+ cells. We found that CD34+ cells from PMF patients, unlike those from normal subjects, presented hypermethylation of CXCR4 promoter CpG island 1. Following incubation with the demethylating agent 5‐Aza‐2′‐deoxycytidine (5‐AzaD), the percentage of PMF CD34+ cells expressing CXCR4 increased 3–10 times, whereas CXCR4 mRNA level increased approximately 4 times. 5‐AzaD‐treated PMF CD34+ cells displayed almost complete reversal of CpG1 island 1 hypermethylation and showed enhanced migration in vitro in response to SDF‐1. These data point to abnormal methylation of the CXCR4 promoter as a mechanism contributing to constitutive migration of CD34+ cells in PMF.

Increased Risk of Lymphoid Neoplasms in Patients with Philadelphia Chromosome–Negative Myeloproliferative Neoplasms

Association of myeloproliferative neoplasm (MPN) with lymphoproliferative neoplasm (LPN) has been occasionally reported. The aim of this study, which included 353 patients with polycythemia vera and 467 with essential thrombocythemia, was to assess whether the risk of developing LPN is increased in MPN patients. Expected numbers of LPN incident cases were calculated based on 5-year age group, gender, and calendar time–specific cancer incidence rates in the general population of the same area. Standardized incidence ratios were computed to estimate the relative risk of developing LPN. Analyses were carried out for the whole series and then separately for essential thrombocythemia and polycythemia vera, gender, and JAK2V617F genotype. With 4,421 person-years, we found 11 patients developing LPN, including four chronic lymphocytic leukemias, five non–Hodgkins lymphomas, and two plasma cell disorders, after a median interval time of 68 months from MPN diagnosis. Cumulative risk to develop LPN at 5 and 10 years was 0.93% (95% confidence interval, 0.39-2.22) and 2.96% (95% confidence interval, 1.52-5.72), respectively. There was a 3.44-fold increased risk of LPN compared with the general population, ranging from 2.86 for plasma cell disorder to 12.42 for chronic lymphocytic leukemia; the risk was significantly increased in JAK2V617F mutated patients (5.46-fold) and in males (4.52-fold). The JAK2V617F mutation was found in lymphoid tumor cells in two of three cases evaluated, indicating that, in some patients, LPN originated in a JAK2V617F mutated common lymphoid-myeloid hematopoietic progenitor cell. We conclude that the risk of developing LPN is significantly increased in MPN patients compared with the general population. (Cancer Epidemiol Biomarkers Prev 2009;18(7):2068–73)

Anti-oxidant enzymes and related elements in term and preterm newborns

Background:  Although oxidative stress‐related diseases mostly affect neonates with extremely low birthweight, healthy preterm newborns might also be at risk of oxidative damages. The aim of the present study was to verify this possibility.

A Sensitive Detection Method for MPLW515L or MPLW515K Mutation in Chronic Myeloproliferative Disorders with Locked Nucleic Acid-Modified Probes and Real-Time Polymerase Chain Reaction

Acquired mutations in the juxtamembrane region of MPL (W515K or W515L), the receptor for thrombopoietin, have been described in patients with primary myelofibrosis or essential thrombocythemia, which are chronic myeloproliferative disorders. We have developed a real-time polymerase chain reaction assay for the detection and quantification of MPL mutations that is based on locked nucleic acid fluorescent probes. Mutational analysis was performed using DNA from granulocytes. Reference curves were obtained using cloned fragments of MPL containing either the wild-type or mutated sequence; the predicted sensitivity level was at least 0.1% mutant allele in a wild-type background. None of the 60 control subjects presented with a MPLW515L/K mutation. Of 217 patients with myelofibrosis, 19 (8.7%) harbored the MPLW515 mutation, 10 (52.6%) with the W515L allele. In one case, both the W515L and W515K alleles were detected by real-time polymerase chain reaction. By comparing results obtained with conventional sequencing, no erroneous genotype attribution using real-time polymerase chain reaction was found, whereas one patient considered wild type according to sequence analysis actually harbored a low W515L allele burden. This is a simple, sensitive, and cost-effective procedure for large-scale screening of the MPLW515L/K mutation in patients suspected to have a myeloproliferative disorder. It can also provide a quantitative estimate of mutant allele burden that might be useful for both patient prognosis and monitoring response to therapy.

The size of duplication does not add to the prognostic significance of FLT3 internal tandem duplication in acute myeloid leukemia patients

The size of duplication does not add to the prognostic significance of FLT3 internal tandem duplication in acute myeloid leukemia patients

Protective effects of the PARP-1 inhibitor PJ34 in hypoxic-reoxygenated cardiomyoblasts

Abstract.To clarify the role of poly(ADP-ribose)polymerase-1 (PARP-1) in myocardial ischemia-reperfusion injury, we explored some effects of PJ34, a highly specific inhibitor of this enzyme, in hypoxic-reoxygenated (HR) H9c2 cardiomyoblasts. Compared to the control, HR cells showed signs of oxidative stress, marked PARP-1 activation, NAD+ and ATP depletion and impaired mitochondrial activity. HR cardiomyoblasts were affected by both necrosis and apoptosis, the latter involving the nuclear translocation of apoptosis-inducing factor. In HR cardiomyoblasts treated with PJ34, oxidative stress and PARP-1 activity were decreased, and NAD+ and ATP depletion, as well as mitochondrial impairment, were attenuated. Above all, PJ34 treatment improved the survival of HR cells; not only was necrosis significantly diminished, but apoptosis was also reduced and shifted from a caspase-independent to a caspase-dependent pathway. These results suggest that PARP-1 modulation by a selective inhibitor such as PJ34 may represent a promising approach to limit myocardial damage due to post-ischemic reperfusion.