Proteome changes in the plasma of myelodysplastic syndrome patients with refractory anemia with excess blasts subtype 2.

The goal of this study was to explore the plasma proteome of myelodysplastic syndrome (MDS) patients with refractory anemia with excess blasts subtype 2 (RAEB-2) in comparison to healthy controls. 20 plasma samples were separated with 2D electrophoresis and statistically processed with Progenesis SameSpots software. 47 significantly differing (P < 0.05) spots were observed, and 27 different proteins were identified by nano-LC-MS/MS. Mass spectrometry-based relative label-free quantification showed a 2-fold increase of the leucine-rich alpha-2-glycoprotein (LRAG) peptide levels in the RAEB-2 group. Changes in the fragments of the inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) protein were observed. Western blot analysis showed no differences in albumin and ITIH4 levels, while increased expression was observed for LRAG in the RAEB-2 group. Quantification using ELISA showed decreased plasma level of alpha-2-HS glycoprotein in the RAEB-2 group. In conclusion, this is the first time that alpha-2-HS glycoprotein and LRAG were proposed as new biomarkers of RAEB-2 and advanced MDS, respectively. Alpha-2-HS glycoprotein, a protein involved in the bone marrow development and previously proposed as a MDS biomarker candidate, was significantly decreased in RAEB-2. Increased expression and changes in modification(s) were observed for LRAG, a protein involved in granulocytic and neutrophil differentiation, and angiogenesis.

Clinico-haematological characteristics in Pakistani patients of primary myelodysplastic syndrome according to World Health Organization classification.

OBJECTIVE: To assess the applicability of WHO classification on a cohort of Pakistani myelodysplastic syndrome (MDS) patients, and determine their epidemiological and clinico-pathological features. STUDY DESIGN: Case series. PLACE AND DURATION OF STUDY: Haematology Department, Shaikh Zayed Hospital, Lahore, from April 2004 to March 2006. METHODOLOGY: Forty six patients of primary MDS diagnosed by World Health Organization (WHO) criteria were included in the study by nonprobability purposive sampling. The cohort was classified accordingly and the epidemiological, clinical and haematological parametres were assessed. Descriptive statistics were used to describe the data. RESULTS: Forty six patients (28 males and 18 females) of primary MDS were included in the study. The mean age was 46.21 years. According to the WHO classification, 12 cases of refractory anaemia, 24 cases of refractory cytopenia with multi lineage dysplasia, 1 case of refractory cytopenia with multi lineage dysplasia and ring sideroblasts, 3 cases of MDS unclassified and 3 cases each of refractory anaemia with excess of blasts I and II were diagnosed. Symptomatic anaemia was seen in 37 cases and pancytopenia was documented in 33 cases. Dyserythropoiesis affected 41 cases. Grade III reticulosis was seen in 7 cases. ALIP was present in 13 cases. CONCLUSION: MDS presented at a young age. Refractory cytopenia with multi lineage dysplasia was the dominant disease category. Further studies are suggested for identifying the cytogenetic abnormalities and del 5q- category.

[Serum proteomics in patients with RAEB myelodysplastic syndromes].

OBJECTIVE: To screen the molecular markers for refractory anemia with excess blasts in transformation (RAEB) in myelodysplastic syndromes (MDS) by serum proteome profiling. METHODS: The serum protein were isolated from patients with RAEB, acute myeloid leukemia or normal subjects by 2-dimensional electrophoresis (2-DE), and the electrophoresis gels were obtained to identify the differentially reacting protein spots. The replica gels of the differentially reacting proteins were analyzed to locate the matching protein spots, which were identified by peptide mass fingerprint based on matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF-MS) and database searching. RESULTS: Seven differentially expressed proteins in RAEB were found by 2-DE. Of the 7 proteins, 4 were identified by MALDI-TOF-MS to have significantly differential expression in RAEB, including dipeptidyl peptidase (DPP/CD26), polymerase (DNA directed) kappa, PRO2044 and an albumin-like protein. CONCLUSION: 2-DE-based serum proteome profiling helps identify serum proteomic biomarkers related to MDS. DDP/CD26 has increased expression in the serum in RAEB subtype MDS, suggesting its possible role in advanced MDS.

Clinicopathological characteristics of erythroblast-rich RAEB and AML M6a in children.

The distinction between RAEB, RAEB-T and AML M6a is difficult when erythroblasts in the bone marrow (BM) exceed 50%. We analyzed 19 children (2 RAEB, 13 RAEB-T and 4 AML M6a) enrolled in a prospective pathological central review in Japan and divided them into two groups according to the myeloblasts percentage among non-erythroid cells in BM: group A (n = 8), 5-19% myeloblasts; group B (n = 11), 20% or more myeloblasts. Their characteristics were very similar except for the number of myeloblasts. The median WBC was in the range of 1.0-5.0 x 10(9) L(-1), the median Hb was around 7.5 g/dL, the median MCV was greater than 90 fL and both group had Auer rods at 60-65%. Severe multilineage dysplasia was observed in most of the patients in two groups. Six with group A and seven with group B treated with AML type chemotherapy achieved complete remission. Five with group A and seven with group B undergoing SCT are alive at a median of 3 years after diagnosis. Erythroblast-rich RAEB and AML M6a in children have similar characteristics and may belong to a single disease entity.

Detection of CD55- and CD59-deficient granulocytic populations in patients with myelodysplastic syndrome.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by absence of CD55 and CD59 from the surface of affected cells. PNH has been associated with myelodysplastic syndromes (MDS). The aim of our study was to estimate the prevalence of the PNH clone in MDS patients by detecting CD55 and CD59 deficiency. We studied 90 MDS patients: 19 patients with RA, 15 with refractory anemia with ringed sideroblasts (RARS), 18 with refractory anemia with excess of blasts (RAEB), 17 with refractory anemia with excess of blasts in transformation (RAEB-t), and 21 with chronic myelomonocytic leukemia (CMML). Twenty healthy individuals were also studied as the control group. We studied the PNH clone on granulocytes of these patients with the aid of flow cytometry. CD55- and CD59-deficient granulocytic populations were detected in 15.5% of MDS patients compared to 2.8% of normal individuals. Among the subgroups of the study, significant difference was present in three cases: (1) between CMML and control, (2) between CMML and RA, and (3) between CMML and RARS. These data indicate a possible association between PNH phenotype and MDS. MDS patients of worse prognosis (CMML) express more strongly the PNH clone compared to those of better prognosis (RA and RARS). Perhaps, the examination of MDS patients for the PNH clone by flow cytometry could provide us with a valuable prognostic tool.

[Angiogenesis in bone marrow of myelodysplastic syndrome patients]. / Angiogeneza w szpiku kostnym u chorych na zespoly mielodysplastyczne.

INTRODUCTION: Angiogenesis is an element of physiological and some pathological processes. Recently various investigators have reported that angiogenesis is associated not only with solid tumors but also with hematological malignancies. OBJECTIVES: The aim of this study was to assess angiogenesis in patients with myelodysplastic syndromes. PATIENTS AND METHODS: We have measured bone marrow microvessel density (MVD) in 43 myelodysplastic syndrome (MDS) patients and in 10 subjects of group control--10 lymphoma patients (their bone marrow was free of disease). It was estimated by immunohistochemical method using anti-CD31 and anti-CD34 monoclonal antibodies. In 14 MDS patients and 6 healthy donors we also measured serum vascular endothelial growth factor (VEGF) by immunoenzymatic method. RESULTS: Higher MVD numbers were found in MDS patients when compared to control bone marrows. The highest number of MVD was in RAEBt and CMML MDS subtypes according to FAB. There was no correlation between MVD and biological features of MDS patients except for the age (negative correlation). Bone marrow microvessel density does not influenced significantly on overall survival. The serum VEGF concentration in MDS patients was higher than in healthy donors, but the difference was not statistically significant. The VEGF levels did not correlate with MVD. CONCLUSIONS: We concluded that angogenesis is enhanced in bone marrow MDS patients though its mechamism is not yet fully understood.

[Case report: RAEB in a patient with rheumatoid arthritis treated with methotrexate and infliximab]. / Anemia refrattaria con eccesso di blasti (AREB) in paziente con artrite reumatoide in trattamento con methotrexate ed infliximab: descrizione di un caso clinico.

Anti TNF-alpha drugs seem to be the new frontier of Rheumatoid Arthritis (RA) therapy. The association infliximab methotrexate has been approved for the treatment of RA not responding to the classic therapy, but the short clinical experience in using antiTNF-alpha molecules brings to segnalation of new risks or adverse events. We describe a case of a patient, treated for many years with classic RA therapy, which developed a refractory anemia after treatment with association infliximab-methotrexate.

Activity of sirolimus in patients with myelodysplastic syndrome--results of a pilot study.

The pathophysiology of the myelodysplastic syndromes (MDS) involves disturbed regulation of angiogenesis, apoptosis, proliferation and differentiation as well as immune surveillance. Increasing data suggest that sirolimus might affect these pathways positively, thus being of possible therapeutic benefit in patients with this disease. Nineteen patients (n = 19) with a median age of 72 years (range 54-80 years) diagnosed with MDS received sirolimus orally with a target blood concentration of 3-12 ng/ml. Sirolimus was administered for a median of 3.7 months (range 0.3-11 months). Three patients [1 x refractory anaemia with excess blasts (RAEB)-2, 1 x RAEB-1, 1 x refractory cytopenia with multilineage dysplasia] showed either a major (1 x platelet, 1 x neutrophil) or a minor (1 x erythroid, 2 x platelet) haematological response according to International Working Group criteria. Major side-effects were hyperlipidaemia (n = 4), stomatitis (n = 3), thrombocytopenia (n = 2) and urinary tract infection (n = 1). These data suggest that sirolimus has activity in a subset of patients with more advanced MDS.

Flow cytometric detection of accelerated telomere shortening in myelodysplastic syndromes: correlations with aetiological and clinical-biological findings.

Using quantitative fluorescence in situ hybridisation and flow cytometry (flow-FISH), we investigated the biological and clinical relevance of telomere length in 55 patients affected by myelodysplastic syndromes (MDS) compared with 55 sex- and age-matched controls. We found that telomere fluorescence in MDS granulocytes, and CD34+ cells did not decline with age as in normal controls and that MDS granulocytes and CD34+ cells had significantly shorter telomeres than healthy controls. A significant higher incidence of cases with intermediate-unfavourable cytogenetics and International Prognostic Scoring System (IPSS) int-2/high-risk group was observed among patients with lower telomere fluorescence. We also found that apoptosis in CD34+ cells was significantly higher in IPSS int-1 low-risk patients when compared with IPSS int-2 high-risk cases and healthy controls and that CD34+ cell telomere fluorescence directly correlated with CD34+ cell apoptosis. Reduced telomere fluorescence was associated with a history of occupational exposure to toxic agents and with worse survival in univariate and multivariate analyses. Our results suggest that flow-cytometry assessment of telomere dynamics may represent a valuable tool in the biological and clinical-prognostic characterisation of MDS disorders.

Relative increase in leukemia-specific DNA in peripheral blood plasma from patients with acute myeloid leukemia and myelodysplasia.

Using loss of heterozygosity (LOH) and X-chromosome inactivation, we compared peripheral blood (PB) plasma with bone marrow (BM) cells in detecting genomic abnormalities in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We detected LOH in the PB plasma of all 45 patients who had cytogenetically documented chromosomal abnormalities (5q-, 7-, +8, 17-, or 20-). BM cells from the same patients showed LOH in 89% of patients with MDS and 70% of patients with AML. Posttherapy samples from 16 of these patients demonstrated complete concordance between LOH and cytogenetics in detecting residual disease in 15 samples. Of the 16 samples, 4 showed LOH in plasma with normal BM morphology. Using X-chromosome inactivation, clonality was detectable in 19 (73%) of 26 BM samples, whereas all PB plasma samples showed clonality. These data support the conclusion that PB plasma is enriched by tumor-specific DNA and can replace BM cells for studying genomic abnormalities.

[Expression of Fas, FasL and Bcl-2 and apoptosis of bone marrow CD34+ cells in patients with myelodysplastic syndrome].

In order to observe the expression of Fas, FasL and Bcl-2 and apoptosis of bone marrow CD34(+) cells in patients with myelodysplastic syndrome (MDS), and to explore the relation between the expression of these antigens and apoptosis, the expression of Fas, FasL and Bcl-2 and apoptosis of bone marrow CD34(+) cell were evaluated by flow cytometry in 26 patients with MDS including 9 cases of refactory anemia (RA), 1 case of RA with ringed sideroblasts (RAS), 9 cases of RA with excess blasts (RAEB) and 7 cases of RAEB in transformation (RAEB-t), 10 patients with acute myeloid leukemia (AML) and 6 control patients with normal bone marrow. The results showed that the expression of Fas and FasL of CD34(+) cells significantly increased in all types of MDS patients compared with control group (P < 0.01). The expression of Bcl-2 on CD34(+) cells in RAEB and RAEB-t patients was much higher as compared with that in control group (P < 0.01), but there was no significant difference between RA/RAS patients and control group (P > 0.05). The expression rates of Fas on CD34(+) cells were almost identical in all kinds of MDS, but there was significant difference on the expression of Bcl-2 (RA/RAS < RAEB < RAEB-t). Apoptosis of CD34(+) cells significantly increased in RA/RAS and RAEB patients compared with control group (P < 0.01), but there was no difference between RAEB-t and control group. Moreover, apoptosis of CD34(+) cells in control much higher than that in AML group (P < 0.01). There was no correlation between the expression of Fas or FasL and apoptosis on CD34(+) cell of MDS patients. Nevertheless, there was a negative correlation between the expression of Bcl-2 and apoptosis. It is concluded that apoptosis of CD34(+) cells was affected by a lot of factors in MDS, in which Bcl-2 is an important factor of depressing apoptosis. During the progress from MDS to AML, apoptosis changes from overgoing to deficiency in CD34(+) cell.

Cyclosporin A therapy for patients with myelodysplastic syndrome: multicenter pilot studies in Japan.

We examined the efficacy of cyclosporin A (CsA) in 50 patients with myelodysplastic syndrome (MDS) consisting from 47 of RA, 1 of RARS, and 2 of RAEB. These patients showed various marrow cell types including hypo-, normo-, and hypercellularity. Patients belonged to the following International Prognostic Scoring System (IPSS) risk groups: 4 of low, 41 of intermediate-1, and 5 of intermediate-2. The median CsA dose was 4.58mg/kg, and treatment responses were classified according to the International Working Group (IWG) criteria. Hematological improvement (HI) was observed in 30 (60%) patients, and all of them were belonged to RA. In the patients with RARS or RAEB, no efficacy was observed. Four (8%) of the responders achieved partial remission (PR) with granulocytes > or = 1500microl(-1), Hb>11g/dl and platelets > or = 100,000microl(-1). Higher response rate (53%) was shown in erythroid lineage (HI-E) compared to platelet (HI-P, 36%) or neutrophil lineage (HI-N, 35%). When we analyzed the correlation between the response to CsA therapy and the karyotype or HLA type, there were significantly more responders with good karyotype or DRB1*1501 than with intermediate/poor karyotypes or with other HLA types. These results indicate the usefulness of CsA therapy for MDS patients with any marrow cellularity, especially for erythroid lineage and patients with good karyotype or DRB1*1501.

Polyclonal hematopoiesis maintained in patients with bone marrow failure harboring a minor population of paroxysmal nocturnal hemoglobinuria-type cells.

Although a minor population of paroxysmal nocturnal hemoglobinuria (PNH)-type blood cells is often detected in patients with aplastic anemia (AA) and refractory anemia (RA), the significance of such cells in the pathophysiology of bone marrow (BM) failure remains obscure. We therefore examined clonality in peripheral blood granulocytes from 118 female patients with AA or myelodysplastic syndrome using the X chromosome inactivation pattern. Clonality, defined as a clonal population accounting for 35% or more of total granulocytes, was confirmed in 22 of 68 (32.4%) AA patients, in 13 of 44 (29.5%) RA patients, in all 4 RA with excess blasts (RAEB) patients, and in 4 patients with PNH. When the frequency of patients with granulocyte clonality was compared with respect to the presence of increased PNH-type cells, the frequency was significantly lower in AA patients with (PNH+; 21.2%) than without (PNH-; 42.9%) increased numbers of PNH-type cells (P =.049). Clonality was absent in granulocytes from the 15 PNH+ RA patients but present in 13 of 29 (44.8%) PNH- RA patients (P =.0013). The absence of clonality in AA and RA patients before treatment was strongly associated with positive response to immunosuppressive therapy (without clonality, 74.4%; with clonality, 33.3%; P =.0031) in all patients as well as in PNH+ patients (without clonality, 96.2%; with clonality, 66.6%, P =.026). These results suggest that AA and RA with a minor population of PNH-type cells are benign types of BM failure with immune pathophysiology that have little relationship to clonal disorders such as RAEB or acute myeloid leukemia.

Empirical examination of the neutrophil criterion (>1500 microl(-1)) currently needed to declare CR in AML.

Currently, NCI guidelines require that the neutrophil count exceed 1500 before complete remission (CR) may be declared in acute myeloid leukemia (AML). We tested the empirical validity of this formulation by comparing event-free survival (EFS) in CR in (a). 305 patients who met the NCI criteria for CR and (b). 36 patients who met all the criteria for CR except that the neutrophil count at what we considered CR was 1000-1499 (lower neutrophil group) at which time they began post-remission therapy. EFS was statistically identical in both groups. We extended these findings to 752 patients, who met the NCI criteria for CR except that some may have had circulating blasts at "CR"; 82 of the 752 were in the lower neutrophil group. These data suggest that the minimum needed to declare CR in AML be changed from 1500 to 1000.