A multicenter cross-sectional study of the quality of life and iron chelation treatment satisfaction of patients with transfusion-dependent ß-thalassemia, in routine care settings in Western Greece.

PURPOSE: To evaluate health-related quality of life (HRQoL) and satisfaction with iron chelation therapy (ICT) of patients with transfusion-dependent ß-thalassemia (TDT) managed under routine care conditions. PATIENTS AND METHODS: This was an observational, multicenter, cross-sectional study conducted in three hospital-based Thalassemia Units of Western Greece. Patients confidentially completed the 36-item short-form (SF-36) and the "satisfaction with ICT" (SICT) instruments to assess HRQoL and ICT satisfaction respectively. RESULTS: One hundred and thirty-one adult TDT patients [74 female, median (IQR) age: 41 (36-47) years] were enrolled. Eighty patients (61.1%) were receiving parenteral ICT, with or without oral chelators (Group I), whereas 51 (38.9%) were only receiving oral ICT (Group II). The median SF-36 physical component summary and mental component summary scores were 76.3 and 75.7 among Group I, and 76.9 and 74.5 among Group II patients, not differing between the two groups. In their majority, Group I (84.6%) and Group II (92.9%) patients reported preferring oral ICT. Moreover, Group I patients reported greater perceived ICT effectiveness (median SICT score: 4.3 versus 4.2; p = 0.039), whereas patients receiving deferasirox-containing ICT reported higher treatment acceptance (median SICT score: 4.0 versus 3.6, p = 0.038) and greater satisfaction with the burden of their ICT (median SICT score: 4.4 versus 3.9, p = 0.033). CONCLUSION: TDT patients prefer to receive oral ICT and are more satisfied of the burden of deferasirox-containing ICT, even though those receiving parenteral ICT are more satisfied by the effectiveness of their treatment. No differences in HRQoL were not noted between patients receiving parenteral versus oral ICT.

Impact of ZBTB7A hypomethylation and expression patterns on treatment response to hydroxyurea.

BACKGROUND: We aimed to clarify the emerging epigenetic landscape in a group of genes classified as "modifier genes" of the ß-type globin genes (HBB cluster), known to operate in trans to accomplish the two natural developmental switches in globin expression, from embryonic to fetal during the first trimester of conception and from fetal to adult around the time of birth. The epigenetic alterations were determined in adult sickle cell anemia (SCA) homozygotes and SCA/ß-thalassemia compound heterozygotes of Greek origin, who are under hydroxyurea (HU) treatment. Patients were distinguished in HU responders and HU non-responders (those not benefited from the HU) and both, and in vivo and in vitro approaches were implemented. RESULTS: We examined the CpG islands' DNA methylation profile of BCL11A, KLF1, MYB, MAP3K5, SIN3A, ZBTB7A, and GATA2, along with γ-globin and LRF/ZBTB7A expression levels. In vitro treatment of hematopoietic stem cells (HSCs) with HU induced a significant DNA hypomethylation pattern in ZBTB7A (p*, 0.04) and GATA2 (p*, 0.03) CpGs exclusively in the HU non-responders. Also, this group of patients exhibited significantly elevated baseline methylation patterns in ZBTB7A, before the HU treatment, compared to HU responders (p*, 0.019) and to control group of healthy individuals (p*, 0.021), which resembles a potential epigenetic barrier for the γ-globin expression. γ-Globin expression in vitro matched with detected HbF levels during patients' monitoring tests (in vivo) under HU treatment, implying a good reproducibility of the in vitro HU epigenetic effect. LRF/ZBTB7A expression was elevated only in the HU non-responders under the influence of HU. CONCLUSIONS: This is one of the very first pharmacoepigenomic studies indicating that the hypomethylation of ZBTB7A during HU treatment enhances the LRF expression, which by its turn suppresses the HbF resumption in the HU non-responders. Its role as an epigenetic regulator of hemoglobin switching is also supported by the wide distribution of ZBTB7A-binding sites within the 5' CpG sequences of all studied human HBB cluster "modifier genes." Also, the baseline methylation level of selective CpGs in ZBTB7A and GATA2 could be an indicator of the negative HU response among the ß-type hemoglobinopathy patients.

LRF/ZBTB7A conservation accentuates its potential as a therapeutic target for the hematopoietic disorders.

Conserved sequences across species have always provided valuable insights to improve our understanding on the human genome's entity and the interplay among different loci. Lymphoma/leukemia related factor (LRF) is encoded by ZBTB7A gene and belongs to an evolutionarily conserved family of transcription factors, implicated in vital cellular functions. The present data, demonstrating the wide-spread and the high overlap of the LRF/ZBTB7A recognition sites with genomic segments identified as CpG islands in the human genome, suggest that its binding capacity strongly depends on a specific sequence-encoded feature within CpGs. We have previously shown that de-methylation of the CpG island 326 lying in the ZBTB7A gene promoter is associated with impaired pharmacological induction of fetal hemoglobin in ß-type hemoglobinopathies patients. Within this context we aimed to investigate the extent of the LRF/ZBTB7A conservation among primates and mouse genome, focusing our interest also on the CpG island flanking the gene's promoter region, in an effort to further establish its epigenetic regulatory role in human hematopoiesis and pharmacological involvement in hematopoietic disorders. Comparative analysis of the human ZBTB7A nucleotide and amino acid sequences and orthologous sequences among non-human primates and mouse, exhibited high conservation scores. Pathway analysis, clearly indicated that LRF/ZBTB7A influences conserved cellular processes. These data in conjunction with the high levels of expression foremost in hematopoietic tissues, highlighted LRF/ZBTB7A as an essential factor operating indisputably during hematopoiesis.

Disease-related anemia in chronic lymphocytic leukemia is not due to intrinsic defects of erythroid precursors: a possible pathogenetic role for tumor necrosis factor-alpha.

BACKGROUND/AIMS: Disease-related anemia in chronic lymphocytic leukemia (CLL) occurs when the obvious causes are excluded while its pathogenesis is still obscure. We investigated its underlying mechanisms in 56 untreated patients with CLL. METHODS: Bone marrow (BM) lymphocytic infiltration was estimated in trephine biopsies. Serum erythropoietin (EPO) and tumor necrosis factor-alpha (TNF-alpha) levels were measured by ELISA. The potential of BM CD34+ to differentiate into erythroid cells was evaluated by methylcellulose-based assays and in liquid cultures supplemented with EPO, SCF, IL-3 +/- TNF-alpha. The response of erythroid precursors to EPO +/- TNF-alpha was assessed by detecting activated key proteins of EPO-EPO receptor signalling pathway using Western Blot and EMSA. RESULTS: Bone marrow lymphocytic infiltration was not exclusively responsible for disease-related anemia and CD34+ cells were intrinsically capable of generating erythroid precursors. Also, no deficiency of serum erythropoietin (EPO) or defective intracellular response of erythroid precursors to EPO +/- TNF-alpha stimulation was observed. Serum TNF-alpha levels were found increased in anemic CLL patients and TNF-alpha appeared to directly inhibit the erythroid development in early stages of erythropoiesis. CONCLUSION: We concluded that CLL-related anemia was not due to intrinsic defects of erythroid precursors, but might result from the direct suppressive effect of TNF-alpha on the erythroid production.

Prevalence of anti-HAV antibodies in multitransfused patients with beta-thalassemia.

AIM: To detect the prevalence of anti-HAV IgG antibodies in adult multitransfused beta-thalassemic patients. METHODS: We studied 182 adult beta-thalassemic patients and 209 controls matched for age and sex from the same geographic area, at the same time. Anti-HAV IgG antibodies, viral markers of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection were evaluated. RESULTS: Anti-HAV IgG antibodies were detected more frequently in thalassemic patients (133/182; 73.1%) than in healthy controls (38/209; 18.2%, P < 0.0005). When we retrospectively evaluated the prevalence of anti-HAV IgG antibodies in 176/182 (96.7%) thalassemic patients, whose medical history was available for the previous ten years, it was found that 83 (47.2%) of them were continuously anti-HAV IgG positive, 16 (9.1%) acquired anti-HAV IgG antibody during the previous ten years, 49 (27.8%) presented anti-HAV positivity intermittently and 28 (15.9%) were anti-HAV negative continuously. CONCLUSION: Multitransfused adult beta-thalassemic patients present higher frequency of anti-HAV IgG antibodies than normal population of the same geographic area. This difference is difficult to explain, but it can be attributed to the higher vulnerability of thalassemics to HAV infection and to passive transfer of anti-HAV antibodies by blood transfusions.

A probable role for trail-induced apoptosis in the pathogenesis of marrow failure. Implications from an in vitro model and from marrow of aplastic anemia patients.

Aplastic anemia (AA) is a syndrome of hematopoietic failure involving increased apoptosis of stem cells. In order to investigate the molecular mechanisms participated in the process of marrow failure, we created an in vitro model of hematopoietic cell suppression, by continuous addition of TNF-alpha and IFN-gamma in an vitro long-term bone marrow culture system. An up-regulation of Fas expression was observed in CD34+ cells in cytokine treated cultures, compared to controls. This was accompanied by significant TRAIL and decreased caspase 3 mRNA expression, whereas the expression of Bcl-2 family members was low (Bcl-xl) or absent (Bcl-2, Bax). The expression of these apoptotic genes was also investigated in aplastic anemia patients. Apart from Fas mRNA expression in total marrow and/or CD34+ cells, TRAIL mRNA expression was found only in CD34+ cells in active disease while in total marrow cell compartment this remains a constant finding even in patients in remission. The above data are in agreement with previous studies proposing a major role for the extrinsic apoptosis pathway in the pathogenesis of aplastic anemia and additionally introduce TRAIL as a probable important molecule in the process.

Expression and inducibility of cytoprotective heat shock proteins in the bone marrow of patients with myelodysplastic syndrome: correlation with disease progression.

We determined the intracellular expression and inducibility of heat shock proteins (Hsps) 72, 73 and 27 in the bone marrow of patients with myelodysplastic syndrome (MDS) and controls. Hsps were overexpressed in MDS marrow especially in advanced disease, providing resistance to induction of apoptosis. These data suggest that Hsps could be implicated in the progression of MDS to acute myeloid leukemia.

Serum lipid profile and hepatic steatosis of adult beta-thalassaemia patients with chronic HCV infection.

OBJECTIVES: The aim of this study was to evaluate the serum lipid profile and to assess the prevalence of hepatic steatosis in adult beta-thalassaemic patients with chronic hepatitis C virus (HCV) infection. METHODS: Thirty-five adult HCV infected, multi-transfused, beta-thalassaemia patients (beta-HCV patients), 63 otherwise normal patients with chronic HCV infection (HCV patients) and 54 beta-thalassaemia patients without chronic viral hepatitis (beta patients) were studied. Total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides, viral markers and liver histology were evaluated. RESULTS: Serum total cholesterol, HDL-C and LDL-C were found at significantly lower levels in beta-HCV and beta patients than in HCV patients. Triglyceride levels were significantly lower in the HCV group compared with the beta group. Nine (25.7%) of the 35 beta-HCV patients had mild hepatic steatosis. Thirteen (23.6%) of 55 HCV patients presented mild and 4/55 (7.3%) moderate hepatic steatosis. None of the beta group presented steatosis. When we compared beta-HCV and HCV patients with steatosis, we found that beta-HCV patients had a lower degree of steatosis (11.1+/-7% vs 22.9+/-17.2%, P=0.021). Multivariate logistic regression analysis showed that the only independent predictor associated with hepatic steatosis in beta-HCV and HCV patients was genotype 3a (OR, 3.61; 95% CI, 1.22-10.71, P=0.021). CONCLUSIONS: Adult beta-thalassaemia patients, compared to other patients with chronic HCV infection, present lower cholesterol levels (total cholesterol, HDL, LDL) and similar frequency but a lower degree of hepatic steatosis. This difference in the degree of steatosis is most likely due to the higher prevalence of genotype 3a in the non-beta-thalassaemia group.

Impaired clonogenic growth of myelodysplastic bone marrow progenitors in vitro is irrelevant to their apoptotic state.

Excessive intramedullary apoptosis has been considered to account for the paradox of hypercellular marrow and refractory cytopenias in myelodysplastic syndrome (MDS). However, a causative relationship of apoptosis to the progenitor's defective clonogenic growth has not been sufficiently demonstrated. We investigated the degree of apoptosis and its contribution to ineffective hematopoiesis in MDS, by assessing the differential clonogenic capacity of purified "apoptotic" and "non-apoptotic" bone marrow progenitors in a short-term semisolid culture system. Although increased apoptosis was indeed detected in MDS bone marrow progenitors, there was no correlation between the existence of apoptosis and culture performance. Non-apoptotic as well as apoptotic CD34+ cells gave similar patterns of growth, both defective compared to normal. The ability of "apoptotic" CD34+ cells to proceed in colony formation as well as the abnormal growth of "non-apoptotic" progenitors are probably pointing towards the need to reconsider the role of apoptosis in the defective clonogenicity of MDS.

Plasma neutrophil gelatinase-associated lipocalin levels are markedly increased in patients with non-transfusion-dependent thalassemia: Lack of association with markers of erythropoiesis, iron metabolism and renal function.

BACKGROUND: Neutrophil Gelatinase-Associated Lipocalin (NGAL) (known as NGAL, Lipocalin 2, Siderocalin, Uterocalin, proteinase-3 and 24p3) is a mammalian small 25-kD peptide that belongs to the lipocalin superfamily, which consists of about 20 small lipoproteins. NGAL was initially discovered as an antibacterial factor of natural immunity and an acute-phase protein. NGAL is also an iron trafficking protein, a member of the non-transferrin-bound iron (NTBI) pool and an alternative to the transferrin-mediated iron-delivery pathway. Of note, NTBI, which is elevated in thalassemic patients, induces cellular toxicity. In this study we investigated the possible association of NGAL with parameters of erythropoiesis, iron metabolism and renal injury in patients with non-transfusion-dependent thalassemia (thalassemia intermedia or TI). PATIENTS AND METHODS: Thirty-five patients with TI, 13 men and 22 women, aged 8-63 years, were included in the study, while, 20 healthy individuals served as controls. Plasma NGAL levels were determined using an immunoenzymatic technique. Erythroid marrow activity was estimated by measuring soluble transferrin receptors (sTfR) levels with a turbidimetric technique. NTBI levels were determined using electrothermal atomic absorption spectrometry. Cystatin C, ß2-microglobulin and hs-CRP concentrations were measured by means of immunonephelometric techniques. RESULTS: The main results of the study showed: a) NGAL levels were significantly higher in patients with TI compared to controls (139.1 ± 86.1 vs 51.2 ± 11.8 µg/L, p<0.0001), without significant effect of splenectomy or hydroxyurea on NGAL levels. Only 4 patients had NGAL levels within the control group range, b) no correlation was found between NGAL levels and either the parameters of erythropoiesis Hb, Hb F, reticulocytes and sTfR (p>0.66, p>0.67, p>0.63 and p>0.81 respectively), or with those of iron metabolism ferritin and NTBI (p>0.90 and p>0.95 respectively). CONCLUSIONS: The increased NGAL levels reported for the first time in TI patients in this study are in agreement with the elevated expression of NGAL observed in TI mouse models. We postulate that the induction of NGAL in these patients may represent either a survival response, facilitating the survival of the less damaged thalassemic erythroid precursors, or a consequence of the abnormal iron regulation in TI.

Inappropriately low erythropoietin response for the degree of anemia in patients with noninsulin-dependent diabetes mellitus.

We investigated erythropoietin (Epo) response in a cohort of diabetic patients with various types of anemia to approach the pathogenesis of some cases of "unexplained" anemia encountered among diabetics. Serum Epo levels were determined totally in 747 evaluable subjects with normal renal and hepatic function, of whom 694 had anemia. Among anemic patients, 237 were diabetics, while among the 53 nonanemic persons, there were also 21 diabetics. Diabetic and nondiabetic subjects were uniformly balanced in relation to their demographic features and were categorized according to the etiology of their anemia. Hemoglobin (Hb) did not differ between diabetic and nondiabetic subjects in all the etiological groups and in the whole population. Diabetic patients had significantly lower serum Epo levels as compared to nondiabetics (36.5+/-61 vs 69.4+/-191 IU/ml, p<0.0001), and this was true for all etiologic groups of anemia with the exception of patients with myeloproliferative disorders and those with megaloblastic anemia. The natural logarithmic (ln)-EpoxHb component was used as an index of response to anemia and was found to be significantly decreased in almost all subgroups of diabetic patients. Serum Epo levels were also negatively correlated with the percentage of glycosylated Hb, HbA1(C) (r=-0.446), and the correlation was stronger with the ln of serum Epo (r=-0.638, p<0.001). Inappropriately low serum Epo level is a uniform feature in patients with type II diabetes mellitus and may represent a constitutive blunted response to anemia or an altered metabolic rate of Epo, probably as a result of abnormal glycosylation of the cytokine.

Defective tumor necrosis factor alpha-induced maturation of monocyte-derived dendritic cells in patients with myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are clonal stem cell disorders, characterized by ineffective and dysplastic hematopoiesis. MDS patients have a defective immune response manifested by increased susceptibility to bacterial infections, autoimmune phenomena, and high incidence of lymphoid malignancies. Presently, we investigated the phenotype and function of monocyte-derived dendritic cells (MoDC) in 23 MDS patients and 15 controls at different stages of differentiation using the maturation stimuli tumor necrosis factor-alpha (TNF-alpha) and LPS. Monocytes from MDS patients showed low potential to differentiate into dendritic cells (DC), as determined by low cell yield and CD1a expression. MDS-MoDCs exhibited low expression of mannose receptor and reduced endocytic capacity. MDS-MoDCs showed a diminished response to TNF-alpha with low CD83, CD80, and CD54 expression and allostimulatory capacity. In patients with 5q syndrome, monocytes and MoDCs were positive for the 5q deletion, suggesting their origin from the malignant clone. Our data indicate that MoDCs in MDS display quantitative and functional abnormalities that may contribute to the defective immune response of these patients.

Impaired generation of bone marrow CD34-derived dendritic cells with low peripheral blood subsets in patients with myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a stem cell disorder characterized by ineffective haematopoiesis and blood cytopenias. The present study investigated the potential of bone marrow CD34(+) progenitors in MDS patients to proliferate and differentiate into dendritic cells (DCs) in a cytokine-supplemented liquid culture system and analysed the status of blood DC subsets in these patients. CD34(+) progenitors had low potential to generate DCs in vitro, as the number of DCs obtained from one CD34(+) cell was significantly lower compared with controls (median value 0.2 vs. 4, P = 0.003). In patients, the survival and proliferation of CD34(+) cells in culture was not correlated to the degree of apoptosis. Phenotypically and functionally CD34(+)-derived DCs were similar in MDS patients and normal subjects. The percentage of both circulating DC subsets in patients was extremely diminished compared with controls (myeloid DC: 0.10 +/- 0.10% vs. 0.35 +/- 0.13%, P < 0.001; plasmacytoid DC: 0.11 +/- 0.10% vs. 0.37 +/- 0.14%, P < 0.001). In cases with the 5q deletion both CD34-derived DCs and blood DCs harboured the cytogenetic abnormality. Our results indicate that, in MDS, the production of DCs is affected by the neoplastic process resulting in ineffective 'dendritopoiesis' with low blood DC precursor numbers. This quantitative DC defect probably contributes to the poor immune response against infectious agents and to the escape of the malignant clone from immune recognition with disease progression towards acute leukaemia.

Increased serum CA-15.3 levels in patients with megaloblastic anemia due to vitamin B12 deficiency.

OBJECTIVES: To estimate the usefulness of serum tumor markers' monitoring, as predictors of gastric cancer in patients with pernicious anemia. PATIENTS AND METHODS: We investigated serum levels of carcinoembryonic antigen (CEA), alpha-fetal protein, cancer antigen (CA)-19.9, CA-125 and CA-15.3 in 50 patients with pernicious anemia and in 24 healthy controls, matched for age and sex. In 38 patients, the evaluation was repeated 1-6 months after the correction of cobalamin deficiency. RESULTS: All patients and controls had normal serum CEA and alpha-FP, and the levels of these markers as well as those of CA-125 and CA-19.9 did not differ between the two groups. All 50 patients, but only 2 controls exhibited increased serum CA-15.3, and the difference between the two groups was very significant (129.4 +/- 84.9 vs. 19.8 +/- 7.3 IU/ml, p < 0.001), while no difference between males and females was found. A thorough clinical examination of all patients, and mammographic study in 18 females did not reveal any finding suspicious of breast cancer. CA-15.3 levels were positively correlated with serum lactate dehydrogenase, and negatively with B(12) and hemoglobin, but they were substantially decreased after the correction of anemia, in all 38 patients tested, and in 33 of them they were restored to normal. After a median follow-up of 34 months, one patient developed a colon cancer, but none showed any sign suspicious of breast cancer. CONCLUSIONS: Serum CA-15.3 shows an aberrant increase in untreated patients with pernicious anemia, which is reversed after the correction of the anemia. The possible origin seems unrelated to mammary tissue, and may be released by the apoptosing bone marrow megaloblastic erythroblasts.

Clinical relevance of balance between type 1 and type 2 immune responses of lymphocyte subpopulations in aplastic anaemia patients.

Immune dysfunction, which leads to the suppression of haemopoiesis by cytokines that are secreted by activated T lymphocytes, is considered to play a key role in the pathogenesis of acquired aplastic anaemia (AAA). We investigated the intracytoplasmic expression of type-1 [interferon gamma (IFN-gamma), interleukin (IL)-2] and type-2 (IL-4, IL-10) cytokines in CD4+ and CD8+ T cells before and after in vitro activation in 16 patients with AAA and 17 normal controls. Untreated or refractory patients had a significantly higher proportion of unstimulated CD4+ and CD8+ T cells that produced IFN-gamma and IL-2 whereas the IL-4 and IL-10 producing T cells did not differ from that of controls, resulting in a shift of IFN-gamma/IL-4 ratio towards a type-1 response. Patients in remission had also increased proportion of IFN-gamma-producing unstimulated CD4+ and CD8+ cells, with a parallel rise of IL-4- and IL-10-producing cells and normal IFN-gamma/IL-4 ratio. These data indicate that, in newly diagnosed and refractory patients with AAA, CD4+ cells are polarized towards a type-1 response that in turn leads to activation of cytotoxic CD8+ cells and finally to haemopoietic stem cell destruction. The type-1 response persists in patients in remission although this effect is compensated by the increase of IL-4 and IL-10 production.