Athanasios Galanopoulos

Effect of lenalidomide therapy on hematopoiesis of patients with myelodysplastic syndrome associated with chromosome 5q deletion

Background Lenalidomide improves erythropoiesis in patients with low/intermediate-1 risk myelodysplastic syndrome and interstitial deletion of the long arm of chromosome 5 [del(5q)]. The aim of this study was to explore the effect of lenalidomide treatment on the reserves and functional characteristics of bone marrow hematopoietic progenitor/precursor cells, bone marrow stromal cells and peripheral blood lymphocytes in patients with low/intermediate-1 risk myelodysplastic syndrome with del(5q). Design and Methods We evaluated the number and clonogenic potential of bone marrow erythroid/myeloid/megakaryocytic progenitor cells using clonogenic assays, the apoptotic characteristics and adhesion molecule expression of CD34+ cells by flow cytometry, the hematopoiesis-supporting capacity of bone marrow stromal cells using long-term bone marrow cultures and the number and activation status of peripheral blood lymphocytes in ten patients with low/intermediate-1 risk myelodysplastic syndrome with del(5q) receiving lenalidomide. Results Compared to baseline, lenalidomide treatment significantly decreased the proportion of bone marrow CD34+ cells, increased the proportion of CD36+/GlycoA+ and CD36−/GlycoA+ erythroid cells and the percentage of apoptotic cells within these cell compartments. Treatment significantly improved the clonogenic potential of bone marrow erythroid, myeloid, megakaryocytic colony-forming cells and increased the proportion of CD34+ cells expressing the adhesion molecules CD11a, CD49d, CD54, CXCR4 and the SLAM antigen CD48. The hematopoiesis-supporting capacity of bone marrow stroma improved significantly following treatment, as demonstrated by the number of colony-forming cells and the level of stromal-derived factor-1α and intercellular adhesion molecule-1 in long-term bone marrow culture supernatants. Lenalidomide treatment also increased the proportion of activated peripheral blood T lymphocytes. Conclusions The beneficial effect of lenalidomide in patients with lower risk myelodysplastic syndrome with del(5q) is associated with significant increases in the proportion of bone marrow erythroid precursor cells and in the frequency of clonogenic progenitor cells, a substantial improvement in the hematopoiesis-supporting potential of bone marrow stroma and significant alterations in the adhesion profile of bone marrow CD34+ cells.

Evaluation of the clinical relevance of the expression and function of P-glycoprotein, multidrug resistance protein and lung resistance protein in patients with primary acute myelogenous leukemia.

The multidrug resistance (MDR) transporter-proteins P-glycoprotein (Pgp), multidrug resistance protein (MRP) and lung resistance protein (LRP) have been associated with treatment failure. The aim of this study was to investigate prospectively the clinical significance of expression and function of the MDR proteins, considering other prognostic factors, such as age, immunophenotype, and cytogenetics. Mononuclear cells of peripheral blood or bone marrow from 61 patients with de novo acute myelogenous leukemia (AML) were analyzed. The monoclonal antibodies JSB1, MRPm6 and LRP56 were used for expression studies. Accumulation and retention studies were performed using the substrates Daunorubicin, Calcein-AM, Rhodamine-123 and DiOC(2) in the presence or absence of the modifiers Verapamil, Genistein, Probenecid, BIBW22S and PSC833. Induction treatment consisted of a 3+7 combination of Ida/Ara-C for patients < or = 60 years of age and a 3+5 Ida/VP-16 combination per OS for patients >60. MDR function was expressed as the ratio of mean fluorescence intensity substrate in the presence of modifier over the substrate alone (resistance index, RI). Patients with advanced age, low CD15 expression and high RI for accumulation of DiOC(2) in the presence of BIBW22S had significantly lower complete remission (CR) rates. No factor was prognostic for event-free survival analysis, which was limited to remitters only. Overall survival was shorter in patients with advanced age, poor prognosis cytogenetics, high CD7 expression, and high RI for Daunorubicin efflux modulated by Verapamil. These results suggest that MDR transporter-proteins have a limited role in the treatment failure of patients treated with Idarubicin-based regimens.

Expression of CD25 antigen on CD34+ cells is an independent predictor of outcome in late-stage MDS patients treated with azacitidine.

Expression of CD25 antigen on CD34+ cells is an independent predictor of outcome in late-stage MDS patients treated with azacitidine

Treatment of patients with myelodysplastic syndrome with amifostine

The efficacy and toxicity of amifostine (300 mg/m(2) three times a week for three consecutive weeks for a maximum of six courses) was evaluated in 12 patients with primary myelodysplastic syndromes. Dose escalation up to 400 mg/m(2) was allowed to patients who failed to respond. Hemoglobin concentration was increased > or = 1.5 g/dl in two (18%) of the 11 anemic patients. These two patients obtained transfusion independence for 20 weeks. Reticulocyte counts and ANC increased > or = 50% of baseline in four (44%) of the nine patients with reticulocytopenia and in three (25%) of the 12 neutropenic patients. Platelet count increased in three (50%) of the six patients with thrombocytopenia. Progenitor growth of CFU-GMs and BFU-Es improved in 8/12 patients. No major side effects were observed. In conclusion amifostine is well tolerated and can promote the growth of primitive hematopoietic progenitors and ameliorate the cytopenias in MDS patients.

High frequency of human leukocyte antigen class II DRB1*1602 haplotype in Greek patients with myelodysplastic syndrome and of DRB1*1501 in the low-risk subgroup

Myelodysplastic syndromes (MDS) comprise a heterogenous group of clonal hematopoietic disorders in which the immune-mediated pathogenetic mechanisms are under investigation. Overrepresentation of human leukocyte antigen (HLA)-DR2 and its serologic split HLA-DR15 has been associated with low-risk MDS in certain ethnic groups and has been proposed as a predictive factor for a favorable response to immunomodulatory treatment. Because the HLA-DRB1*15 haplotype does not predominate in the Greek population, we investigated the frequency of HLA-DRB1 alleles among 114 patients of Greek origin suffering from various types of MDS: 36 refractory anemia (RA), 24 refractory anemia with ringed sideroblasts (RARS), 19 refractory anemia with excess of blasts (RAEB), 14 refractory anemia with excess of blasts in transformation (RAEB-t), 14 chronic myelomonocytic leukemia, and 7 hypoplastic MDS patients. HLA-DRB1 molecular typing was performed with polymerase chain reaction-sequence specific oligonucleotides and results were compared with that from a previously reported control Greek population. HLA-DRB1*1602 was the only allele that was significantly overrepresented in Greek MDS patients as a whole, whereas HLA-DRB1*1501 allele frequency was significantly higher in Greek patients with low-risk myelodysplasia. Our results suggest the possible value of HLA-DR15 and HLA-DR16 as determinants for immunomodulatory interventions, at least for Greek patients with low-risk MDS.

The Stat3/5 signaling biosignature in hematopoietic stem/progenitor cells predicts response and outcome in myelodysplastic syndrome patients treated with azacitidine

Purpose: Azacitidine is the mainstay of high-risk myelodysplastic syndromes (MDS) therapy, but molecular predictors of response and the mechanisms of resistance to azacitidine remain largely unidentified. Deregulation of signaling via Stat3 and Stat5 in acute myeloid leukemia (AML) is associated with aggressive disease. Numerous genes involved in cell signaling are aberrantly methylated in MDS, yet the alterations and the effect of azacitidine treatment on Stat3/5 signaling in high-risk MDS have not been explored. Experimental Design: We assessed longitudinally constitutive and ligand-induced phospho-Stat3/5 signaling responses by multiparametric flow cytometry in 74 patients with MDS and low blast count AML undergoing azacitidine therapy. Pretreatment Stat3/5 signaling profiles in CD34+ cells were grouped by unsupervised clustering. The differentiation stage and the molecular properties of the CD34+ G-CSF–inducible Stat3/5 double-positive subpopulation were performed by flow cytometry and quantitative real-time PCR in isolated MDS progenitors. Results: The pretreatment Stat3/5 signaling profiles in CD34+ cells correlated strongly with response and cytogenetics and independently predicted event-free survival. We further identified a CD34+ G-CSF–inducible Stat3/5 double-positive subpopulation (DP subset) whose pretreatment levels were inversely associated with treatment response and cytogenetics. The kinetics of the DP subset followed the response to azacitidine and the disease course, whereas its molecular characteristics and cellular hierarchy were consistent with a leukemia propagating cell phenotype. Conclusions: Our findings provide a novel link among Stat3/5 signaling and MDS pathobiology and suggest that the Stat3/5 signaling biosignature may serve as both a response biomarker and treatment target. Clin Cancer Res; 22(8); 1958–68. ©2015 AACR.

Macrophage Inflammatory Protein‐1 alpha (MIP‐1α) is over‐expressed in a cohort of patients with myelodysplastic syndromes

To the Editor: Macrophage inflammatory protein-1 alpha (MIP1a) is a low molecular weight chemokine that belongs to the RANTES family and is primarily associated with cell adhesion and migration, being chemotactic for monocytes, T lymphocytes, natural killer cells, immature dendritic cells and granulocytes (1). Due to its activating effect on premature osteoclasts MIP-1a correlated with lytic bone disease in multiple myeloma (2). The MIP-1a has also been shown to inhibit the proliferation of primitive progenitors and has been implicated in the pathogenesis of acute and chronic myeloid leukaemia as well as adult T-cell leukaemia (3–6). Maciejewski et al. have reported very significant increases in the levels of MIP-1a RNA transcripts in the bone marrow nucleated cells from patients with aplastic anaemia and myelodysplastic syndromes (MDS) compared to normal individuals, suggesting that the expression of MIP-1a in bone marrow may reflect dysregulated cytokine production and activation of the immune system that may possibly contribute to disease progression (7). However, there is no information for the serum levels of MIP-1a in patients with MDS. The aim of this study was the evaluation of MIP-1a serum levels in MDS patients and the correlation with clinical and laboratory data. Therefore, MIP-1a was measured in the serum of 62 patients with MDS and 20 normal, ageand gender-matched individuals (control group) using an ELISA method (Quantikine ELISA, R & D Systems, Minneapolis, MN, USA). After veinpuncture, serum was separated within 4 h and stored at )70 C until the day of measurement. The MDS patients had a median age of 76.5 yr (range: 45– 91 yr). Thirty-three patients were males and 29 were females. The type of MDS according to recent WHO classification was as follows: 25 patients had refractory anaemia (RA), 20 patients had refractory cytopenia with multilineage dysplasia (RCMD), five patients had refractory anaemia with ringed sideroblasts (RARS), one patient had RCMD-RS, seven patients had refractory anaemia with excess of blasts type-I (RAEB-I), and four patients had RAEB-II. No patient had any kind of infection at the time of measurement and their medical records were reviewed to exclude any chronic disease or osteoporosis. Differences between patients and controls were evaluated using the Wilcoxon rank sum test. Associations between serum levels of MIP-1a and clinical or laboratory data were examined by the Kruskal–Wallis test, while the Spearman Rank correlation test was employed to examine relationships between various parameters. Differences between patients and controls were evaluated using the Wilcoxon rank sum test. All P values were two sided and confidence intervals refer to 95% boundaries. The MIP-1a serum levels presented no significant difference between patients and controls (median values ± SD: 29.21 ± 9.48 pg/mL vs. 25.10 ± 4.14 pg/mL, for patients and controls, respectively, P > 0.1). However, four MDS patients (6.4%), two with RA and two with RAEB-II, had at least a twofold higher concentration of MIP-1a in their serum compared to the mean value of control group. We found no correlation between MIP-1a levels and clinical or laboratory data of the patients, such as haemoglobin, neutrophil and platelet count, cytogenetic abnormalities, bone marrow blast count, lactate dehydrogenase serum levels, age, gender, number of transfusion requirements of red blood cells units, presence of organomegaly, IPSS or disease progression. Therapeutic decisions in patients with MDS are very complex due to the heterogeneity of this group of haematological disorders. The dilemma that confronts the management of MDS is illustrated by the presence of only one agent, which has been recently approved by the USA Food and Drug Administration with an indication for high-risk MDS, 5-azacytidine. Supportive care, low-intensity Eur J Haematol 2005: 75: 85–86 All rights reserved Copyright Blackwell Munksgaard 2005

Evidence for regulation of oxidative stress by latent membrane protein 1 oncoprotein in patients with low-grade leukemic B cell lymphoma with latent Epstein - Barr virus infection

Abstract The role of latent Epstein–Barr virus (EBV) infection in the pathogenesis of low-grade B cell non-Hodgkin lymphoma (B-NHL) has not been studied. We therefore investigated the incidence of latent EBV infection in a group of patients with leukemic low-grade B-NHL, as well as the incidence of viral latent membrane protein 1 (LMP1) oncoprotein expression in the same patient group. Furthermore, in an attempt to elucidate the role of this viral oncoprotein in non-EBV-related lymphomas, we correlated the expression of LMP1 with the level of oxidative stress, a parameter related to apoptosis. In the present study we detected lower levels of oxidative stress in the sera of LMP1-positive patients. This possibly implies an anti-apoptotic role of this viral oncoprotein in low-grade B cell lymphomas. However, LMP1 expression status did not affect expression of the major anti-apoptotic gene BCL-2.

Leukemic transformation in patients with myelodysplastic syndromes after treatment with granulocyte colony-stimulating factor.

Myelodysplastic syndromes (MDS) constitute bone marrow malignancies occurring mainly in older individuals and associated with acute myeloid leukemia (AML) transformation in 30 – 40% of cases [1]. In order to assess long-term survival and the AML transformation rate, in 1997 the International MDS Risk Analysis Workshop developed the International Prognostic Scoring System (IPSS), which divides patients with MDS into four risk groups: low, intermediate-1, intermediate-2 and high. Determining the prognosis of MDS is crucial in the eff ort toward defi ning the best treatment for each group [2]. When patients with MDS develop cytopenias and/or blood product transfusion dependency, treatment with growth factors may be initiated according to most treatment guidelines. Th ese include epoetin or darbepoetin and colony-stimulating factors, either alone or in combination, acting in synergy. A number of trials have been published, with erythroid response rates of around 40% in patients with lower-risk disease using International Working Group (IWG) criteria for response [3 – 5]. Some studies have shown a clear synergistic eff ect between the two drugs [6 – 8]. Th e negative impact of treatment with growth factors is widely discussed in the literature relating to solid tumors. Th ere are studies indicating that granulocyte colony-stimulating factor (G-CSF) may increase the risk of AML evolution in aplastic anemia [9] and severe congenital neutropenia [10,11], while other studies suggest a favorable response of patients with MDS to treatment including G-CSF [12]. Th e aim of this retrospective study was to assess the eff ect of treatment with G-CSF on leukemic transformation in 92 consecutive patients with MDS treated in two hematology units in Athens, Greece, between 1996 and 2005. Th e medical records of these patients were retrospectively reviewed. Bone marrow biopsies were reevaluated and diagnosis confi rmed. We used the French – American – British (FAB) classifi cation for MDS instead of the more recent World Health Organization (WHO) classifi cation for practical reasons and due to the presence of patients with chronic myelomonocytic leukemia (CMML). We excluded patients with refractory anemia with excess blasts in transformation (RAEB-T) ( n 16), as well as patients who were considered non-evaluable ( n 5) and

Intravascular B-cell lymphoma with leukemic presentation: case report and literature review

Intravascular lymphoma is an extremely rare, disseminated, and aggressive extranodal CD20+ non‐Hodgkin’s lymphoma characterized by the presence of lymphoma cells only in the lumina of small vessels. We report a 72‐year‐old woman with a diagnosis of intravascular lymphoma presented with splenomegaly and leukemic appearance in the peripheral blood smear. Her clinical course was rapidly deteriorated before the initiation of specific chemotherapy and finally died due to multiorgan insufficiency. Bone marrow biopsy revealed a characteristic infiltration of CD5, CD10 B‐cell lymphoma. To our knowledge, this is the first reported case of a CD5, CD10 intravascular B‐cell lymphoma with leukemic presentation in peripheral blood with multiple cytogenetic aberrations.