Risk Myelodysplastic Syndrome Patients Research Articles

Multivariate time‐dependent comparison of the impact of lenalidomide in lower‐risk myelodysplastic syndromes with chromosome 5q deletion

The impact of lenalidomide treatment on long-term outcomes of patients with lower risk myelodysplastic syndromes (MDS) and chromosome 5q deletion (del(5q)) is unclear. This study used time-dependent multivariate methodology to analyse the influence of lenalidomide therapy on overall survival (OS) and acute myeloblastic leukaemia (AML) progression in 215 patients with International Prognostic Scoring System (IPSS) low or intermediate-1 risk and del(5q). There were significant differences in several relevant characteristics at presentation between patients receiving (n=86) or not receiving lenalidomide (n=129). The 5-year time-dependent probabilities of OS and progression to AML were 62% and 31% for patients receiving lenalidomide and 42% and 25% for patients not receiving lenalidomide; differences were not statistically significant in multivariate analysis that included all variables independently associated with those outcomes (OS, P=0·45; risk of AML, P=0·31, respectively). Achievement of RBC transfusion independency (P=0·069) or cytogenetic response (P=0·021) after lenalidomide was associated with longer OS in multivariate analysis. These data clearly show that response to lenalidomide results in a substantial clinical benefit in lower risk MDS patients with del(5q). Lenalidomide treatment does not appear to increase AML risk in this population of patients.

Biosimilar epoetin in elderly patients with low-risk myelodysplastic syndromes improves anemia, quality of life, and brain function

The myelodysplastic syndromes (MDS) are a group of clonal hematopoietic disorders characterized by bone marrow failure and a risk of progression to acute myeloid leukemia (AML). Anemia affects the course of disease, quality of life (QOL), and cognitive function of MDS patients. Erythroid-stimulating agents (ESAs) are effective; however, not all patients respond to ESAs. To evaluate the effectiveness of a biosimilar epoetin α (Binocrit) for the treatment of anemia in low-/intermediate-1 risk MDS patients and to evaluate the impact of ESAs on QOL and on cognitive function, 24 consecutive patients aged over 65years were treated with Binocrit at 40,000IU once a week for 12weeks and were followed for at least 3months. Responsive patients continued with 40,000IU once a week for a further 12weeks. Changes in QOL were assessed by the Functional Assessment of Cancer Therapy-Anemia (FACT-An), while cognitive assessment was carried out by mini-mental state examination (MMSE). All patients completed 12weeks of therapy. Sixteen patients (66.67%) achieved an erythroid response (ER), 15 patients (62.5%) became transfusion independent and remained free from transfusion requirement for at least 3months, while two patients had reduction in transfusion requirement of at least four RBC transfusions/8weeks compared with the pretreatment transfusion requirement. Seven patients were nonresponders (29.1%), of whom four patients were low risk and three intermediate-I risk. Seven transfusion-independent patients were low risk, and eight were intermediate-1 risk. Median hemoglobin (Hb) values were significantly higher after treatment in responders (p < 0.001). ER was maintained after 24weeks. Statistically significant positive correlations between improvement in Hb and variations in patients' mini-mental (Spearman's Rho = 0.54, p < 0.01) and FACT-An scores (Spearman's Rho = 0.59, p < 0.003) were demonstrated. This preliminary study shows that Binocrit is promising for the treatment of anemia of MDS patients. ER positively correlates with improvements in patients' cognitive status and positive changes in QOL.

Recombinant human erythropoietin in very elderly patients with myelodysplastic syndromes: results from a retrospective study

Myelodysplastic syndromes (MDS) are common in elderly patients. Recombinant human erythro-poietin (rHuEPO) has been widely used to treat anemia in lower risk MDS patients, but few data are known about rHuEPO treatment in the very elderly patient group. In order to investigate the role of rHuEPO treatment in terms of response, overall survival (OS), and toxicity in a very elderly MDS patient group, 93 MDS patients treated with rHuEPO when aged ≥80 years were selected among MDS cases enrolled in a retrospective multicenter study by the cooperative group Gruppo Romano Mielodisplasie (GROM) from Jan 2002 to Dec 2010. At baseline, median age was 82.7 (range 80-99.1) with a median hemoglobin (Hb) level of 9 g/dl (range 6-10.8). The initial dose of rHuEPO was standard (epoetin alpha 40,000 IU/week or epoetin beta 30,000 IU/week) in 59 (63.4 %) patients or high in 34 (36.6 %) (epoetin alpha 80,000 IU/week) patients. We observed an erythroid response (ER) in 59 (63.4 %) patients. No thrombotic event was reported. Independent predictive factors for ER were low transfusion requirement before treatment (p = 0.004), ferritin <200 ng/ml (p = 0.017), Hb >8 g/dl (p = 0.034), and a high-dose rHuEPO treatment (p = 0.032). Median OS from rHuEPO start was 49.3 months (95 % CI 27.5-68.4) in responders versus 30.6 months (95 % CI 7.3-53.8) in resistant patients (p = 0.185). In conclusion, rHuEPO treatment is safe and effective also in the very elderly MDS patients. However, further larger studies are warranted to evaluate if EPO treatment could be worthwhile in terms of quality of life and cost-efficacy in very old patients.

Absence of aberrant myeloid progenitors by flow cytometry is associated with favorable response to azacitidine in higher risk myelodysplastic syndromes

In intermediate-2 (Int-2) and high risk patients with myelodysplastic syndromes (MDS), treatment with azacitidine is associated with hematological improvement and prolonged overall survival (OS) in patients who respond to therapy. However, only half of the patients who are treated will benefit from this treatment. It is a major challenge to predict which patients are likely to respond to treatment. The aim of this study was to investigate the predictive value of immunophenotyping for response to treatment with azacitidine of Int-2 and high risk MDS patients. Bone marrow aspirates were analyzed by flow cytometry in 42 patients with Int-2 and high risk MDS, chronic myelomonocytic leukemia, or low blast count acute myeloid leukemia before treatment and after every third cycle of azacitidine. A flow score was calculated using the flow cytometric scoring system (FCSS). The presence of myeloid progenitors with an aberrant immunophenotype was significantly associated with lack of response (p = 0.02). A low pretreatment FCSS was associated with significantly better OS compared with a high pretreatment FCSS (p = 0.03). A significant decrease in FCSS was observed in patients with complete response after three cycles azacitidine compared to patients with progressive disease (p = 0.006). Absence of aberrant myeloid progenitor cells at baseline and/or a decrease in the FCSS during treatment identified Int-2 and high risk MDS patients who are likely to respond to treatment with azacitidine.

Absence of aberrant myeloid progenitors by flow cytometry is associated with favorable response to azacitidine in higher risk myelodysplastic syndromes.

Background: In intermediate-2 and high risk patients with myelodysplastic syndromes (MDS), treatment with azacitidine is associated with hematological responses and prolonged overall survival in patients who respond to therapy. However, only half of the patients that are treated will benefit from this treatment. It is a major challenge to predict which patients are likely to respond to treatment. The aim of this study was to investigate the predictive value of immunophenotyping for response to treatment with azacitidine of Int-2 and high risk MDS patients. Methods: Bone marrow aspirates were analyzed by flow cytometry in 42 patients with Int-2 and high risk MDS, chronic myelomonocytic leukemia or low blast count acute myeloid leukemia before treatment and after every third cycle of azacitidine. A flow score was calculated using the flow cytometric scoring system (FCSS). Results: The presence of myeloid progenitors with an aberrant immunophenotype was significantly associated with lack of response (p=0.02). A low pretreatment FCSS was associated with significantly better overall survival compared with a high pretreatment FCSS (p=0.03). A significant decrease in FCSS was observed in patients with complete response after three cycles azacitidine compared to patients with progressive disease (p=0.006). Conclusions: Absence of aberrant myeloid progenitor cells at baseline and/or a decrease in the FCSS during treatment identified Int-2 and high risk MDS patients who are likely to respond to treatment with azacitidine. © 2014 Clinical Cytometry Society.

Distribution of serum erythropoietin levels in lower risk myelodysplastic syndrome cases with anemia

International guidelines for myelodysplastic syndrome (MDS) state that the standard therapy for lower risk MDS patients with symptomatic anemia of serum erythropoietin (EPO) <500 IU/L is erythroid-stimulating agents (ESAs). The objective of this study is to examine the distribution of EPO levels in lower risk MDS patients, and to inquire into the relationship of EPO distribution to hemoglobin levels and transfusions. Twenty cases of lower risk MDS (low or intermediate-1 by the International Prognostic Scoring System) with hemoglobin level <90 g/L at our institution were enrolled. Eight received more than two units of transfusions per month. Median hemoglobin level was 78 g/L. EPO levels ranged between 26.4 and 11300 IU/L (median 645 IU/L), including 10 cases (50 %) with >500 IU/L. EPO levels were inversely correlated to hemoglobin levels, especially in the cases without transfusion support (p < 0.001, R = 0.92). The rate of the cases with EPO <500 IU/L was significantly higher in the group without transfusion than the others (p = 0.020). Considering that, in Japan, the indication for transfusion is around 70 g/L of hemoglobin for chronic diseases, it may be possible to improve anemia in a subset of lower risk MDS cases by administration of ESAs before transfusions are required.

Review Of Published Survival Evidence For Iron Chelation Therapy (ICT) In Patients With Myelodysplastic Syndromes (MDS)

Iron overload due to frequent blood transfusions in patients with myelodysplastic syndrome (MDS) is associated with complications, including a risk of cardiac disease and transformation to acute myeloid leukaemia (AML). Iron chelation therapy (ICT) with deferasirox and desferrioxamine is an essential part of the management of iron overload in low and intermediate prognostic risk MDS patients [NCCN Clinical Practice Guidelines in Oncology: Myelodysplastic Syndromes; Bennett 2008]. However, despite the risks of cardiac complications and AML, prior to 2009 there was little published evidence that ICT could improve survival outcomes in patients with MDS. A PubMed and ASH abstract search (conducted July 2013) has revealed a growing body of evidence indicating increased survival of a median of 2 – 6 years in transfusion-dependent MDS patients treated with ICT, relative to patients without ICT (see Table).All the published evidence for survival benefit is in lower IPSS risk MDS patients, with a significant survival benefit seen in the sub-group of patients with non-RARS (Leitch 2012). The main limitation of the studies is that they are all based on retrospective or prospective observational study designs; hence have a risk of selection bias, although many utilize multivariate techniques to control for confounding factors. Furthermore, none of the studies assess survival associated with type of ICT although there is an ongoing RCT (TELESTO) addressing the impact of deferasirox on OS in low risk MDS patients. In addition, it would be useful to explore whether the observational data from each study could be pooled to assess OS outcomes in transfusion-dependent lower risk MDS, by MDS sub-group and by type of ICT. In conclusion, there is now extensive evidence of an association between ICT and survival improvement in transfusion-dependent lower prognostic risk MDS patients. Disclosures:Tolley:Tolley Health Economics: Consultancy. Off Label Use: ICT for patients with iron overload due to blood transfusion in patients with MDS. Vieira:Novartis: Employment. Strickson:Tolley Health Economics: Consultancy. Kundishora:Novartis: Employment.

The New CD95 Ligand Inhibitor APG101 Leads To Decreased Apoptosis and Improved Erythroid Differentiation In Primary CD34+ Cells From Patients With Low Risk Myelodysplastic Syndrome (MDS)

IntroductionMyelodysplastic syndromes (MDS) are a heterogeneous group of clonal myeloid malignancies characterized by ineffective hematopoiesis and an increased risk of transformation into acute myeloid leukemia. Particularly early stage MDS are at least in part characterized by an increased apoptosis of myeloid and erythroid progenitors that causes peripheral cytopenia. APG101 is a glycosylated fusion protein consisting of the extracellular domain of human CD95 (Fas receptor) and the Fc domain of human IgG1. APG101 effectively binds to the CD95 ligand (CD95L) expressed on effector cells as well as to functionally active ligand in solution, by that blocking the interaction between CD95 and its ligand. The aim of our study was to evaluate whether APG101 treatment of primary CD34+ reduces the apoptotic rate and improves the differentiation capacity of these cells. MethodsBone marrow cells were obtained during routine bone marrow aspiration after all patients gave their written informed consent. Isolated primary CD34+ cells from 11 MDS patients were cultured in complete supplemented IMDM medium for 6 days with increasing concentrations of APG101 (1 µg/mL, 3 µg/mL, 10 µg/mL, 30 µg/mL, 100 µg/mL, 200 µg/mL, 300 µg/mL). After incubation time, cells were multicolor- stained with the following dye combination: Annexin-FITC + CD235a-PE + CD34-PECy7 + CD71-APC + 7-AAD and analyzed immediately on a flow cytometer (FACSCanto, BD Bioscience, Heidelberg, Germany). Analysis of raw FACS data was done with the FACSDiva software. To analyze the differentiation capacity of CD34+ progenitors, methylcellulose assays were performed in parallel to the aforementioned experiments. However, due to limited cell numbers, colony assays were performed on 9 MDS patients only. Cells were cultured in triplicates with increasing concentrations of APG101 for 14 days. Colonies were counted and the mean number of colonies was determined. ResultsTreatment of differentiating CD34+ cells with APG101 led to a decreased apoptosis in both CD34+ cells and CD71+ cells, respectively, indicated by decreased Annexin-FITC fluorescence. Interestingly, this effect was particularly seen at low APG101 concentrations (maximum of 10 µg/ml), while the effect was abrogated at higher APG101 concentrations. The anti-apoptotic effect was more pronounced in low risk MDS patients compared to high risk MDS patients. No effect was seen when the CD235a+ fraction of cells was analyzed. With regard to colony formation, an improvement of erythroid differentiation, indicated by an increase in CFU-E, was found in 3 out of 4 low risk patients (less than 5% blasts in the bone marrow). No effect was seen on erythroid differentiation in high risk patients (more than 5% blasts in the bone marrow). ConclusionAPG101 shows promising in vitro activity in viable CD34+ cells with regard to inhibition of apoptosis and promotion of differentiation. The observation that the anti-apoptotic effect was more pronounced in low risk MDS patients as compared to high risk MDS patients supports the concept of increased apoptosis particularly in early stage MDS progenitors. Although the numbers in the differentiation experiments are small, we found a promising effect of APG101 on CFU-E formation at lower doses in patients with less than 5% bone marrow blasts. Moreover, the same dose-dependent effect was observed in the apoptosis assays. Since the activation of the CD95 pathway seems not only to be involved in apoptosis induction, but is also required for terminal erythroid differentiation in normal hematopoiesis, this dose-dependent effect might particularly reflect these ambivalent roles of CD95 and its ligand in both MDS and healthy hematopoiesis, repsectively. Disclosures:Kunz:APOGENIX GmbH: Employment. Fricke:APOGENIX GmbH: Employment.

Identification Of Chromosomal Abnormalities By Microarray-Based Genomic Profiling As Compared To Karyotyping In Patients With Low and Intermediate-1 Risk Myelodysplastic Syndromes Within a Prospective Clinical Trial: A Study On Behalf Of The HOVON89 Study Group

Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal hematopoietic disorders, characterized by ineffective hematopoiesis resulting in cytopenias and a highly variable clinical course. Cytogenetics are routinely used as one of the diagnostic, prognostic and therapeutic markers in the clinical management of MDS (Greenberg et al. Blood 120:2454,2012). Although karyotyping is generally considered as the gold standard in the cytogenetic characterization of MDS, 40-60% of the patients exhibit a normal karyotype. Intrinsically, the resolution of karyotyping is limited by its capacity to detect only those copy number changes that are microscopically visible (5-10 Mb in size). In contrast, microarray-based genomic profiling analyses allow a genome-wide detection of copy number alterations (CNAs), down to 100 kb in size, and regions of copy neutral loss of heterozygosity (CNLOH). Such analyses also overcome some of the other major limitations of karyotyping such as low success rate due to inadequate metaphase yield and/or poor banding quality. We have compared karyotyping and fluorescence in situ hybridization (FISH) with microarray-based genomic profiling with respect to the detection yield for genetic abnormalities in bone marrow samples from lower risk MDS patients.We used the HOVON89 study-cohort, a prospective phase II randomized multicenter study to assess the efficacy of lenalidomide with or without erythropoietin and granulocyte-colony stimulating factor in patients with low/intermediate-1 risk MDS; www.trialregister.nl; NTR1825; EudraCT nr.: 2008-002195-10. Inclusion target of the study is 200 low/intermediate-1 risk MDS patients (134 enrolled, inclusion ongoing). Data regarding cytogenetics, FISH and microarray were obtained in a fully blinded fashion for 68 MDS patients. For microarray-based genomic profiling we used the recently launched high resolution CytoScan HD Array (Affymetrix) platform. The following interpretation criteria were applied: (i) the threshold for CNAs was set at >5 Mb, (ii) inclusion of <5 Mb CNAs segments that coincide with known cancer genes as reported on www.sanger.ac.uk and (iii) the threshold for CNLOH was set at >10 Mb and to telomere. Karyotyping and interphase FISH were performed using standard cytogenetic methods. In all 4 patients where karyotyping revealed no metaphases interphase FISH was performed.Thirty-six of the 68 (53%) MDS patients had an abnormal microarray profile. Of interest, in 13 of these 36 patients no abnormalities were observed by karyotyping and/or FISH. All these abnormalities observed by microarray only, involved focal <5 Mb CNAs (containing e.g. the TET2, RUNX1 and DNMT3A genes) and regions of CNLOH (coinciding with 1p, 2p, 4q, 7q, 11p, 11q, 12q, 14q and 18q), which are all out of the scope of karyotyping and FISH. All CNAs identified by karyotyping and FISH were also observed by microarray-based genomic profiling, including a case with loss of 5q in 5% of the cells and loss of 7q in 9% of the cells as observed by interphase FISH and another case with loss of 5q in 3 of 20 the analyzed metaphases by karyotyping. These observations demonstrate the high sensitivity of the CytoScan HD array platform for the identification of CNAs in (small) subclones. As expected balanced translocations such as t(3;3)(q21;q26) and t(2;14)(q37;q22) present in 2 of the patients in our cohort were not identified by microarray-based genomic profiling. This study will be extended to all patients to be included in the HOVON89 trial.In conclusion, we demonstrate that in the present cohort of 68 patients, microarray-based genomic profiling allows the identification of almost all copy number abnormalities also observed by karyotyping and FISH. In addition, we show that microarray-based genomic profiling allows the detection of potential prognostic relevant abnormalities (focal CNAs and CNLOH) which would have remained undetected by karyotyping and FISH. The predictive and/or prognostic value of these novel CNAs and CNLOH will be evaluated within the ongoing prospective clinical HOVON89 trial in lower risk MDS. Disclosures:No relevant conflicts of interest to declare.

Oral Rigosertib (ON 01910.Na) Treatment Produces An Encouraging Rate Of Transfusion Independence In Lower Risk Myelodysplastic Syndromes (MDS) Patients; A Genomic Methylation Profile Is Associated With Responses

▪Rigosertib, a novel orally bioavailable small molecule inhibitor of PI3-Kinase and PLK1 pathways, was tested in a phase II study in lower risk (intermediate-1 or low risk per IPSS classification) transfusion-dependent MDS patients. This randomized, two-arm study administered rigosertib (560 mg bid) either intermittently (2 out of 3 weeks) or continuously to transfusion-dependent patients (4 units RBC transfusions over 8 weeks). Erythrocyte stimulating agents (ESAs) were allowed on study. Forty eight patients have been enrolled as of August 2nd, 2013 and another 12 patients will be enrolled. Continuous dosing was stopped after 9 patients due to a higher urinary toxicity resulting in 39 patients receiving intermittent dosing. Overall the drug was well tolerated, except for reversible grade 3 urinary toxicity (dysuria, hematuria, cystitis, and urinary urgency) in 6/48 (12%) patients while 17/48 (35%) experienced grade 2 urinary toxicity. Notably, no significant treatment emergent myelosuppression was evident in these patients. Dosing has been modified to a total daily dose of 840 mg (560 mg am/280 mg afternoon) to improve urinary tolerability. Other measures to manage urinary symptoms include hydration, sodium bicarbonate and dose reduction or interruption. Of 33 patients (3 with del5q) on intermittent dosing treated for at least 8 consecutive weeks, 15 (45%) achieved transfusion independence (TI or no RBC transfusion for at least 8 consecutive weeks) lasting 8 to 53+ weeks (median=17 weeks). Intent-to-treat analysis showed 17/48 (35%) patients achieved TI. Twelve of 15 responding patients were refractory to prior treatment with ESAs. Fourteen of 15 responding patients (11 out of 12 ESA refractory) received concomitant ESAs, suggesting an effect of rigosertib on ESA resistance or potential synergy with ESA. These effects are being explored in additional trials now in planning.There was no obvious correlation of response to karyotype or other classifications. We hypothesized that DNA methylation profiles could help explain the differences between responders and non-responders. Pre-therapy bone marrow mononuclear cells from 32 patients (including 4 from the prior Phase I study previously reported in ASH 2011) were analyzed using the Illumina 450K methylation array platform. Seven had complete response or CR (TI + increase in Hb >2Gm/dL), 10 had partial response or PR (TI without Hb increase) and 15 had no response or NR. Supervised hierarchical clustering by methylation intensity demonstrated a distinct profile associated with complete responders. Bisulfite sequencing (which allows quantification of multiple consecutive CpGs in an amplicon) of several differentially methylated loci confirmed the Illumina 450K data. In general, hypermethylation of a group of genes was associated with responders. Functional annotation of the hypo and hypermethylated genes which best distinguished CRs from NRs showed that the genes most affected by methylation were related to regulation of transcription followed by genes involved in cell-cell adhesion, inflammatory response, apoptosis and proliferation. Additional patient samples are being analyzed to confirm these results. In this interim analysis the observed correlation of hematological response to genomic methylation status suggests the possibility of preselecting patients likely to benefit from treatment with rigosertib. ConclusionsIntermittent dosing of oral rigosertib is well tolerated and active in producing transfusion independence in lower risk MDS patients. A genomic methylation signature, once confirmed in prospective studies, may allow pre-selection of responders. Disclosures:Raza:Onconova Therapeutics: Research Funding. Al-Kali:Onconova Therapeutics: Research Funding. Tibes:Onconova Therapeutics: Research Funding. Spitzer:Onconova Therapeutics: Research Funding. Wilhelm:Onconova Therapeutics: Employment, Equity Ownership. Mukherjee:Onconova Therapeutics: Research Funding.

Validation Of a Multi-Diagnostic Approach Including Flow Cytometry To Identify Specific Risk Categories Within Low and Intermediate-1 Risk Myelodysplastic Syndromes Within a Prospective Clinical Trial: A Study On Behalf Of The HOVON89 Study Group

Myelodysplastic syndromes (MDS) constitute a heterogeneous group of hematopoietic stem cell disorders, characterized by ineffective hematopoiesis resulting in cytopenias and variable risk of acute myeloid leukemia (AML). To make an accurate distinction between specific risk categories in MDS, especially in low and intermediate risk MDS, a multi-diagnostic approach is recommended. To verify the efficacy of multiple diagnostic tools in MDS we used the HOVON89 study-cohort (a prospective phase II randomized multicenter study to assess the efficacy of lenalidomide with or without erythropoietin and granulocyte-colony stimulating factor in patients with low-intermediate-1 risk MDS; trial registered at www.trialregister.nl as NTR1825; EudraCT nr.: 2008-002195-10.Inclusion target of the study is 200 low/intermediate-1 risk MDS patients (134 enrolled, inclusion ongoing). We collect data on cytomorphology (CM), conventional cytogenetics (CCG), fluorescence in situ hybridization (FISH) and microarray-based genomic profiling. In addition, we performed flow cytometric (FC) analysis according to European LeukemiaNet guidelines (Van de Loosdrecht et al., Haematologica 2009 and Leukemia 2012). Current CM results (N=98) identified: 8 refractory anemia (RA); 16 refractory anemia with ringed sideroblasts (RARS); 43 refractory cytopenia with multilineage dysplasia with/without ringed sideroblasts (RCMD/RCMD-RS); 16 refractory anemia with excess blast-1 (RAEB-1), 5 chronic myelomonocytic leukemia-1 (CMML-1) and 10 patients with isolated del(5q). CCG analysis (N=101) indicated 2 very good risk, 84 good risk, 13 intermediate risk and 2 poor risk patients according to the IPSS-R risk categories (Greenberg et al., Blood 2012). In addition, interphase FISH analysis (N=72) was normal in 15 patients, in 6 patients the del(5q) was confirmed. From 68 MDS patients data from both CCG and microarray were available. Microarray-based genomic profiling identified genomic abnormalities such as copy neutral loss of heterozygosity and small (<5 Mb) copy number alterations coinciding with a cancer gene in 13 patients with normal CCG. As expected in one patient the balanced translocation t(3;3)(q21;q26) was not identified by microarray-based genomic profiling.FC analysis (N=82) evaluated aberrancies with regard to count, marker expression level and lineage infidelity marker expression on myeloid progenitors, B cell progenitors, maturing myeloid/monocytic and erythroid cells. Current, validated FC-scoring systems identified 60/81 (Della Porta et al., Haematologica 2012) and 61/81 (Wells et al., Blood 2003) patients with MDS. Both scoring systems do not evaluate dyserytropoiesis and dysmegakaropoiesis, thereby possibly not recognizing RA, RARS or RCMD patients with only dyserytropoiesis and dysmegakaropoiesis. We integrated both scoring systems into one score (Van de Loosdrecht et al., Leukemia 2012, and JNCCN 2013). Patients were divided into 3 categories: not likely MDS (A), signs of dysmyelopoiesis (B) and fitting MDS (C). This score diagnosed 69/78 patients as probably or likely MDS by FC (B or C, figure 1). Remarkably, patients scored as ‘category A' only displayed dyserytropoiesis and/or dysmegakaryopoiesis by CM. FC identified dyserytropoiesis in these patients, however, a consensus erytroid flow score is not yet validated. In addition, FC identified different risk categories within the patient group with no genetic abnormalities (based on CCG, FISH and microarray-based genomic profiling; data not shown). [Display omitted] In conclusion, this is the first prospective study in low/int-1 risk MDS that validates FC as a valuable diagnostic tool in MDS (sensitivity of FC in this cohort: 88%). FC only failed to recognize some patients with only dyserytropoiesis and dysmegakaryopoiesis by CM, not evaluated by the current scoring system. Thirteen patients with unilineage dysplasia by CM had multilineage dysplasia by FC. We postulate that RA/RARS patients with multi-lineage dysplasia by FC may have clinical features of RCMD patients and therefore a higher risk on transformation to AML. Clinical follow-up data are expected within 1.5 year.In near future, a multi-diagnostic approach may i) identify risk categories within well defined IPSS-R subgroups, ii) predict risk on transformation and iii) select patients who might benefit from new emerging drugs for low-int-1 risk MDS. Disclosures:No relevant conflicts of interest to declare.

Hedgehog Pathway Is Deregulated In Myelodysplastic Syndrome Progenitor Bone Marrow Cells

The sonic hedgehog (Shh) pathway is a regulatory network involved in the development of cancer. The Hh ligands (Sonic Hh, Indian Hh, and Desert Hh) bind to the receptors Patched1 and Patched2, resulting in the inhibition of their repression on Smoothened (Smo). Smo is a seven-transmembrane protein, which inhibits Suppressor of Fused (SUFU), resulting in the stabilization and activation of the Gli family of transcription factors, followed by a transcriptional response to the “canonical Hh signaling”. SUFU is a negative regulator of the Hedgehog signaling pathway, through the restriction of Gli activity through cytoplasmic sequestration. Germline mutations in SUFU are involved in medulloblastoma. Dysregulation of Shh signaling has been reported to be involved in several malignancies, including chronic myeloid leukemia.To our knowledge, however, only one study has been carried out in myelodysplastic syndrome (MDS) and acute leukemia so far, and the role of this pathway in leukemogenesis is still unknown. Recently we showed an increased protein expression of Sonic and Dessert Hedgehog in addition to increased mRNA levels of PTCH and SMO in MDS bone marrow. Here, we investigated whether this increase was capable of activating Hedgehog pathway in total and in progenitor bone marrow cells.Bone marrow (BM) samples were collected from 39 MDS (25 low-risk, 14 high-risk, according WHO 2008) patients and 26 healthy donors. CD34+ cells were isolated from peripheral blood of 9 healthy donors and 20 (12 low-risk and 8 high risk) MDS patients. Relative expressions of PTCH, SMO, SUFU and GLI1 were obtained by Real time-PCR.Increased expression observed in ligands and receptors were not sufficient to upregulate the activity of Hedgehog pathway in MDS total bone marrow. The downstream effector GLI1 was under expressed in the disease as median [min-max] as follows: healthy donors 1.04[0.02-4.01]; low risk MDS 0.21[0.03-2.26]; high risk MDS 0.07[0.03-1.48] healthy donors vs low risk p=0.007; healthy donors vs high risk p=0.006. Furthermore, SUFU expression was similar in healthy donors 3.3[0.35-7.84] and in myelodysplastic (low risk 3.76[0.75-13, 2] and high risk 1.8[0.58-16.4]) bone marrows. Different from total bone marrow, the receptors Patched and SMO showed decreased expression in MDS CD34+ cells: Patched healthy donors 2.24[0.74-4.36]; low risk 0.6[0.1-3.34]; high risk 0.98[0.29-1.22] and SMO healthy donors 1.18[0.62-2.16]; low risk 0.79[0,11-1.87]; high risk 0.86[0.37-1.02]; healthy donors vs high risk MDS p=0.049. SUFU was overexpressed (healthy donors 1.44[0.75-2.39]; low risk 2.77[0.93-4.11]; high risk 1.68[1.07-3.43]; healthy donors vs low risk MDS p=.005). However, myelodysplastic progenitors had GLI1 overexpression (healthy donors 1.09[0.6-2.91]; low risk 2.93[0.75-14.52]; high risk 1.32[0.71-2.58]; healthy donors vs low risk p=0.03), indicating an activation of the Shh pathway in MDS progenitors.Recently, Kobune et al (Blood Cancer J. 2012 Sep 7) showed activation of Hedgehog signaling in primary CD34+ blasts from acute myeloid leukemia and MDS, as those cells expressed IHh, PTCH, SMO, GLI1 and GLI. Taken together, these results and ours led us to conclude that the Hedgehog pathway is activated in MDS. Since Shh pathway is being explored as a new opportunity for targeted therapies against tumors, our study suggests that the inhibition of this pathway could be strategic in the control of leukemic stem cell and MDS treatment. Disclosures:No relevant conflicts of interest to declare.

Azacitidine Treatment For Patients With Myelodysplastic Syndromes and Acute Myeloid Leukemia Harboring Chromosome 3q Abnormalities

BackgroundKaryotype is one of the key prognosis factors in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Chromosome 3q abnormalities, including aberrant expression of EVI-1, represent a small subset of patients and are associated with a poor outcome in both MDS and AML patients treated with conventional treatment. Azacitidine is the current standard of care for high-risk MDS and is also active in AML. Given that AZA may provide responses in patients with poor chances of response to conventional treatment as patients with monosomy 7 and given the strong epigenetic modifications related to EVI-1 dysregulation, AZA may represent an interesting option for patients with 3q abnormalities. Therefore, we analyzed the outcome of patients with Chromosome 3q abnormalities treated with Azacitidine. Patients and methodsThis is a retrospective multicenter study. Patients were included if 1/ Diagnosis of MDS, CMML, or AML, including therapy related neoplasms 2/ harbored chromosome 3q abnormalities and/or EVI-1 overexpression by PCR 3/ received at least one cycle of azacitidine for either frontline or relapse treatment. Response was evaluated according IWG 2006 criteria. This cohort of patients was compared to 406 high risk MDS and AML patients without 3q abnormalities included in the French AZA compassional program. ResultsA total of 184 patients were analyzed. Median age was 64 years. According to WHO classification, patients presented RA/RCMD/RAEB-1 28% of cases, RAEB-2/CMML-2 31% of cases, and AML 41% of cases. Median bone marrow blast count was 15%. Cytogenetics were inv(3)/t(3;3) 22%, del(3q)/monosomy 3 26%, t(3q26;x) 20%, EVI-1 overexpression without 3q abnormality 14%, t(3q21;x) 4% and other 3q abnormalities 14%. 58% of patients had complex karyotype. Azacitidine was used frontline for 67% of patients, 33% being previously treated with intensive chemotherapy. Median number of cycle of azacitidine was 5 (1-33). Overall response rate was 47% (Complete or Partial response 27%). 31 patients (25% of patients younger than 70y) bridged to allogeneic transplantation. With a median follow-up of 16 months, median overall survival was 10.5 months. Younger age (below 65y, 13m vs 10m, p=0.052), low bone marrow blast count (below 20%, 13m vs 8m, p=0.001), presence of t(3q21;x) (74 vs 10 months, p=0.008), few cytogenetic abnormalities (below 3, 14m vs 9m, p=0.003), and use of Azacitidine frontline (13m vs 8 m, p=0.029) were associated with a better survival. Long term survival remains limited with a 3 years survival probability below 10%. Interestingly, baseline characteristics and prognosis of patients with EVI-1 overexpression without 3q cytogenetic abnormality were comparable with patients with 3q26 cytogenetic abberation. Then, we compared our cohort with MDS/AML patients without 3q abnormalities, their overall response rate was 30% (41% for HR MDS, 15% for AML, including 11% CR/PR), and median OS was 10.5 months (15 months for HR MDS and 7 months for AML). When we merged the 2 cohorts and performed multivariate analyses, presence of 3q abnormality had no prognosis impact on either response or survival. ConclusionAzacitidine seems to limit the dismal outcome associated with 3q abnormalities and thus represents a good alternative to conventional chemotherapy including for patients eligible for allogeneic transplantation. Moreover, our data showed that 3q group is heterogeneous with a favorable outcome for the small subset of patients with 3q21 abnormalities. Disclosures:Prebet:CELGENE: Honoraria. Off Label Use: Use of azacitidine in acute myeloid leukemia.

A Phase II Expansion Study Of Vorinostat In Combination With Idarubicin and Cytarabine For Patients With Acute Myelogenous Leukemia (AML) With FLT3 Molecular Alterations

BackgroundThe combination of vorinostat, idarubicin and cytarabine (IA+vorinostat) is associated with high response rate in patients with newly diagnosed acute myelogenous leukemia (AML) or higher-risk myelodysplastic syndromes (MDS) (JCO 2012;30:2204). In that study, presence of FLT3-ITD was associated with 100% overall response rate (ORR) in 11 patients. To confirm the efficacy of this combination, we extended the phase II study to treat 2 additional cohorts: one for patients with newly diagnosed (untreated cohort) and the other with relapsed and refractory (R/R cohort) AML or higher-risk MDS with FLT3 alteration (both ITD and D835 mutation). MethodsPatients with the above diagnosis, ages 15 to 65 years, with appropriate organ function (measured cardiac ejection fraction ≥ 50%, serum creatinine ≤ 2 mg/dl, total bilirubin ≤ 2 mg/dl, and GPT/GOT ≤ 2.5 x upper limit of normal) whose eastern cooperative group (ECOG) defined performance status ≤ 2 were eligible for the study. Study treatment comprised of vorinostat 500 mg orally three times a day (days 1 to 3), idarubicin 12 mg/m2 intravenously (IV) daily x 3 days (days 4 to 6), and cytarabine 1.5 g/m2 IV as a continuous infusion daily x 3 - 4 days (days 4 to 7). Patients in remission could be treated with five cycles of consolidation therapy with lower dose combination and up to 12 months of maintenance therapy with single-agent vorinostat. ResultUntreated cohort included 26 patients, whereas 13 patients were treated in R/R cohort (total 39 patients). Thirty six patients had de novo AML, 1 had de novo MDS and 2 had therapy-related AML. For the R/R patients, the median number of prior therapies was 3 (range: 1-6). The median age of the entire study group was 49 (range: 19-64) and 17 (44%) were male. Among the patients whose cytogenetic result were available, 20 (51%) patients had normal and 15 (39%) had abnormal karyotype. By Medical Research Council (MRC) criteria, 30 (77%) patients had intermediate risk and 9 (23%) had poor risk karyotype. Thirty three (85%) patients had FLT3-ITD only, 4 (10%) had both FLT3-ITD and D835 mutation, and 2 (5%) had D835 mutation only. Seventeen (44%) patients had NPM1 mutation.In untreated cohort (N = 26), CR and CRp were documented in 21 (80%) and 2 (8%) patients, respectively (ORR = 88%). In R/R cohort (N = 13), overall response (OR) was observed in 4 (30%) patients (CR in 2 [15%] and CRp in 2[15%]). Of those 4 patients who had OR in R/R cohort, 2 patients were refractory to other high-dose cytarabine-based regimen. The median duration of CR or CRp was 9.2 months (range: 0.1-48.4) in untreated cohort and was 2.9 months (range: 1.6-4.7) in R/R cohort. Twelve (46%) patients in the untreated cohort were bridged to stem cell transplant (SCT) while they were in 1st CR. None of the patients in R/R cohort were bridged to SCT. No difference in response was observed in 1) younger (Age < 60) vs. older patients, 2) normal vs. abnormal karyotype, 3) intermediate vs. poor risk cytogenetics by MRC criteria, 4) presence of RAS mutation, 5) presence of NPM1 mutation, or 6) de novo vs. therapy-related disease.The median overall survival (OS) was 21.7 months (95% CI: 8.1-35.3) in the untreated cohort and was 4.9 months (95% CI: 0.1-10.4) in the R/R cohort. Early treatment related mortality (defined by the death within 4 weeks of the induction) was documented in 1 (4%) patient in the untreated cohort and 2 (15%) patients in the R/R cohort. Toxicity profiles were similar to that reported in the original phase II study (JCO 2012;30:2204). DiscussionVorinostat in combination with IA provides high response rate and durable remission in previously untreateed AML or higher risk MDS patients with FLT3 alteration but is less effective in patients with R/R disease. Phase III randomized study of IA+/- vorinostat in previously untreated AML patients is ongoing (SWOG S1203). Disclosures:Off Label Use: vorinostat in MDS and AML. Cortes:Ambit: Research Funding; Astellas: Research Funding; Argo: Research Funding; Novartis: Research Funding.

High Cereblon Expression In Lower Risk Myelodysplastic Syndromes With 5q Deletion Is Associated With The Efficacy Of Lenalidomide

IntroductionThe binding of immunomodulatory drugs (IMiDs), including lenalidomide, to CRBN is associated with cytotoxicity of IMiDs used in the treatment of multiple myeloma, myelodysplastic syndromes (MDS) and lymphomas. Cereblon (CRBN) is named for its putative role in cerebral development, especially in memory and learning. CRBN forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1), cullin-4 (CUL4) and regulator of cullin 1 (ROC1). This complex regulates DNA repair, DNA replication and transcription. CRBN is a primary target of thalidomide teratogenicity (Ito et al., Science 2010; 327:1345-1350). Down-regulation of the CRBN expression is associated with the development of marked IMiDs resistance in human multiple myeloma cells (Zhu et al., Blood 2011; 118: 4771-4779; Lopez Girona et al., Leukemia 2012; 26: 2326-2335; Heintel et al., Br J Haematol 2013; 161: 695-700; Broyl et al., Blood 2013; 121: 624-627; Lodé et al., Br J Haematol 2013; Jul 17. doi: 10.1111/bjh.12478). CRBN expression is thus required for the antimyeloma activity of IMiDs. AimsTo gain insight into the role of CRBN in lower risk myelodysplastic syndromes with or without 5q deletion and into the mechanisms of lenalidomide action, we study CRBN expression in these two groups of lower risk MDS patients and in healthy controls. We further study the expression of DDB1 and IRF4 (interferon regulatory factor 4, one of target genes of CRBN). MethodsMononuclear cells were isolated both from bone marrow [23 low risk MDS patients with 5q- deletion, 37 low-risk MDS patients with normal chromosome 5 (non5q-) and 24 healthy controls] and from peripheral blood [38 patients with 5q- , 52 non 5q- patients and 25 healthy controls] by Ficoll-Paque PLUS gradient separation, and washed with phosphate-buffered saline. The rest of the red cells were lysed. Total RNA was isolated using RNA isolation solvent and complementary DNA was synthesized from total RNA using SuperScript II reverse transcriptase. Relative levels of the CRBN, DDB1 and IRF4 mRNAs were determined by TaqMan-based quantitative real-time PCR and by calculation to the level of housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The experiments were performed in duplicate. Informed consent was obtained from all patients and healthy controls. Evaluation of 2-ddCt indicates the fold change in gene expression relative to the control. ResultsThe median of CRBN mRNA levels in total RNA isolated from peripheral blood mononuclear cells was the highest (3.6) in lower risk MDS with 5q deletion, lower (1.5) in non5q- MDS and lowest (1.2) in healthy controls. Similar results were obtained in bone marrow mononuclear cells (medians 3.3 in 5q- syndrome, 2.2 in non5q- MDS and 1.3 in healthy controls). The differences between the 3 groups were statistically significant (p˂0.05, Mann-Whitney test) with the exception of non5q- MDS in comparison with healthy controls in mononuclear cells of peripheral blood. Very similar results were obtained in the analysis of DDB1 and IRF4 mRNAs levels, which correlated with CRBN levels. We analyzed CRBN mRNA levels before and in the course of the lenalidomide treatment of 5q- low risk MDS (6 patients). We obtained high CRBN mRNA levels before and during the treatment by lenalidomide in all lenalidomide responders (4 patients). We found a sharp decrease of CRBN mRNA in mononuclear cells of bone marrow in two patients who stopped responding to lenalidomide therapy and subsequently progressed to higher risk MDS. A/G polymorphism located at -29 nt of the 5x UTR of CRBN does not act as a biomarker of response to treatment with lenalidomide in 5q- syndrome. Conclusions5q- low risk MDS patients have the highest levels of CRBN mRNA in comparison to lower risk myelodysplastic syndromes with normal chromosome 5 and to healthy controls. DDB1 and IRF4 mRNAs levels correlate with CRBN levels. High levels of CRBN mRNA were detected in all lenalidomide responders during the course of the therapy. Significant decrease of CRBN levels during treatment by lenalidomide is associated with loss of response to treatment and disease progression. Similarly to the treatment of multiple myeloma, these results suggest that high levels of CRBN mRNA are necessary for the efficacy of lenalidomide in low risk 5q- patients.Supported by the MH CR grant NT/13836-4/2012, PRVOUK-27/LF1/2 and by the MH CR project for development of research institute (UHKT). Disclosures:No relevant conflicts of interest to declare.

Efficacy and Toxicity Of Decitabine Versus CHG Priming Regimen In Patients With Higher Risk Myelodysplastic Syndrome

▪ ObjectivesDecitabine has been approved for the treatment of myelodysplastic syndrome (MDS). CHG regimen (low-dose cytarabine and homoharringtonine in combination with granulocyte colony-stimulating factor (G-CSF)) has been reported to be effective in higher risk MDS patients. The comparative efficacy of these 2 forms of treatment in MDS is unknown. The objective of the current study was to compare the efficacy and toxicity of decitabine and CHG regimen in higher risk MDS. MethodsWe compared lower intensity decitabine with CHG regimen in higher risk MDS (International Prognostic Scoring System (IPSS) intermediate- or high-risk or chronic myelomonocytic leukemia (CMML)). Decitabine was given 20 mg/m2 intravenously over 1 hour daily for 5 days. CHG chemotherapy consisted of an intravenous continuous infusion of low-dose cytarabine (25 mg/d) (days 1–14) and homoharringtonine (1 mg/d) (days 1–14) in combination with G-CSF (300 μg/d) by subcutaneous injection from day 0 until peripheral neutrophil count recovery to 2.0× 109/L. The complete remission (CR), overall response (OR), overall survival and toxicity was compared between the two groups. ResultsA total of 132 patients with higher risk MDS were enrolled in this study, 70 cases in decitabine group and 62 cases in CHG group. The CR rate was 25.7% with decitabine, compared to 32.3% with CHG regimen (p=0.538). The overall response rate (including CR, partial remission (PR), marrow CR and hematological improvement (HI)) between the two groups was also not significantly different (68.6% with decitabine and 58.1% with CHG regimen) (p = 0.209). Overall survival was not significantly different between the two groups (median survival: 18.2 months in decitabine group vs 15.3 months in CHG group; p=0.176). Grade 4 neutropenia occurred more frequently with decitabine (58.3 %) than with CHG regimen (32.3 %) (p = 0.004). Grade 4 thrombocytopenia was also more frequently with decitabine (61.4 %) than with CHG regimen (38.7 %) (p = 0.009). ConclusionsBoth decitabine and CHG regimens were effective in treating patients with higher risk MDS. The two regimens did not differ in terms of clinical responses. However, decitabine showed severer hematological toxicities than CHG regimen. This trial was registered at www.clinicaltrials.gov as ChiCTR-ONC-11001501. Disclosures:No relevant conflicts of interest to declare.

Myocardial Tissue Characterization By Cardiac MR Imaging In Myelodysplastic Syndromes

IntroductionMagnetic Resonance Imaging (MRI) provides unique insight regarding tissue characterization in the heart. We reported the baseline MRI findings at the end of the recruitment in the MIOMED (Myocardial Iron Overload in MyElodysplastic Diseases) study. In particular, we evaluated the distribution of iron overload in the whole left ventricle (LV) and he presence of myocardial fibrosis in patients with myelodysplastic syndromes (MDS); the association with LV function was also investigated. No data are available in the literature about this issue. MethodsMIOMED is an observational, MRI multicentre study in low and intermediate-1 risk MDS patients who have not received regular iron chelation therapy. Out of the 51 MDS patients enrolled, 48 underwent the baseline MRI exam. Mean age was 71.7±8.5 years and 17 patients were females. MIO was assessed using a multislice multiecho T2* approach. Biventricular function parameters were quantified by cine sequences. Myocardial fibrosis was evaluated by late gadolinium enhancement acquisitions. ResultsWe found 27 (56.3%) patients with no MIO (all 16 segmental T2* values >20 ms). The remaining patients showed an heterogeneous MIO (some segments with T2* values >20 ms and other segments with T2* values <20 ms) and of them 2 (9.5%) showed a global T2* value <20 ms, indicating significant MIO. A reduced LV ejection fraction (EF) was found in the 29.5% of cases and a reduced RV EF in the 23.3%. There was not a significant association between heart T2* values and LV EF.Myocardial fibrosis was detected in the 35.9% of the patients. Three patients showed an ischemic pattern and one of them had a transmural fibrosis in the LV apical region. Out of the 3 patients with an ischemic pattern, only one patient had a positive history for a previous myocardial infarction. The majority of the patients had two or more foci of myocardial fibrosis, involving more frequently the septal segments. Patients with myocardial fibrosis were significantly older (75.4±7.9 vs 68.9±7.6 yrs; P=0.019). Global heart T2* and LV volumes were not significantly different between patients with and without fibrosis. The LV EF was lower in fibrotic patients but the statistical significance was not reached (58.4±11.7 vs 64.8±8.9 %; P=0.067). ConclusionsAlthough a significant heart iron was found only in two cases, nearly half the patients had abnormal T2* values in at least one myocardial segment. This finding underlines the importance to use a multislice approach in order to perform an early diagnosis and prevent a more diffuse iron distribution by chelation therapy. This goal could be critical in patients with myocardial fibrosis that seems to be a relative common findings in the old MDS patients. In fact, an underlying heart damage as represented by fibrosis could make the hearts of the old MDS patients more sensitive to lower levels of accumulated iron. Disclosures:No relevant conflicts of interest to declare.

Characterization Of T Follicular Helper Cells In Patients With Low Risk Myelodysplastic Syndromes

A subpopulation of CD4+helper T cells, T-follicular helper cells (TFH), are characterized by the surface expression of CXCR5, ICOS, and PD1, the transcription factor Bcl-6, and produce mainly IL-21, but also IL-17, IL-4, and IFN-γ. They represent the major population that helps B cells to turn into plasma cells and they are implicated in the pathogenesis of different autoimmune diseases. Peripheral CXCR5+CD4+ T helper cells (p-TFH) are the circulating component of TFH. p-TFH cells have been extensively studied in the context of inflammation and autoimmunity. Patients with systemic lupus erythematosus and rheumatoid arthritis have increased p-TFH. Immune dysregulation characterizes low risk myelodysplastic syndromes (MDS). The expression of p-TFH in patients in low risk MDS has not been previously evaluated. To examine the expression of p-TFH in this disease we isolated peripheral blood mononuclear cells (PBMCs) from patients with low risk MDS (n=20, Refractory Anemia,RA; and Refractory Cytopenias with Multilineage Dysplasia, RCMD) and ten healthy, age- matched controls. Written informed consent was obtained from all study subjects. PBMCs were left untreated or activated with PMA and Ionomycin, in the presence of Brefeldin A, for 5 hours. Subsequently, cells were stained with the surface markers CXCR5, CD4, CD45RO, ICOS and intracellular IL-21, and analyzed by flow cytometry. Patients with MDS show decreased CD4+ICOS+ cells compared to healthy controls (5,34±1,44% vs 8,69±4,08% respectively, p=0.028). The median percentages of CXCR5+CD4+ cells (estimated on CD4+ cells) before stimulation were lower in MDS patients although not statistically significant different from the control subjects. The expansion of the CXCR5+CD4+ population after stimulation was higher in MDS patients compared to healthy individuals (ratio of stimulated to unstimulated CXCR5+CD4+ cells of MDS patients: 1.44±0.47 vs control: 1.23± 0.32, p=0.027). MDS patients at diagnosis showed increased median levels of IL-21 producing CXCR5+CD4+ cells compared to patients previously treated with erythropoietin (EPO), (21,34±10,14% vs 17,55±12,91, respectively). Additionally, patients with RA showed increased CXCR5+CD4+IL-21+ cells compared to the RCMD patients (22,39±11,76% vs 17,84±10,70%, respectively). Collectively, the percentage of IL-21 producing CXCR5+CD4+ cells did not differ significantly between the MDS and control groups (20,57± 10,12% vs 19,86± 11,03%, respectively). The presence of marrow fibrosis did not affect the differences observed and described above. The p-TFH cell subset was identified in every case as a memory component of CD4+ T helper cells, as previously described. Although the number of patients analyzed is limited, our results suggest that low risk MDS patients show a trend of lower CXCR5+CD4+ cells compared to age–matched control subjects. EPO treatment eliminates IL-21 production from CXCR5+CD4+ cells compared to treatment-naïve patients. Further analysis of a larger pool of subjects along with the examination of the specific transcription factor Bcl-6 will reveal the role of p-TFH cells in low risk MDS. Disclosures: No relevant conflicts of interest to declare.

Preventing Infections in Higher-Risk Myelodysplastic Syndrome Patients Treated with Hypomethylation Agents

Hypomethylation agents became the standard of care for patients with high-risk myelodysplastic syndrome (MDS). While long-term benefits of azacitidine (AZA) and decitabine (DEC) were demonstrated in multiple studies, methods to enhance patients’ safety during therapy with those agents are pending. The causative correlations between drug administration and non-life-threatening complications such as injection-site erythema or gastrointestinal (GI) discomfort are obvious. However, infections, which are the most common life-threatening complication among higher risk MDS patients, are frequent even in those receiving therapy other than hypomethylation agents, including supportive care solely. Therefore, the contribution of hypomethylation therapy to infection risk is difficult to determine. Herein, data regarding infectious complications, their prevalence, risk stratification, and methods of prevention will be reviewed.

Expression of autophagy related gene Beclin1 in myelodysplastic syndrome patients and its significance

This study was aimed to explore the contribution of autophagy associated gene Beclin1 in the prognosis of myelodysplastic syndrome (MDS) by detecting the expression level of Beclin1 in bone marrow mononuclear cells (BMNC) from 40 MDS patients, 14 non-malignant anemia patients and 25 AML patients. The expression of Beclin1 mRNA was detected by real-time quantitative polymerase chain reaction (qRT-PCR). At the same time, the Western blot was used to analyze the expression of Beclin1 proteins. The results showed that the expression of Beclin1 in low risk MDS patients and non-malignant anemia patients was both significantly higher than that in acute myeloid leukemia patients (P < 0.01). And more interestingly, the Beclin1 mRNA expression in MDS group was negatively correlated with World Health Organization classification-based prognostic system (WPSS) score (r = -0.495). It is concluded that the expression of Beclin1 in the patients with MDS is higher than that in AML patients, and negatively correlated with WPSS scores. Beclin1 is a potential biomarker for predicting prognosis of the patients with MDS.