Danijela Lekovic

Increased risk of venous thromboembolism in patients with primary mediastinal large B-cell lymphoma

BACKGROUND Primary mediastinal large B-cell lymphoma (PMBCL) is a rare subtype of diffuse large B-cell lymphoma, arising in the mediastinum. Compression of large mediastinal vessels is common in patients with PMBCL, predisposing these patients to venous thrombosis. The incidence of this complication is still unknown. MATERIALS AND METHODS We conducted a retrospective chart review of 42 consecutive patients diagnosed with PMBCL at a Clinic for hematology, Clinical Center of Serbia between 1999 and 2009 to identify the frequency and risk factors for venous thromboembolic disease (VTE) and its correlation with survival. RESULTS In the subgroup of patients with thrombosis (VTE subgroup) were 15/42 patients (35.7%): 14 patients had deep venous thrombosis and 1 had pulmonary embolism. Ten patients had a thrombosis at the moment of diagnosis PMBCL, while in five thrombosis occurred during the course of the disease. According to hemostasis variables, patients in the VTE subgroup had significantly higher fibrinogen (P=0.02) and D-Dimer (P<0.001). Also, patients in the VTE subgroup had significantly larger diameter of mediastinal tumor mass (P=0.01) and the incidence of syndrome venae cava superior (P=0.009). The median survival rate in the VTE subgroup was 45 months (95% CI; 22 to 68 months) while in the NVTE it was 93 (95% CI; 46 to 140 months), (log rank P=0.058). CONCLUSIONS VTE is a common complication in PMBCL patients and according to our results negatively influences survival. Use of antithrombotic prophylaxis may be considered together with chemotherapy, especially in those with bulky mediastinal tumor mass.

Thrombotic events in acute promyelocytic leukemia

INTRODUCTION Thrombotic events (TE) appear to be more common in acute promyelocytic leukemia (APL) than in other acute leukemias, with reported prevalence ranging from 2 to 10-15%. MATERIALS AND METHODS We retrospectively analyzed the data on TE appearance in 63 APL patients. RESULTS TE occured in 13 (20.6%) cases, four arterial (6.3%) and nine venous (14.3%). TE were more frequently diagnosed after initiation of weekly D-dimer monitoring (7 TE during 20 months vs 6 during 76 months, P=0.032). Patients with and without venous thrombosis were significantly different regarding female/male ratio (P=0.046), PT (P=0.022), aPTT (P=0.044), ISTH DIC score (P=0.001), bcr3 (P=0.02) and FLT3-ITD (P=0.028) mutation. The most significant risk factor for venous TE occurrence in multivariate analysis was FLT3-ITD mutation (P=0.034). PAI-1 4G/4G polymorphism was five times more frequent in patients with venous TE than without it (P=0.05). Regarding risk factors for arterial TE we failed to identify any. CONCLUSIONS We have demonstrated that APL-related TE rate is higher than previously reported and that weekly D-dimer monitoring might help to identify patients with silent thrombosis. Moreover, our study suggests a possible relationship between venous TE occurrence and several laboratory findings (PT, aPTT, ISTH DIC score, bcr3 isoform, FLT3-ITD mutation and PAI 4G/4G). Prophylactic use of heparin might be considered in patients with ISTH DIC score<5, bcr3 isoform, FLT3-ITD mutation and PAI 4G/4G.

Microarray and Proteomic Analyses of Myeloproliferative Neoplasms with a Highlight on the mTOR Signaling Pathway

The gene and protein expression profiles in myeloproliferative neoplasms (MPNs) may reveal gene and protein markers of a potential clinical relevance in diagnosis, treatment and prediction of response to therapy. Using cDNA microarray analysis of 25,100 unique genes, we studied the gene expression profile of CD34+ cells and granulocytes obtained from peripheral blood of subjects with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). The microarray analyses of the CD34+ cells and granulocytes were performed from 20 de novo MPN subjects: JAK2 positive ET, PV, PMF subjects, and JAK2 negative ET/PMF subjects. The granulocytes for proteomic studies were pooled in 4 groups: PV with JAK2 mutant allele burden above 80%, ET with JAK2 mutation, PMF with JAK2 mutation and ET/PMF with no JAK2 mutation. The number of differentially regulated genes was about two fold larger in CD34+ cells compared to granulocytes. Thirty-six genes (including RUNX1, TNFRSF19) were persistently highly expressed, while 42 genes (including FOXD4, PDE4A) were underexpressed both in CD34+ cells and granulocytes. Using proteomic studies, significant up-regulation was observed for MAPK and PI3K/AKT signaling regulators that control myeloid cell apoptosis and proliferation: RAC2, MNDA, S100A8/9, CORO1A, and GNAI2. When the status of the mTOR signaling pathway related genes was analyzed, PI3K/AKT regulators were preferentially up-regulated in CD34+ cells of MPNs, with down-regulated major components of the protein complex EIF4F. Molecular profiling of CD34+ cells and granulocytes of MPN determined gene expression patterns beyond their recognized function in disease pathogenesis that included dominant up-regulation of PI3K/AKT signaling.

Prognostic significance of new biological markers in chronic lymphocytic leukaemia

INTRODUCTION Chronic lymphocytic leukaemia is a disease with heterogeneous clinical course and outcome. Some patients have a progressive course of the disease and require therapy immediately after the diagnosis, while others have a stable form without the need for treatment. Recently, two new biological markers, the expression of CD38 antigen and ZAP-70 have shown independent significance in the prognosis in CLL patients. OBJECTIVE The aim of our study was to evaluate the clinical value of CD38 antigen and ZAP-70 expression as predictors of the disease progression and to analyze the correlation of these markers with other B-CLL prognostic markers. METHODS We assessed the expression of CD38 antigens by flow cytometry on peripheral blood samples and the expression of ZAP-70 by immunohistochemistry on formalin-fixed bone marrow (BM) biopsies in 40 newly diagnosed B-CLL patients. Disease progression was defined by the period elapsed from diagnosis to the time to first treatment (TFT). RESULTS Expression of CD38 antigen correlated positively with ZAP-70 expression (Pearson, r = 0.476; p = 0.002). Also, correlation analysis results showed that a positive expression of CD38 and ZAP-70 statistically significantly correlated with unfavourable classical prognostic parameters, such as advanced Binet stage C, diffuse BM infiltration, increased lactate-dehydrogenase and beta-2 microglobulin serum levels. Patients with positive expression of CD38 antigen and ZAP-70 had a shorter TFT (log rank, 0.003 vs. 0.049). CONCLUSION Both new biological markers were shown to have an exceptional significance in the prediction of prognosis in CLL patients.

Angiogenic factors are increased in circulating granulocytes and CD34+ cells of myeloproliferative neoplasms

It has been shown that angiogenesis and inflammation play an important role in development of most hematological malignancies including the myeloproliferative neoplasm (MPN). The aim of this study was to investigate and correlate the levels of key angiogenic molecules such as hypoxia‐inducible factor‐1α (HIF‐1α), vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in peripheral blood and bone marrow cells of MPN patients, along with JAK2V617F mutation allele burden and effects of therapy. HIF‐1α and VEGF gene expression were decreased, while eNOS mRNA levels were increased in granulocytes of MPN patients. Furthermore, positively correlated and increased VEGF and eNOS protein levels were in negative correlation with HIF‐1α levels in granulocytes of MPN patients. According to immunoblotting, the generally augmented angiogenic factors demonstrated JAK2V617F allele burden dependence only in granulocytes of PMF. The angiogenic factors were largely reduced after hydroxyurea therapy in granulocytes of MPN patients. Levels of eNOS protein expression were stimulated by Calreticulin mutations in granulocytes of essential thrombocythemia. Immunocytochemical analyses of CD34+ cells showed a more pronounced enhancement of angiogenic factors than in granulocytes. Increased gene expression linked to the proinflammatory TGFβ and MAPK signaling pathways were detected in CD34+ cells of MPN patients. In conclusion, the angiogenesis is increased in several cell types of MPN patients supported by the transcriptional activation of inflammation‐related target genes, and is not limited to bone marrow stroma cells. It also appears that some of the benefit of hydroxyurea therapy of the MPN is mediated by effects on angiogenic factors.

Predictors of survival and cause of death in patients with essential thrombocythemia.

Standard risk stratification for overall survival (OS) in patients with essential thrombocythemia (ET) is based on advanced age and history of thrombotic events. Recently, International Prognostic Score for ET (IPSET) incorporated also leukocytosis in prognostic model. The aim of this study was to establish additional risk factors for OS in ET patients.

Diagnostic Challenges during Pretreatment Long-term Follow-up in a Patient with FIP1L1-PDGFRA-positive Eosinophilia

Obtaining a precise characterization of eosinophilia is crucial, as successful treatment relies on the underlying etiology of the disease. Platelet-derived growth factor receptor alpha-related disorders were first specified in 2008 as a distinct group of clonal eosinophilic disorders with exceptional responsiveness to imatinib. We herein present the case of a man with myeloid neoplasm and eosinophilia in whom a definitive diagnosis could not be adequately made based on histopathological features who was ultimately diagnosed only after extensive molecular analyses and successfully treated with imatinib. In addition, we discuss the diagnostic and therapeutic approaches to treating patients presenting with eosinophilia.

The emergence of non-secretory multiple myeloma during the non-cytotoxic treatment of essential thrombocythemia: a case report.

IntroductionThe emergence of multiple myeloma as a second malignancy in patients with essential thrombocythemia is extremely rare. Several cases have been published so far, pointing out the impact of a cytotoxic effect during treatment of essential thrombocythemia on the development of multiple myeloma.Case presentationWe report the case of a 52-year-old Caucasian man who presented to our hospital because of leukocytosis, a slightly decreased hemoglobin level and thrombocytosis. After a complete hematological work-up, essential thrombocythemia was diagnosed. The patient was included in a multicenter clinical study, treated with anagrelide and his platelet counts were maintained in the normal range for more than 3 years. A sudden drop in his hemoglobin level with normal leukocyte and platelet count occurred at the same time as a back pain. Magnetic resonance imaging of his spine revealed the existence of a pathological fracture of Th4, the collapse of the upper edge of Th7 and osteolytic lesions of multiple thoracic vertebrae. Repeated hematological examinations, including bone biopsy with immunohistochemistry, disclosed diagnosis of multiple myeloma of the non-secretory type.ConclusionsTo the best of our knowledge this is the first published case in which multiple myeloma developed during the treatment of essential thrombocythemia with the non-cytotoxic drug anagrelide. Our attempts to find a common origin for the coexistence of multiple myeloma and essential thrombocythemia have not confirmed the genetic basis of their appearance. Further studies are needed to determine the biological impact of this coexistence.

TLR4 and RAGE conversely mediate pro-inflammatory S100A8/9-mediated inhibition of proliferation-linked signaling in myeloproliferative neoplasms

PurposePreviously, the family of S100A proteins has been found to be associated with inflammation and myelopoiesis and to be able to induce or support myeloproliferation during chronic inflammation. Here, we studied the inflammatory myeloid-related proteins S100A4, S100A8, S100A9 and S100A12 in myeloproliferative neoplasms (MPNs) in order to assess the involvement of chronic inflammation in the pathogenesis of MPN.MethodsWe analyzed the S100A4, S100A8, S100A9 and S100A12 mRNA and protein levels in the bone marrow and circulation of 140 patients with MPN and 15 healthy controls using Western blotting, microarray-based mRNA expression profiling and ELISA assays, respectively. In addition we performed functional studies on the proliferation-related AKT and ERK1/2 signaling pathways in MPN-derived granulocytes using Western blotting and proteomic analyses.ResultsWe found that the S100A mRNA levels were increased in MPN patient-derived circulatory CD34+ cells, and that their protein expression levels were also augmented in their granulocytes and bone marrow stroma cells, depending on the JAK2V617F mutation allele burden. We also found that calreticulin (CALR) mutations were related to reduced S100A8 plasma levels in primary myelofibrosis (PMF). The S100A8 plasma levels were found to be increased in MPN, the S100A9 plasma levels in PMF and essential thrombocythemia (ET), and the S100A12 plasma levels in polycythemia vera (PV). These S100A plasma levels showed a positive correlation with the systemic inflammation marker IL-8, as well as with the numbers of leukocytes and thrombocytes, depending on the JAK2V617F mutation status. Additionally, we found that heterodimeric S100A8/9 can inhibit the AKT pathway in MPN-derived granulocytes mediated by the Toll-like receptor 4 (TLR4), depending on the CALR mutation status. Conversely, we found that blocking of the receptor for advanced glycation end products (RAGE) increased the S100A8/9-mediated inhibition of AKT signaling in the MPN-derived granulocytes. Moreover, we found that heterodimeric S100A8/9 generally induced TLR4-mediated ERK1/2 dephosphorylation proportionally to the JAK2V617F mutation allele burden. TLR4/RAGE blocking prevented the S100A8/9-mediated inhibition of ERK1/2 phosphorylation in PV.ConclusionsFrom our data we conclude that the S100A8 and S100A9 granulocyte and plasma levels are increased in MPN patients, along with inflammation markers, depending on their JAK2V617F mutation allele burden. We also found that S100A8/9-mediated inhibition of the proliferation-related AKT and ERK1/2 signaling pathways can be decreased by CALR mutation-dependent TLR4 blocking and increased by RAGE inhibition in MPN.

Proliferative characteristics of Philadelphia-negative myeloproliferative neoplasms - clinical implications.

Philadelphia‐negative myeloproliferative neoplasms (Ph−MPN) are characterized by overproduction of one or more blood cell lines.