Search

Intensive chemotherapy/radiotherapy in cancer, especially with hematologic malignancies, causes cellular injury and suppression of inflammatory responses, which increase the risks of neutropenia and febrile episodes. Absolute neutrophil count < 1 ? 109/L is considered neutropenia, with absolute neutrophil count < 0.5 ? 109/L or < 1 ? 109/L that is expected to decrease to < 0.5 ? 109/L in the next 48 hours considered severe neutropenia, while absolute neutrophil count < 0.1 ? 109 is referred as profound neutropenia. Febrile episodes are usually defined as oral temperature > 38.3?C or two consecutive readings > 38.0?C lasting more than 1 hour. Although there is the possibility of non-infection-caused febrile neutropenia, most episodes are caused by infections. Febrile neutropenia is a clinical emergency that requires prompt management. Despite advances in therapy in recent years, febrile neutropenia remains a common complication in chemotherapy causing serious clinical results, including death. The administration of empirical antibacterial therapy has been successful in the management of febrile neutropenia since its launching 50 years ago. The wide application of broad-spectrum antibiotics has effectively decreased the mortality of febrile neutropenia patients. Neutropenic patients who remain febrile despite 4-7 days of broad-spectrum antibacterial therapy are at a high risk of invasive fungal infection. Empirical antifungal therapy with Amphotericin B or Caspofungin in persistently febrile neutropenic patients and other high-risk patients has shown to reduce the risk of invasive fungal infection by 50 - 80% and the risk of fungal infection-related mortality by 23- 45%. Lipid formulations which improve the therapeutic ratio of the traditional formulation are available

Introduction: The impact of activated blood and endothelial cells on the thrombosis in myeloproliferative neoplasms (MPN) has not yet been clarified. We prospectively analyzed correlation between circulating leukocyte-platelet aggregates and soluble selectins to thrombosis occurrence in MPN, in the context of standard and cardiovascular risk factors, and different clinical and biological characteristics. Methods: Flow cytometric analysis of neutrophil-platelet (Neu-Plt) and monocyte-platelet (Mo-Plt) aggregates in peripheral blood, as well as quantification of soluble E-/L-/P-selectins by enzyme immunoassay, was performed on 95 newly diagnosed MPN patients. Results: During the follow-up, thrombosis occurred in 12.6% MPN patients (arterial 9.4%, venous 3.2%), with a mean time of 39 months. The overall incidence rate of main thrombotic events was 4.36 per 100 patient-years. The incidence of arterial hypertension (HTA) was significantly higher in patients with thrombosis, compared to those without thrombosis (P

Introduction: Erythrocytosis represents elevated hemoglobin and hematocrit levels above the range of normal values. Primary erythrocytosis - polycythemia vera, is characterized by increased erythrocyte production, due to a disorder at the level of the multipotent stem cell in the bone marrow. On the other hand, secondary erythrocytosis (SE) is the result of bone marrow stimulation by an external factor. Aim: The aim of our study was to determine parameters which are significant in differentiating SE from primary erythrocytosis - polycythemia vera (PV). Materials and methods: This is a retrospective study involving 108 patients with SE and 111 patients with PV, who were diagnosed and treated at the Clinic of Hematology of the Clinical Center of Serbia (CCS), in the period: December 2005 - November 2018.From the patient records, the following data were extracted: demographic characteristics, laboratory parameters, spleen size, total red cell mass, serum erythropoietin (EPO) level, and spontaneous growth of the BFU-E colony. Results: Patients with SE were younger, with a predominance of the male gender and with significantly higher serum EPO values than patients with PV. Patients with PV had significantly higher values of BFU-E, leukocyte and platelet count, spleen size, and LDH level than patients with SE. Total red cell mass analysis did not show a differential diagnostic significance. Conclusion: Findings of normal spleen size, normal leukocyte and platelet count, normal serum LDH level, and elevated EPO, in patients, refer to the diagnosis of secondary erythrocytosis, while the findings of splenomegaly, leukocytosis, thrombocytosis, elevated serum LDH level, decreased EPO, and the presence of spontaneous BFU-E colony speak in favor of the diagnosis of polycythemia vera.

Regarding diagnosis of polycythemia vera (PV), discussion persists about hemoglobin (Hb) and/or hematocrit (Hct) threshold values as surrogate markers for red cell mass (RCM) and the diagnostic impact of bone marrow (BM) morphology. We performed a retrospective study on 290 patients with PV (151 males, 139 females; median age 65 years) presenting with characteristic BM features (initial biopsies, centralized evaluation) and endogenous erythroid colony (EEC) formations. This cohort included (1) a group of 229 patients when following the 2008 versus 256 patients diagnosed according to the 2016 World Health Organization (WHO) guidelines, all presented with increased RCM; (2) masked PV patients with low Hb (n = 143)/Hct (n = 45) recruited from the 2008 WHO cohort; (3) a cohort of 17 PV patients with elevated diagnostic Hb/Hct levels but low RCM; and (4) nine PV patients with increased RCM, opposing low Hb/Hct values. All patients were treated according to current PV guidelines (phlebotomies 87%, hydroxyurea 79%, and acetylsalicylic acid 87%). Applying the 2016 WHO criteria significantly increased concordance between RCM and Hb values compared with the 2008 WHO criteria (90 vs. 43% in males and 83 vs. 64% in females). Further analysis of the WHO 2016 PV cohort revealed that increased RCM is associated with increased Hb/Hct (93.8/94.6%). Our study supports and extends the diagnostic impact of the 2016 revised WHO classification for PV by highlighting the importance of characteristic BM findings and implies that Hb/Hct threshold values may be used as surrogate markers for RCM measurements.

Increased angiogenesis in BCR-ABL1 negative myeloproliferative neoplasms (MPNs) has been recognized, but its connection with clinical and molecular markers needs to be defined. The aims of study were to (1) assess bone marrow (BM) angiogenesis measured by microvessel density (MVD) using CD34 and CD105 antibodies; (2) analyze correlation of MVD with plasma angiogenic factors including vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8; (3) examine the association of MVD with clinicopathological and molecular markers. We examined 90 de novo MPN patients (30 polycythemia vera (PV), primary myelofibrosis (PMF), essential thrombocythemia (ET)) and 10 age-matched controls. MVD was analyzed by immunohistochemistry "hot spot" method, angiogenic factors by immunoassay and JAK2V617F, and CALR mutations by DNA sequencing and allelic PCR. MVD was significantly increased in MPNs compared to controls (PMF gt PV gt ET). Correlation between MVD and plasma angiogenic factors was found in MPNs. MVD was significantly increased in patients with JAK2V617F mutation and correlated with JAK2 mutant allele burden (CD34-MVD: rho = 0.491, p lt 0.001; CD105-MVD: rho = 0.276, p = 0.02) but not with CALR mutation. MVD correlated with leukocyte count, serum lactate dehydrogenase, hepatomegaly, and splenomegaly. BM fibrosis was significantly associated with CD34-MVD, CD105-MVD, interleukin-8, and JAK2 mutant allele burden. JAK2 homozygote status had positive predictive value (100%) for BM fibrosis. Patients with prefibrotic PMF had significantly higher MVD than patients with ET, and we could recommend MVD to be additional histopathological marker to distinguish these two entities. This study also highlights the strong correlation of MVD with plasma angiogenic factors, JAK2 mutant allele burden, and BM fibrosis in MPNs.

Introduction: Philadelphia-negative myeloproliferative neoplasms (Ph-MPN) are characterized by overproduction of one or more blood cell lines. Methods: We studied the proliferative characteristics of 91 patients with de novo Ph-MPN. Colony-forming cells (CFC) and endogenous colonies (EC), from bone marrow (BM) and/or peripheral blood (PB), were analyzed by colony assay based on methylcellulose. The level of circulating CD34(+) cells was determined by flow cytometry. Results: The total number of PB CFC in primary myelofibrosis (PMF) was increased compared to the control sample (P lt 0.01) and essential thrombocythemia (ET) (P lt 0.05). The highest number of BM and PB EC was observed in polycythemia vera (PV) (P lt 0.01). Increased levels of CD34(+) cells characterized early-prefibrotic (57%) and advanced-fibrotic PMF (90%) as compared to PV (34%) and ET (32%) (P lt 0.01). In the whole Ph-MPN group, the total number of PB CFC (P lt 0.01), PB EC (P lt 0.05), and CD34+ cells (P lt 0.01) correlated with the degree of BM fibrosis. Higher levels of circulating CD34(+) cells in PMF correlated with the total number of PB EC (P lt 0.05) and degree of BM fibrosis (P lt 0.01). Conclusions: Exploration of the PB proliferative characteristics of Ph-MPN on diagnosis may be helpful in revealing early-prefibrotic PMF. Monitoring the levels of circulating CD34(+) cells may provide a sensitive indicator of fibrotic evolution in PV and PMF.

Purpose We compared the gene expression profile of peripheral blood CD34 + cells and granulocytes in subjects with chronic myeloid leukemia (CML), with the accent on signaling pathways affected by BCR–ABL oncogene. Methods The microarray analyses have been performed in circulating CD34 + cells and granulocytes from peripheral blood of 7 subjects with CML and 7 healthy donors. All studied BCR–ABL positive CML patients were in chronic phase, with a mean value of 2012 ± SD of CD34 + cells/μl in peripheral blood. Results The gene expression profile was more prominent in CML CD34 + cells (3553 genes) compared to granulocytes (2701 genes). The 41 and 39 genes were significantly upregulated in CML CD34 + cells ( HINT1 , TXN , SERBP1 ) and granulocytes, respectively. BCR–ABL oncogene activated PI3K/AKT and MAPK signaling through significant upregulation of PTPN11 , CDK4 / 6 , and MYC and reduction of E2F1 , KRAS , and NFKBIA gene expression in CD34 + cells. Among genes linked to the inhibition of cellular proliferation by BCR–ABL inhibitor Imatinib, the FOS and STAT1 demonstrated significantly decreased expression in CML. Conclusion The presence of BCR–ABL fusion gene doubled the expression quantity of genes involved in the regulation of cell cycle, proliferation and apoptosis of CD34 + cells. These results determined the modified genes in PI3K/AKT and MAPK signaling of CML subjects.

Duplication of the long arm of chromosome 1 (1q) is widely reported in human neoplasia, including the myelodysplastic syndromes (MDS). So far, it has not been described as a single aberration in the chronic myelomonocytic leukemia (CMML), a subtype of MDS. Rather, trisomy 1q was always a part of complex chromosome changes affecting the subtypes of MDS other than CMML. We report on a patient with CMML with an unbalanced translocation of the entire 1q onto the short arm of chromosome 14 as a sole cytogenetic abnormality. Fluorescence in situ hybridization (FISH) analysis with an α-satellite probe for the paracentric region of the long arm of chromosome 1 confirmed the presence of trisomy 1q in a derivative chromosome, der(14)t(1;14)(q12;p11). The discrepant results between the metaphase cytogenetics (100% abnormal) and interphase cytogenetic (71% nuclei with 3 signals) suggest that trisomy 1q, even in the absence of additional cytogenetic changes, has a sufficient leukemogenic potential to confer a proliferative advantage on hematopoietic cells committed to monocyte stemline both in vitro and in vivo. The literature data on partial and complete trisomy 1q in CMML is reviewed.

The effect of Tiazofurin (TR) on the in vitro growth of bone marrow (BM) and peripheral blood (PB) leukemic progenitors was investigated in 29 patients. Nineteen of the patients were suffering the blast crisis of chronic myeloid leukemia (bcCML) and ten patients refractory acute myeloid leukemia (AML). PB and BM mononuclear cells were cultured in methylcellulose alone or with concentrations of TR ranging between 10 and 200 microM. TR produced a dose dependent inhibition of colony forming unit (CFU)-blast growth in all the samples tested from BM and PB. The most effective concentrations of TR used were 150 and 200 microM, while concentrations of less than 50 microM TR were not adequate for 50% inhibition of cell growth (IC50). Differences were found in the response of CFU-blasts to TR related to the type of underlying leukemia. Inhibition of CFU-blast growth was more pronounced in bcCML than in AML in both the BM and PB samples. The concentration of TR required to induce IC50 in bcCML was 50 microM, while the same effect in AML required a concentration of 150 microM. Analysis of the control samples also revealed that CFU-blasts from bcCML produced smaller numbers of colonies, though these differences were not statistically significant. It has therefore been demonstrated that TR has strong in vitro anti-leukemic activity, more pronounced in bcCML than in refractory AML. We thus feel this study gives further rationale for the clinical application of TR, and would strongly support this.

Splenic venous thrombosis is a rare disease in which an underlying hypercoagulable state can often be found. A 27-years old female patient with recurrent mesenteric venous and splenic thrombosis as a severe complication of an association of resistance to activated protein C and essential thrombocythemia is presented in this report. Establishing the diagnosis of essential thrombocytosis was particularly difficult because this was the case of the so called "silent" myeloproliferative disorder. The number of thrombocytes was almost normal before the splenectomy performed because of the splenic venous thrombosis. Thus, spontaneous growth of erythroid and megakaryocyte colonies in vitro and the clinical course of the disease were the clues for establishing the diagnosis, because the number of thrombocytes reached the values over 1500?109/l after only 1.5 years of the follow-up. The case of this patient was interesting particularly from the surgical point of view because of the management strategy.

Objectives 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. Methods After the median follow-up of 7 yr, in 244 consecutive ET patients, 32 deaths were documented (13.2%). The 5- and 10-yr OS was 95.9% and 79.7%, respectively. Considered additional risk factors at diagnosis of ET were the presence of arterial hypertension, diabetes, hyperlipidemia, and smoking attitude. Results The main cause of death in 75% of patients was cardiovascular (CV) comorbidity. Patients with CV risk factors had increased risk of death (HR = 2.33). Cox regression model identified age, leukocytosis, presence of CV risk factors, and previous thrombosis as unfavorable predictors of survival. Based on these parameters, four risk groups were defined, with significantly different survivals (P

We have investigated growth in vitro of bone marrow megakaryocytic progenitors (CFU-Mk) in 7 patients with paroxysmal nocturnal haemoglobinuria (PNH) to determine the sensitivity of CFU-Mk to complement. Bone marrow light density mononuclear cells were exposed to fresh or heat-inactivated AB human serum in the presence of medium or isotonic sucrose solution. We found that the proliferative activity of bone marrow CFU-Mk in PNH patients was significantly lower than in controls. In addition, the number of CFU-Mk in PNH bone marrow cells exposed to isotonic sucrose and complement was reduced to 25% of that in PNH cells exposed to isotonic sucrose without complement. In conclusion, our finding showed an increased sensitivity of CFU-Mk in PNH bone marrow cells to complement, supporting the hypothesis that the PNH defect is present at the level of CFU-Mk.

Spontaneous colony formation from bone marrow megakaryocyte progenitors (BMsCFU-Mk) was studied in 24 patients with essential thrombocythaemia (ET), 20 patients with reactive thrombocytosis (RT), 20 patients with polycthaemia rubra vera with thrombocytosis (PRVtr), 16 patients with chronic myeloid leukaemia with thrombocytosis (CMLtr) and 18 normal control subjects (C). The culture medium which was used in the methylcellulose assay in vitro contained 30% of plasma from a single patient with hereditary haemochromatosis. Remarkable BMsCFU-Mk growth was recorded in all patients with ET but in none with RT or in C. BMs-CFU-Mk were present in 11/20 patients with PRVtr and 7/16 patients with CMLtr. Spontaneous bone marrow erythroid progenitors (BMsBFU-E) were also determined in these patients. BMsBFU-E were found in 21/24 patients with ET and none in the patients with RT and C. All patients with PRVtr and one patient with CMLtr showed BMsBFU-E. We conclude that our implementation of the in vitro methylcellulose assay allows the BMsCFU-Mk to be used as an unequivocal test for discrimination between ET and RT which has not been shown in previously published studies. In addition, we present evidence that in 10 patients BMsCFU-Mk and/or BMsBFU-E growth in the test persisted after long-lasting haematological remission.

Abstract 3856 The gene and protein expression profiles in myeloproliferative neoplasms (MPN) may reveal gene markers of a potential clinical function in diagnosis and prediction of response to therapy. Using cDNA microarray analysis, involving 25,100 unique genes, we studied the gene expression profile of hematopoietic CD34+ progenitor cells and granulocytes obtained from peripheral blood of patients with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) compared with healthy individuals. The microarray analyses of the hematopoietic progenitor cells and granulocytes have been performed on 9 patients with ET, 8 patients with PV, 4 patients with PMF and 8 healthy donors. The granulocytes for proteomic studies have been 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. We focused our analysis to hematopoiesis related genes. In the collected patient samples, the increased number of granulocytes allowed for further validation by protein analysis of microarray gene expression suggested from less differentiated hematopoietic progenitor cells. Folate receptor 3 (FOLR3), constitutively secreted in hematopoietic tissues, has increased protein levels in granulocytes of JAK2V617F homozygous PV as well as mRNA levels in hematopoietic progenitor cells of patients with PV. The enzyme matrix metallopeptidase 9 (MMP9), involved in IL-8-induced mobilization of hematopoietic progenitor cells from bone marrow, also has significantly increased protein levels in granulocytes of PV patients with increased JAK2 mutation allele burden. In addition, Ras-related C3 botulinum toxin substrate 2 (RAC2) protein level, essential for erythropoiesis, is increased specifically in PV granulocytes with JAK2V617F homozygosity. Moreover, RAC2 gene expression is significantly increased in hematopoietic progenitor cells of PV, with no changes in its granulocytes. Although, like PV, RAC2 gene expression was also increased in ET and PMF hematopoietic progenitor cells compared to healthy individuals, in granulocytes of ET and PMF patients with JAK2 mutation RAC2 protein levels were decreased, contrary to the elevated level in PV. Furthermore, inconsistent with JAK2V617F homozygous PV patients, granulocytes of ET and PMF with the JAK2 mutation exhibit FOLR3 protein at levels lower than the ET and PMF with no JAK2 mutation. Investigating the extent to which these genes participate in the complex molecular and cellular mechanisms of MPN will likely lead to new insights of malignancy development. In conclusion, molecular profiling of hematopoietic progenitor cells and granulocytes of MPN patients revealed gene expression patterns that are beyond their recognized function in disease pathogenesis and can be related to patients' clinical characteristics with imminent prognostic relevance. Disclosures: No relevant conflicts of interest to declare.

Acquired amegakaryocytic thrombocytopenia (AATP) in adults is a rare disorder characterized by severe thrombocytopenia and decreased or absent megakaryocytes in an otherwise normal bone marrow. We present a 44-yr-old man in whom the diagnosis of AATP was established in January 2001. Immunophenotyping of the peripheral blood lymphocytes showed a relative increase in the subpopulation of gamma/delta T-cell receptor (TCR) positive (gamma/delta TCR(+)) and (CD4, CD8) negative T lymphocytes, and PCR suggested a monoclonal pattern of TCR gamma chain gene rearrangement. Cytogenetic examination of his bone marrow cells showed a normal male karyotype but RT-PCR analysis revealed a BCR-ABL (p210) fusion transcript. The inhibition of CFU-Mk growth mediated by the patient's T lymphocytes indicated that the pathogenic mechanism for AATP could be an immunological attack on megakaryocyte progenitors where the gamma/delta TCR-positive T lymphocytes are directly involved. The case emphasizes the complex association of T-lymphocyte monoclonal proliferation and AATP.

Splenic venous thrombosis is a rare disease in which an underlying hypercoagulable state can often be found. A 27-years old female patient with recurrent mesenteric venous and splenic thrombosis as a severe complication of an association of resistance to activated protein C and essential thrombocythemia is presented in this report. Establishing the diagnosis of essential thrombocytosis was particularly difficult because this was the case of the so called "silent" myeloproliferative disorder. The number of thrombocytes was almost normal before the splenectomy performed because of the splenic venous thrombosis. Thus, spontaneous growth of erythroid and megakaryocyte colonies in vitro and the clinical course of the disease were the clues for establishing the diagnosis, because the number of thrombocytes reached the values over 1500 x 10(9)/l after only 1.5 years of the follow-up. The case of this patient was interesting particularly from the surgical point of view because of the management strategy.

Publisher Summary This chapter presents a study in which the effect of the peptide LD78 (the human homologue of murine macrophage inflammatory protein-1α (MIP-1α) that shows 747 amino acid sequence homology with MIP-1α) on bone marrow acute myeloid leukemia (AML) progenitor cells as measured by in vitro CFU-AML growth is investigated. The results demonstrate the inhibitory effect of LD78 on AML cell growth of bone-marrow-derived progenitors, probably mediated by an effect on AML cell proliferation. This suggests that the normal inhibitory control mechanisms mediated by LD78 are still intact in AML progenitor cells. The observed inhibitory effect of LD78 in CFU-AML growth was not related to the type of AML as evaluated by the FAB criteria for classification of AML. The effect of MIP-1α on the proliferation of T-lymphocytes seems to be mediated in part by the inhibition of IL-2 production. The study demonstrates that LD78 is more active on AML progenitors than on AML cell proliferation. Inhibition of the AML cells, although less than that of the progenitors, indicates that more limited activity of LD78 on more mature leukemic cells is present in AML.

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) has recently been shown to inhibit proliferation of immature hemopoietic progenitors. In addition, significant inhibition of early and mature leukemic progenitors in acute myeloid leukemia (AML) has been obtained with MIP-1 alpha. We performed a study of 25 AML patients at diagnosis to evaluate the effect of a human homolog of MIP-1 alpha (LD78) on bone marrow (BM) and peripheral blood (PB) leukemic progenitors (colony-forming unit-AML [CFU-AML]) and AML cell proliferation. A methylcellulose culture system was used for CFU-AML and incorporation of 3H-TdR for AML cell proliferation. We found that LD78 inhibits CFU-AML colony formation up to 100% for the BM in 14/16 samples studied with the average maximal inhibition of 62.7 +/- 9.1% and up to 100% for the PB in 12/13 samples studied with the average maximal inhibition of 71.4 +/- 9.9%. In addition to this, LD78 inhibited AML cell proliferation up to 60% for the BM in 10/18 samples studied with the average maximal inhibition of 17.8 +/- 3.5%, and up to 87.1% for the PB cell proliferation in 10/16 samples studied with the average maximal inhibition of 27.5 +/- 6.8%. Our results have shown that LD78 is more active on AML progenitors than on AML cell proliferation. Inhibition of the AML cells, although less than that of the progenitors, indicates that more limited activity of LD78 on more mature leukemic cells is present in AML.