Konstantinos L. Bourantas

Prolonged administration of erythropoietin increases erythroid response rate in myelodysplastic syndromes: a phase II trial in 281 patients

Summary. Treatment with recombinant human erythropoietin (rHuEpo) improves anaemia in approximately 20% of patients with myelodysplastic syndromes (MDS). We investigated the potential advantage of a prolonged administration of rHuEpo to achieve higher erythroid response rates (RR) in 281 MDS patients: 118 with refractory anaemia (RA), 77 with refractory anaemia and ringed sideroblasts (RARS), 59 with refractory anaemia with excess of blasts and blast count < 10% (RAEB‐I), and 27 with RAEB and blast count between 11–20% (RAEB‐II). rHuEpo was given subcutaneously at a dose of 150 U/kg thrice weekly, for a minimum of 26 weeks. Response to treatment was evaluated after 12 and 26 weeks of therapy. The overall RR was 45·1%; the RR for RA, RARS, RAEB‐I and RAEB‐II were 48·3%, 58·4%, 33·8% and 13% respectively. A significant increase in RR was observed at week 26 in RA, RARS and RAEB‐I patients, as the response probability increased with treatment duration. The RR was higher in the good cytogenetic prognostic group and serum Epo level of > 150 U/l at baseline predicted for non‐response. The median duration of response was 68 weeks and the overall risk of leukaemic transformation was 21·7%. These results suggest that prolonged administration of rHuEpo produces high and long‐lasting erythroid RR in MDS patients with low blast counts, particularly in those with pretreatment serum Epo levels of < 150 U/l and good cytogenetic prognosis.

CpG methylation analysis of the MEG3 and SNRPN imprinted genes in acute myeloid leukemia and myelodysplastic syndromes

Methylation is now established as a fundamental regulator of gene transcription. To investigate this in haematologic malignancies, we evaluated the aberrant promoter methylation of two imprinted genes (MEG3 and SNRPN) in 43 MDS and 42 AML patients. MEG3 hypermethylation occurred in 15 MDS patients (34.9%), and in 20 AML patients (47.6%). SNRPN hypermethylation was observed in 15 MDS patients (34.9%), and in 21 AML patients (50%). There were no significant correlations between WHO subtype, WPSS score, karyotype, haemoglobin levels, white blood cell count, platelet count and CpG methylation of any gene. MEG3 hypermethylation was associated with significantly reduced overall survival in individuals with AML (HR=1.98, p=0.04), while SNRPN CpG methylation was not associated with survival (HR=0.94, p=0.87). In addition, no association between survival and aberrant MEG3 (HR=2.15, p=0.072) or SNRPN methylation (HR=1.08, p=0.85) was observed in patients MDS. Our findings suggest that these genes are abnormally methylated in AML and MDS patients, and methylation of MEG3 confers worse overall prognosis. The MEG3 methylation status may serve as a useful biomarker in leukemia.

Acute phase proteins and interleukins in steady state sickle cell disease.

Abstract: To identify a possible acute phase response during the steady state of sickle cell disease, we estimated the serum alterations of acute phase proteins, β2‐microglobulin (β2M), κ and λ light chains, interleukins (ILs) and tumor necrosis factor‐α (TNFα) in 21 patients. Increased concentrations of C‐reactive protein (CRP) were found in 5 patients, alpha‐1‐acid‐glycoprotein (AGP) in 3, alpha‐1‐antitrypsin (AAT) in 8, ceruloplasmin (CER) in 2, alpha‐2‐macrogiobulin (AMG) in 14 and decreased haptoglobin (HPT) and transferrin (TFR) in 11 and 9, respectively. Increased β2M was found in 10 patients and κ and λ light chains in 11. IL‐1β, IL‐2, IL‐4, IL‐10 and TNFα were not detected in any of the patients. However, significantly increased values of IL‐6 and sIL‐2r were found. This study has demonstrated increased serum levels of some of the acute phase proteins in patients during the steady state of sickle cell disease. This may be a result of a subclinical vaso‐occlusion which in turn leads to a covert inflammatory response. Cytokines, and in particular IL‐6, produced after this response, seem to be responsible for the high levels of acute phase proteins in the steady state of this disease.

Serum interleukin (IL)-1, IL-2, sIL-2Ra, IL-6 and thrombopoietin levels in patients with chronic myeloproliferative diseases

A number of growth factors are involved in clonal haematopoietic expansion and their clinical significance in patients with chronic myeloproliferative diseases requires further evaluation. Using enzyme‐linked immunosorbent assays, we analysed serum levels of interleukin (IL)‐1a, IL‐1b, IL‐2, IL‐6, the soluble IL‐2 receptor alpha (sIL‐2Ra), and thrombopoietin (TPO), in 25 individuals with myelofibrosis with myeloid metaplasia (MMM), 40 with essential thrombocythaemia (ET), eight with polycythaemia vera (PV), 10 patients with chronic myeloid leukaemia (CML) and 27 normal controls. These were correlated with clinicopathological characteristics including overall survival, and histopathological bone marrow features, including angiogenesis. The serum derived from patients with MMM, ET, PV and CML contained significantly higher IL‐2 and sIL‐2Ra than healthy subjects, while IL‐6 levels were higher only in MMM and CML than controls. IL‐2, sIL‐2Ra and IL‐6 levels were raised during the transformation phase of CML, during progression of MMM to AML, and ET and PV to myelofibrosis (P < 0·001). There was a positive correlation between IL‐2, sIL‐2Ra, IL‐6 and angiogenesis in bone marrow samples. Cytokines may be useful markers for predicting clinical evolution, reflecting increased angiogenesis. This requires further evaluation to guide diagnostic and therapeutic options.

The role of cytokines in sickle cell disease.

Abstract Sickle cell disease (SCD) is characterized by chronic hemolysis, frequent infections, and recurrent occlusions of microcirculation, which cause painful crises and result in chronic organ damage and failure. Occlusions of the microcirculation and infections are important factors that stimulate the production of cytokines and acute-phase proteins. Cytokines seem to be involved with several possible mechanisms in the pathogenesis of vasoocclusive phenomena in SCD: vascular endothelial activation, induction of red-cell adhesiveness to vascular endothelium, induction of neutrophil adhesiveness to endothelium, development of vascular intimal hyperplasia, platelet activation, endothelin-1 production, and dysregulation of endothelial apoptosis. Cytokines are also thought to be involved in the regulation of hemopoiesis, the inhibition of immune functions, and the development of growth deficits. Investigation of cytokines in SCD patients will elucidate the pathogenesis of the disease and its complications and may help in assessing disease severity and prognosis.

Serum ferritin, transferrin and soluble transferrin receptor levels in multiple sclerosis patients

Over the last few years, increased evidence has supported the role of iron dysregulation in the pathogenesis of multiple sclerosis (MS), as iron is essential for myelin formation and oxidative phosphorylation. We studied indices of iron metabolism, such as serum iron, ferritin, transferrin and soluble transferrin receptor (sTFR) levels in 27 MS patients. Seven patients had chronic progressive active disease (CP-A), six had chronic progressive stable (CP-S), ten had relapsing—remitting active (RR-A) and four had relapsing—remitting stable (RR-S) disease. sTFR levels were found to be significantly higher in CP-A (P=0.021) and RR-A (P= 0.004) patients than in controls. sTFR levels were also elevated in CP-S patients but did not reach significance (P=0.064). sTFR values in RR-S patients were comparable to those found in controls (P=0.31). Ferritin levels were significantly elevated only in CP-A patients (P= 0.002). Patients of the CP group had significantly higher ferritin values than the RR patients (P= 0.004). Haemoglobin values as well as iron and transferrin levels were within normal limits in all patients. In conclusion, the increased serum sTFR and ferritin levels in nonanaemic MS patients with active disease reflect the increased iron turnover. The mild elevation of sTFR levels in CP-S patients may indicate active inflammation with ongoing oxidative damage that is not detectable by history or examination.

Promoter Hypermethylation of the MEG3 (DLK1/MEG3) Imprinted Gene in Multiple Myeloma

BACKGROUND Methylation represents the most studied epigenetic modification and results in the silencing of genes involved in various processes such as differentiation and cell-cycle regulation. MEG3 represents an imprinted gene maternally expressed in humans that encodes a nontranslated product. In this survey, we studied the methylation status of the specific gene in multiple myeloma (MM). PATIENTS AND METHODS Twenty-one patients with MM (17 with immunoglobulin [Ig] G, 3 with IgA, and 1 with IgM) were evaluated using methylation-specific polymerase chain reaction (after DNA bisulphite modification). RESULTS Promoter hypermethylation was observed in 12 (57.14%) bone marrow samples and in 9 of 14 (64.28%) available peripheral blood samples. A correlation with disease stage was also observed and also with the disease subtype (IgG, 64.7%; IgA, 0; IgM, 100%). CONCLUSION We conclude that promoter hypermethylation of the differentially methylated region of the MEG3 imprinted gene is observed in patients with MM.

The prolyl-hydroxylase EGLN3 and not EGLN1 is inactivated by methylation in plasma cell neoplasia

EGLN1 and EGLN3 are members of the egg‐laying‐defective 9 (EglN) prolyl‐hydroxylases which during normoxia catalyse hydroxylation of the hypoxia‐inducible factor (HIF)‐1α, thereby promoting its ubiquitination by a complex containing the von Hippel–Lindau (VHL) tumour suppressor. EGLN3 also has pro‐apoptotic activity in some cell types. Analyses of a well‐characterised series of cases of plasma cell dyscrasias, including multiple myeloma (MM), Waldenström’s macroglobulinaemia (WM) and monoclonal gammopathy of undetermined significance (MGUS) surprisingly demonstrated that the CpG island of EGLN3, and not EGLN1, is frequently methylated in these disorders. Multiple myeloma patients with a methylated EGLN3 promoter showed trends towards an increased risk of death, bone lytic lesions, anaemia, advanced stage of disease and the presence of extramedullary disease. Those individuals with methylation in the EGLN3 CpG island also had significantly lower albumin levels. These data suggest that the prolyl‐hydroxylases may be a novel class of potential tumour suppressors in plasma cell neoplasia that warrant further investigation with regard to their potential utility as biomarkers. Moreover, we observed that EGLN3 is also methylated at high frequency in B‐cell lymphoma subtypes, implying that loss of EGLN3 is an important epigenetic event not only in plasma cell neoplasias but also in B‐cell neoplasias.

Serum levels of IL-6 and its soluble receptor (sIL-6R) in Waldenström's macroglobulinemia

Abstract: Background: Interleukin‐6 (IL‐6) is a multifunctional cytokine that plays roles in the immune response, inflammation and hematopoiesis. Serum IL‐6 levels have reported to reflect disease severity and high tumor burden in multiple myeloma (MM) patients and to correlate with several other laboratory parameters. Serum‐soluble IL‐6 receptor (sIL‐6R) plays an agonist role in IL‐6 signaling, enhancing its biological activity tenfold. Purpose–Methods: We measured IL‐6 and sIL‐6R levels in 11 patients (7 male, 4 female, mean age 66.9 yr) with Waldenströms macrobulinemia (WM) using a commercially available enzyme‐linked immunoassay in order to investigate their biological role and to find any possible relationship with disease severity, tumor burden or response to treatment. Results: Serum IL‐6 and sIL‐6R concentrations at diagnosis were significantly higher than in healthy controls (Mann–Whitney U‐test, p<0.001 and p<0.01, respectively). Patients who were effectively treated had a significant reduction in IL‐6 levels (p=0.017). With regard to sIL‐6R levels, no specific tendency was observed. In some of the responsive patients the levels increased whereas in others they decreased. No correlation was found between IL‐6 and sIL‐6R levels at diagnosis (p=0.9, r=0.036) or after treatment (p=0.083, r=0.3). Conclusions: Our results suggest that IL‐6 may be a marker reflecting tumor burden, disease severity and response to treatment in WM. With regard to sIL‐6R, we believe that it does not seem to be of much value, and its role remains to be clarified. However, future studies are needed to confirm and further extend the present results.

Angiogenesis in chronic myeloproliferative diseases detected by CD34 expression

Increased bone marrow angiogenesis estimated as bone marrow microvessel density (MVD), or as serum angiogenic factor levels and/or immunohistochemical expression of these factors in bone marrow biopsy has been demonstrated in a variety of hematological disorders including chronic myeloproliferative diseases (MPDs). The aim of this study was to investigate the MVD in 25 cases of myelofibrosis with myeloid metaplasia (MMM). MVD was estimated by CD34 immunohistochemical expression in bone marrow biopsies. A control group of 27 patients without bone marrow disease, eight cases of polycythemia vera (PV), 41 cases of essential thrombocythemia (ET) and nine cases of chronic myeloid leukemia (CML) were also studied. Moreover, in cases with MMM, MVD was correlated with clinical, laboratory, histological parameters and the outcome of the patients. Our study confirmed a significantly higher degree of angiogenesis in MMM, PV, ET and CML compared with controls (P < 0.001, P = 0.0007, P < 0.001 and P = 0.0008, respectively). Angiogenesis was higher in MMM than PV, ET and CML cases (P < 0.001, P < 0.001 and P = 0.008). Increased angiogenesis was correlated with hypercatabolic symptoms in MMM patients (P = 0.009). No correlation with other clinicopathological parameters or clinical outcome was found. However, definitive conclusions regarding the prognostic value of increased angiogenesis may require additional follow‐up and a larger group of patients.