Myelodysplastic Syndromes Group Research Articles

NIX-mediated mitochondrial autophagy in pathogenesis of myelodysplastic syndrome anemia

Objective: To investigate the change of NIX level of bone marrow nucleated red blood cells in anemia patients with myelodysplastic syndromes (MDS), to explore the significance of NIX-mediated mitochondrial autophagy in the pathogenesis of MDS anemia. Methods: A total of 54 patients with MDS diagnosed in the Department of Hematology of General Hospital, Tianjin Medical University from July 2015 to July 2016 were enrolled into the MDS group, 33 cases of immune thrombocytopenia or idiopathic leukopenia as controls.The level of NIX, the number of mitochondria, mitochondrial membrane potential, the level of reactive oxygen species (ROS) in GlycoA(+) nucleated red blood cells were measured by flow cytometry; the level of NIX mRNA was measured by PCR. Results: (1) The expression of NIX in GlycoA(+) nucleated red blood cells in high-risk MDS patients (0.61±0.24) was significantly lower than that in controls (0.79±0.16, P=0.027), and lower than that in low-risk MDS patients (0.81±0.15, P=0.011), while there was no significant difference between the controls and low-risk MDS patients. The expression of NIX mRNA in GlycoA(+) nucleated red blood cells in high-risk MDS group (0.36±0.09) was lower than that in the controls (1.44±0.41, P=0.027) and that in the low-risk group (1.02±0.22, P=0.012); there was no significant difference between the controls and the low-risk group. (2) The number of mitochondria in GlycoA(+) nucleated red blood cells in high-risk MDS patients (937.17±707.85) was significantly higher than that in the controls (513.49±372.33, P=0.019) and that in low-risk MDS patients (461.74±438.02, P=0.008); while there was no significant difference between low-risk MDS patients and the controls. (3) The level of mitochondrial membrane potential in GlycoA(+) nucleated red blood cells in high-risk MDS patients (0.33±0.18) was significantly lower than that in the controls (0.61±0.32, P=0.001) and that in low-risk MDS patients (0.61±0.34, P=0.001); with no significant difference between low-risk MDS patients and the controls. (4)The level of ROS in GlycoA(+) nucleated red blood cells in high-risk MDS patients (438.65±322.83) was significantly higher than that in the controls (242.77±136.87, P=0.006), and higher than that in low-risk MDS patients (197.40±95.07, P=0.001); no significantly different between low-risk MDS patients and the controls. (5) The number of mitochondria in GlycoA(+) nucleated red blood cell was positively correlated with the percentage of ring sideroblast (r=0.457, P=0.028) in the MDS patients.(6) The number of mitochondria in GlycoA(+) nucleated red blood cells was negatively correlated with the concentration of hemoglobin (r=-0.521, P=0.009) in high-risk MDS patients, but not correlated with the concentration of hemoglobin in low-risk MDS patients. Conclusion: NIX level is reduced in nucleated red blood cells of high-risk MDS patients, which leads to impaired mitochondrial autophagy, increased damaged mitochondria and apoptosis of nucleated red blood cells, thus related with anemia.

Cardiac iron overload in chronically transfused patients with thalassemia, sickle cell anemia, or myelodysplastic syndrome.

The risk and clinical significance of cardiac iron overload due to chronic transfusion varies with the underlying disease. Cardiac iron overload shortens the life expectancy of patients with thalassemia, whereas its effect is unclear in those with myelodysplastic syndromes (MDS). In patients with sickle cell anemia (SCA), iron does not seem to deposit quickly in the heart. Our primary objective was to assess through a multicentric study the prevalence of cardiac iron overload, defined as a cardiovascular magnetic resonance T2*<20 ms, in patients with thalassemia, SCA, or MDS. Patient inclusion criteria were an accurate record of erythrocyte concentrates (ECs) received, a transfusion history >8 ECs in the past year, and age older than 6 years. We included from 9 centers 20 patients with thalassemia, 41 with SCA, and 25 with MDS in 2012-2014. Erythrocytapharesis did not consistently prevent iron overload in patients with SCA. Cardiac iron overload was found in 3 (15%) patients with thalassemia, none with SCA, and 4 (16%) with MDS. The liver iron content (LIC) ranged from 10.4 to 15.2 mg/g dry weight, with no significant differences across groups (P = 0.29). Abnormal T2* was not significantly associated with any of the measures of transfusion or chelation. Ferritin levels showed a strong association with LIC. Non-transferrin-bound iron was high in the thalassemia and MDS groups but low in the SCA group (P<0.001). Hepcidin was low in thalassemia, normal in SCA, and markedly elevated in MDS (P<0.001). Two mechanisms may explain that iron deposition largely spares the heart in SCA: the high level of erythropoiesis recycles the iron and the chronic inflammation retains iron within the macrophages. Thalassemia, in contrast, is characterized by inefficient erythropoiesis, unable to handle free iron. Iron accumulation varies widely in MDS syndromes due to the competing influences of abnormal erythropoiesis, excess iron supply, and inflammation.

Levels and Significance of Serum Interleukin-35 and the New Regulatory T Cells -iTR35 Cells in Myelodysplastic Syndrome Patients

To investigate the significance of serum interleukin-35 level and the new regulatory T cells -iTR35 cells in patients with myelodysplastic syndrome (MDS). Twenty three cases of newly diagnosed MDS were enrolled in this study from January 2014 to January 2016 in Department of Hematology of The First Hospital of Quanzhou in Fujian Province. According to MDS International Prognostic Scoring System (IPSS), the 23 patients were divided into 4 groups: high-risk (n=4), intermediate risk-2 (n=10), intermediate risk-1 (n=5) and low-risk group(n=4). Twenty healthy people of routine physical examination were used as control during the same period. Enzyme Linked Immunosorbent assay(ELISA) and flow cytometry(FCM) were used to detect the expression level of serum IL-35, the proportion of iTR35 cells, and the expression levels of associated molecules such as IL-12p35,IL-27EBl3 respectively. The proportion of CD4+ CD25+ Foxp3+ Treg cells in peripheral blood of MDS patients was significantly higher than that in controls (P<0.01). The proportion of iTR35 cells was also higher than that in controls(P<0.01). However, the proportion of CD4+ CD25- Foxp3+ T cells was not significantly different between 2 groups (P>0.05). The level of serum IL-35 in MDS patients was significantly higher than that in control group (P<0.05). The expression levels of IL-12p35 and IL-27EBl3 in the Treg cells were also significantly upregulated than those in control group(P<0.05), the expression levels of IL-35, IL-12p35 and IL-27EBl3 in MDS group positively correlated with the proportion of iTR35 cells(r=0.92, 0.99 and 0.52, P<0.05, respectively). IL-35 level and the proportion of iTR35 cells in 4 groups of MDS showed significantly difference in general term, no significant difference was found in IL-35 level between the high-risk group and intermediate risk-2 group (P>0.05), but the IL-35 levels in both groups were significantly higher than those in intermediate risk-1 group and the low-risk group (P<0.05), and the level in the intermediate risk-2 group was significantly higher than that in the intermediate risk-1 group and the low-risk group (P<0.05), while there was not different between the intermediate risk-1group and the low risk group (P>0.05). The proportion of iTR35 cells was not significantly different between the high-risk group and the intermediate risk-2 group. The proportions of iTR35 cells in the high-risk group and the intermediate risk-2 group were higher than those in the intermediate risk-1 group and the low-risk group respectively (P<0.05), but there was no differentce in population of iTR35 between the intermediate risk-1 group and the low-risk group (P>0.05). The imbalance between IL-35 level and iTR35 cells propertion may play an important role in the development of MDS, which possibly to provides a new theoretical basis for the study of MDS immune targeting therapy.

Clinical and Biological Role of Accumulation of the NPM-1 Splice Variant R2 in AML, MDS and sAML

BackgroundAcute myelogenous leukemia (AML) represents a heterogeneous group of myeloid malignancies harboring different chromosomal abnormalities, gene mutations, and epigenetic modifications. Recent clinical and biological studies indicate that myelodysplastic syndromes (MDS) and AML could be considered as part of the same continuous disease spectrum rather than as distinct disorders. NPM1 is a multifunctional protein involved in both biological and pathological processes controlling development, cell proliferation, ribosome biogenesis, transformation and genomic stability. It interacts with many cellular proteins, including ARF and the tumor suppressor p53. Recently, we found that high expression of the NPM1 splice variant R2, which encodes a truncated form of NPM1, may provide prognostic value for CN-AML patients.AimsTherefore, our aim was evaluation of NPM1 R2 splice variant significance for MDS and sAML cases, as well as assignment if different expression levels of R2 might have influence on the expression pattern of each of the components of the ARF-MDM2-p53-p21 signaling pathway and additional downstream molecules (miR-34a, miR-34b and miR-34c). In order to determine the impact of NPM1 R2 on NPM1 localization and to compare it with the NPM1mut effect, transfection analyses and IHC stainings were performed.MethodsNPM1 R2, CDKN2A (encoding ARF), MDM2, TP53 and CDKN1A(encoding p21) genes expression levels were assessed for 128 samples (58 AML, 62 MDS and 8 sAML) using qRT-PCR. Additionally, expression level of miR-34a (n=29), miR-34b (n=20) and miR-34c (n=20) was measured in CD33+ cells derived from AML patient samples. WI-38 fibroblasts and HEK-293 cells were transfected with constructs containing eGFP-tagged NPM1-R2, NPM1-mut and NPM1-wt under a cytomegalovirus promoter, stained and visualized with confocal microscope. Immunochemistry analysis was performed for NPM1 in 23 AML bone marrow smears.ResultsNPM1 R2 expression levels differed between AML, sAML, MDS and healthy volunteers (HV) groups and were significantly higher in AML, sAML and MDS groups compared to HVs (median 0.023 vs 0.005, p<0.001, 0.025 vs 0.005, p<0.001 and 0.017 vs 0.005, p<0.001, respectively). CDKN2A, MDM2, TP53 and CDKN1A expression analysis in these sample groups showed also significant differences. Expression of TP53 was elevated in groups with high R2 expression in comparison to groups with low R2 expression in AML and MDS patients (median 0.01 vs 0.005, p<0.001 and 0.007 vs 0.004, p<0.001, respectively). Moreover, we found strong positive correlation of R2 expression with TP53 expression in AML (r=0.77, p<0.001) and MDS (r=0.68, p<0.001). We observed elevated expression of miR-34c in HVs group compared to AML (0.11 vs 0.07, p<0,001) and trend to decreased expression of miR-34a in AML in comparison with HVs. No differences were found in miR-34a, miR-34b and miR-34c expression between groups with high or low R2 expression.Transfection analyses showed various localization of each eGFP-tagged NPM1 forms. NPM1-wt localized mainly in nucleoli, NPM1-R2 was detected in the nucleoplasm and nucleoli, whereas eGFP-NPM1-mut displayed cytoplasmic localization. However, the IHC stainings for AML samples revealed that in cases with high R2 expression we were able to determine a cytoplasmic localization of NPM1 even in the absence of its concomitant mutation.ConclusionsThe elevated level of NPM1 R2 splice variant in AML, sAML and MDS groups versus HVs suggests that R2 might play some role in neoplasia process also in early stages of this hematological malignancy. Transfection analyses established that NPM1 R2 mostly localizes in the nucleoplasm, where it might interact with other proteins e.g. ARF and p53. Nucleolar localization of this NPM1 form might be determined both by lack of nucleolar localization signal present in the wt form of NPM1 and nuclear export signal occurring in mutated NPM1. Moreover, strong positive correlation between R2 and TP53 expression was found in AML and MDS groups suggesting biological link between these transcripts. In summary, the expression of NPM1 R2 might be of biological importance for AML as well as for transformation of MDS into sAML.This work was supported by National Centre for Science Grant HARMONIA (UMO-2013/10/M/NZ5/00313). DisclosuresGrzasko:Celgene: Honoraria; Munipharma: Honoraria; Janssen: Honoraria.

Observational Study of Magnetic Resonance Imaging Monitoring in Patients with Aplastic Anemia (AA) and Low or Int-1 Risk of Myelodysplastic Syndromes(MDS) Complicated with Iron Overload

▪Objective: To observe the clinical characters and magnetic resonance imaging (MRI) T2* change in patients with aplastic anemia (AA) and low or int-1 risk of myelodysplastic syndromes(MDS) complicated with iron overload.Methods:This is a prospective study. Patients with AA and low and intermediate-1 risk factors of MDS complicated with iron overload were enrolled. Clinical parameters and T2* of liver, heart and pancreas were collected regularly.Results: Of all the 92 recruited patients (54 males and 38 females), the median age was 47.5 (15~18) year-old, median disease duration was 26 (3~407) months. There were 54 AA patients and 38 MDS patients. SF was related to liver T2* and pancreatic T2*(AA group r=-0.476,-0.509, respectively, P<0.05; MDS group r=-0.401, -0.565, respectively, P<0.05) in both groups, but not related to cardiac T2* in either group. SF level was correlated with transfusion amount in AA patients (r=0.403, P<0.05), but not in MDS patients (r=0.234, P>0.05. Of the 20 patients who received iron-chelation therapy, 3 (15%) had diarrheas, 2 (10%) decreased in hemoglobin, 2 (10%) decreased in platelet, 2 (10%) had increased creatinine, 1 (5%) had allergic dermatitis, but the symptoms relieved after drug withdrawal. 2 patients died of bone marrow failure during the follow-up period. 38 patients finished the half-year evaluation. Those (18 cases) who chelated adequately had significant decrease in SF (3354.9±3287.3 vs 2119.3±1742.0 ng/ml, P=0.044). Meanwhile an increase in hemoglobin level (83.5±30.0 vs 96.7±31.6 g/L, P=0.01) was noticed in those with decreased SF. 26 patients finished one year follow-up, those (15 cases) who chelated adequately had significant decrease in SF (3583.9±3543.8 vs 1693.9±1532.5 ng/ml, P=0.006), increase in cardiac T2* (25.0±13.4 vs 32.3±15.7 ms, P=0.015) and left ventricular ejection fraction (LVEF) (63.6±8.6 vs 67.1±5.2 %, P=0.028) , whereas no such changes were found in those did not chelate adequately. No change was found either in chelated or non-chelated groups in hemoglobin, WBC count, platelet count, SGPT, TBil, creatinine level, liver or pancreas T2* value. For those with decreased SF, even without chelation, increase in hemoglobin level (91.1 ±29.9 vs 108.4±31.4 g/L, P=0.046),cardiac T2* (25.5±13.7 vs 33.1±15.9 ms, P=0.014)) and LVEF level (63.6±8.6 vs 67.2±5.4 %, P=0.027) were also documented.Conclusion: SF was related to liver T2* and pancreatic T2* in both AA and MDS groups. SF was relevant to transfusion amount in patients with AA but not MDS. Adequate iron chelation can decrease the SF level, and might bring improvement in cardiac T2* and LVEF level. Decrease of SF even without chelation might also benefit patients. DisclosuresNo relevant conflicts of interest to declare.

Interest of the XN-10® Analyzer to Screen for Myelodysplastic Syndromes on Complete Blood Count (CBC) Especially with Hemoglobin Levels and Neut-WX

A prospective study was performed over one year at Nantes University Hospital in France, in order to investigate whether suspected myelodysplastic syndromes (MDS) could be detected on a complete blood count (CBC), the most rapid laboratory investigation. Indeed, the recently developed XN-10® (Sysmex, Kobe, Japan), provides novel CBC parameters witch could be useful to discriminate such patients from normal samples or from cytopenia of other etiology.Seventy-nine patients were enrolled in the study, for whom a diagnosis of MDS was concluded based on CBC, bone marrow smears examination and karyotype. All patients were free of treatment, including transfusions, at inclusion. They were 40 men and 39 women with a median age of 77,9 years (range 36,4-92,4). CBC were performed on a Sysmex analyzer XN-10®, including investigation of reticulocytes and fluorimetric analysis of platelets. For comparison with normal values, results from 776 healthy samples, for which CBC were performed on the same analyzer and generated no flag, were used. All had parameters within the normal range according to age. The classical parameters of hemoglobin level, Mean Corpuscular Volume (MCV), reticulocytes, platelets and neutrophil counts were recorded. In addition, the extra-parameters, immature reticulocytes fraction (IRF%), platelets by fluorescence (PLT-F) and immature platelets fraction (IPF%), were taken into account. The neutrophils median position on the three axes as well as their dispersion (Neut-WX) were also measured by the analyser.The primary end-point was to discriminate between MDS and healthy patients and the secondary end-point was to distinguish MDS with excess blasts, MDS with multilineage dysplasia and MDS with single lineage dysplasia within the MDS group and by comparison with controls. According to the WHO 2016 classification, 27 patients in the cohort had MDS with excess blasts, 26 MDS with multilineage dysplasia (among whom 7 had ring sideroblasts [RS], group 2), 16 MDS-RS and single lineage dysplasia, 7 MDS with single lineage dysplasia and 3 MDS with isolated del(5q). Forty-four patients had a normal karyotype and 28 displayed anomalies classically reported in MDS, including 5 complex karyotypes. Among the latter, 4 were associated with MDS with excess blasts.Both classical and extra parameters indeed showed significant differences between the subgroups tested. Among the whole group of MDS patients, a number of parameters of all lineages were statistically different from the healthy cohort. The median level of hemoglobin was 9,8 g/dL (range 4,7-14,9), (p<0,0001), the median MCV 104,3 fL (range 75,4-123,9; p<0,0001), reticulocyte counts 44,3x109/L (range 8-165,9; p=0,041) and IRF% 16,7% (range 2,4-50,9; p<0,0001). An hemoglobin value below 11,5 g/dL was strongly suggestive of MDS with a sensitivity of 81% and specificity of 100%. The median platelet count was 164x109/L (range 8-505; p<0,0001) and median IPF% 8,8% (1,2-42; p<0,0001). Among leukocyte parameters, the MDS median neutrophil count was significantly lower at 2,15x109/L (range 0,17-13,67; p<0,001) and the Neut-WX value increased above 350. The latter, by itself, allowed to make a diagnosis of MDS with a sensitivity of 73,1% and a specificity of 96,9%.When considering the three MDS subgroups of MDS with excess blasts, multilineage or single lineage dysplasia, although each of them was significantly different from controls for hemoglobin levels, MCV, IRF% and neutrophil counts (p<0,0001), they could not be discriminated by these parameters. In the subgroup of MDS with single lineage dysplasia, platelet counts were similar to those of controls, yet significantly higher than for MDS with excess blast or with multilineage dysplasia (p=0,004 and p=0,029 respectively).Taken together, this study demonstrates that a simple CBC allows to screen for MDS using thresholds of 11,5 g/dL for hemoglobin and of 350 for Neut-WX. Blood smear examination should be performed in this situation even if the XN-10® analyzer does not raise an alarm, especially in unknown older patients. DisclosuresNo relevant conflicts of interest to declare.

Immune Dysfunction in Patients with MDS Is Partially Corrected in EPO-Treated Patients: Differences According to WHO Classification

Background: The immune system has been shown to be involved in the pathogenesis of myelodysplastic syndromes (MDS), and is also affected by the disease. Recombinant erythropoietin (rHuEPO), or in general, erythroid stimulating agents (ESAs) have become a standard treatment for anemic patients with MDS. They were found to improve anemia, quality of life, and possibly survival. We have previously demonstrated that EPO has effects on cellular and humoral immunity and specifically, on immune function in patients with multiple myeloma (MM). Here we report our findings demonstrating the effect of ESAs on T cell (CD4+, CD8+ and CD4+CD25+) number and function in patients with MDS.Patients and Methods: We examined three groups: healthy subjects ('Control', 20 participants), MDS patients without ESA treatment ('MDS', 13), and MDS patients treated with an ESA ('MDS+EPO', 17). All diagnosed patients gave informed consent as approved by our IRB. Cell numbers were evaluated with flow cytometry. In a subset of patients, cell activation was assessed in response to phytohemagglutinin (PHA) by examining CD69 expression in both CD4+ and CD8+ cells. The co-stimulatory marker, CD28, and the inhibitory marker CTLA-4 (CD152) were evaluated as well. We also examined World Health Organization (WHO) subgroups, refractory anemia (RA) and RA with ringed sideroblasts (RARS) versus more advanced disease.Results: CD4+ and CD8+ T cell levels are reduced and increased respectively in MDS patients compared to control, and these changes are reversed in MDS+EPO (Table 1, CD4+, p<0.01; CD8+, p=0.05). The CD4+:CD8+ ratio (Table 1) is reduced and nearly equalized in MDS (1.16), but approaches that of the control (2.24) in MDS+EPO (1.94). CD4+CD25+ T cell numbers (including regulatory T cells), were lower in MDS patients and improve in the MDS+EPO group (Table 1). In vitro activation of T cells (CD4+CD69+ and CD8+CD69+) achieves an approximately 15-fold increase in healthy subjects. MDS patients without EPO sustained only a 7.17 fold increase in CD4+ activation versus 13.64 fold for the MDS+EPO group (p<0.01); for CD8+ T cells, 10.20 fold (MDS) versus 18.56 fold (MDS+EPO, p<0.01). The expression of the co-stimulatory marker CD28 was decreased in both CD4+ and CD8+ T cells in MDS, and approached normal in MDS+EPO in CD4+ T cells (Table 1). There was no significant change in inhibitory CTLA-4 (CD152) expression among the groups (not shown). Subgroup analysis demonstrated that ESA has a similar effect on CD4+ and CD8+ cells and their ratio in both RA/RARS and more advanced disease, similar to those of the whole cohort (Table 2, green). On the other hand, some parameters were affected by ESA only in one subgroup (Table 2, blue): The ESA effect on CD4+CD25+ cells was evident only in patients with advanced disease (Table 2, blue). ESA affected CD4+ and CD8+ cell stimulation (CD69) in RA/RARS, similar to that seen in the whole cohort (Table 2, blue). Of note, in more advanced disease, CD4+ and CD8+ cells achieved stimulation in the MDS group not treated with ESA, with no difference between MDS and MDS+EPO. This finding needs to be further addressed in larger cohorts and with additional markers of activation.Conclusions: MDS patients display T-cell abnormalities that are improved upon EPO treatment. MDS is a heterogeneous disease where the immune system both affects and is affected by the disease. As such, treatment with ESAs might ameliorate not only the anemia, but also the immune deficiencies and perhaps the disease itself. Future studies will clarify the immunomodulatory role of ESA in the various stages of MDS. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Azacitidin Is Effective in the Therapy Related Myelodysplastic Syndrome.

Introduction:Myelodysplastic syndromes (MDS) are clonal hematopoietic neoplasms characterized by abnormal maturation of precursor cells which often translates into peripheral blood cytopenias and a high rate of transformation to acute myeloid leukemia (AML) due to accumulation of genetic alterations.The AZA-001 trial showed azacitidine (AZA) significantly prolonged median overall survival compared with conventional care regimens (24.5 vs 15.0 months; P=0.0001).AZA is standard first-line treatment for Intermediate-2 and High-risk myelodysplastic syndrome patients who are not immediate candidates for allogeneic stem cell transplantation, but this study included no cases of therapy related MDS (t-MDS).T-MDS is known to have poor prognosis, therefore it is very important to analyze the outcome of patients with t-MDS treated in the front-line with AZA.Methods: We studied newly diagnosed 29 MDS patients who were treated by AZA in our hospital from July, 2010 to April, 2016, retrospectively. AZA was given subcutaneously at 75 mg/m2 per day for 5 or 7 days every 28 days.Results: We analyzed 29 MDS patients. According to the WHO classification, there were 12 RA, 15 RCMD, 10 RAEB-1, 2 RAEB 2 and 1 MDS-U. The median age was 70 year (range 49-88), and men was 12 (41.3%). There were 12 de novoMDS cases (41.3%) and 17 t-MDS cases (58.6%). All of the t-MDS patients had previously received chemotherapy (17 patients, 100%) and 9 patients had also received radiotherapy (9 patients, 53%).Very poor risk group was 47.1% (9/17) in t-MDS group compared to 25.0% (3/12) in de novo MDS group (P=0.26).Median follow up time was 11.4 months (range 1.4-47.8). Twenty five patients (86.3%) were treated by AZA for 5 days. Four patients (13.7%) were treated by AZA for 7 days, but all 4 patients decreased the dosing period to 5 days due to unacceptable toxicity. AZA was given for a median of 4 cycles (range 1-33).In 29 MDS patients, 1-year overall survival (OS) was 60.5% (95% CI, 38.7-76.7%) and 1-year PFS was 40.1% (95% CI, 18.8-60.6%). After a median follow-up of 11.4 months, median OS was 18.7 months (95% CI, 9.4-21).One-year OS was 59.3% in t-MDS group compared to 63.6% in de novo MDS group (P=0.294). 1-year PFS was 38.4% in t-MDS group compared to 40.4% in de novo MDS group (P=0.626).One-year OS was 37.5% in very poor risk karyotype group (R-IPSS) compared to 74.6% in not very poor risk karyotype group (P=0.000748). 1-year PFS was 43.2% in very poor risk karyotype group compared to 39.0% in not very poor risk karyotype group (P=0.594).Focusing on t-MDS group, 1-year OS was 46.9% in very poor risk karyotype group (8/17 47%) compared to 74.1% in not very poor risk karyotype group (9/17 53%) (P=0.054). 1-year PFS was 48.0% in very poor risk karyotype group compared to 26.0% in not very poor risk karyotype group (P=0.339).Conclusions: In our study, 1-year OS in all MDS patients was 60.5%. It was slightly poor prognosis than 1-year OS in AZA-001 trial (about 70%).Our study include t-MDS cases (58.6%). Additionally, AZA was given for a median of 4 cycles in our study but 6 cycles in the AZA-001 trial. It showed severe patient's background of our study. These difference may cause the lower median OS and poorer prognosis.There trended to be more patients who had very poor risk karyotype in t-MDS group, but there was no significant difference between t-MDS and de novoMDS for the 1-year OS and PFS.Azacitidin is effective in the therapy related myelodysplastic syndrome. DisclosuresYokoyama:Chugai: Consultancy. Mishima:Chugai: Consultancy. Nishimura:Chugai: Consultancy. Terui:Yanssen: Honoraria. Hatake:Kyowa Kirin: Honoraria, Research Funding; Chugai: Research Funding; Otsuka: Consultancy; Meiji-Seika: Consultancy.

Increased expression of HERG K+ channels contributes to myelodysplastic syndrome progression and displays correlation with prognosis stratification

Objectives: Human ether-a-go-go-related gene (HERG) K+ channels are shown to be aberrantly expressed in a variety of cancer cells where they play roles in contributing to cancer progression. Myelodysplastic syndromes (MDS) are a group of clinical heterogeneous disorders characterized by bone marrow failure and dysplasia of blood cells. However, the involvement of HERG K+ channels in MDS development is poorly understood. Methods: The expression of HERG K+ channels in untreated MDS, acute myeloid leukemia (AML) patients and the control group was detected by flow cytometry. The roles of HERG K+ channels in regulation of SKM-1 cell proliferation, apoptosis, and cell cycle were determined by CCK-8 assay and flow cytometry, respectively. Results: We found that expression of HERG K+ channels in MDS patients was significantly higher than controls and was lower than AML. Percentage of HERG K+ channels on CD34+CD38− cells gradually increased from controls to high-grade MDS subtypes. And HERG K+ channel levels showed an ascending tendency from low-risk to high-risk MDS group. In addition, the CCK-8 assay, apoptosis and cell cycle analysis were performed and showed that blockage of HERG K+ channels decreased the proliferation of MDS cells but rarely had effects on cell apoptosis and cell cycle distribution. Conclusion: Our study demonstrated that HERG K+ channels might be a potential tumor marker of MDS. These channels were likely to contribute to MDS progression and were helpful for predicting prognosis of MDS. Inhibition of HERG K+ channels might be a novel therapeutic measure for MDS.

Analysis of hematopathology and alteration of JAK1/STAT3/STAT5 signaling axis in experimental myelodysplastic syndrome

Hematological disorders like myelodysplastic syndrome (MDS) may arise due to cumulative dysregulation of various signalling pathways controlling proliferation, differentiation, maturation and apoptosis of bone marrow cells. This devastating bone marrow condition can be due to consequential abnormalities in haematopoiesis as well as its supportive microenvironment. Although mutations related to JAK/STAT pathway are common in myeloproliferative neoplasms, further studies are required to fully explore the myelodysplastic scenario regarding the concerned pathway. In this study, we have investigated the JAK-STAT signalling pathway which inevitably plays a crucial role in haematopoiesis. MDS was mimicked in a mouse model with an induction of ENU in adult mice. The bone marrow of the control and MDS groups of animals were subjected to a variety of tests, including cell morphology study in peripheral blood and bone marrow, cytochemistry and histochemistry of bone marrow smears, karyotyping and flowcytometric expression analysis of the phosphorylated forms of proteins like JAK1, STAT3 and STAT5 (denoted as pJAK1, pSTAT3 and pSTAT5) and the phenotypic expression of proteins like CD45 and CD71. The results revealed that the morphology of the blood and bone marrow cells were dysplastic compared to the affected blast populations of different lineages. The expression of common leucocyte antigen CD45 was less in comparison to the expression of transferrin receptor CD71 which was increased in the ENU induced MDS mouse model. Moreover, we have observed an upregulated expression of JAK1 followed by STAT5. Therefore, we can conclude that downregulation of CD45 may have helped in the upregulation of JAK-STAT signaling and CD71 expression. This aberrant signaling may be among one of the activated signaling axes that lead to affected hematopoietic lineages in Myelodysplastic syndrome.

Value of a Panel Fluorescence in Situ Hybridization in Three Kinds of Hematological Malignancies

To evaluate the role of a panel fluorescence in situ hybridization (Panel-FISH) for the detection of common cytogenetic abnormalities in patients with chronic lymphoblastic leukemia (CLL), multiplemyeloma (MM) and myelodysplastic syndrome (MDS). Three panels of probes were used to perform FISH assays in 46 patients with CLL, 53 with MM and 93 with MDS. Their results were compared with that obtain by conventional cytogenetic examination. The panel FISH detection in CLL and MM groups showed significantly higher sensitivity in revealing chromosomal abnormalities than that in conventional cytogenetics (73.8% vs 9.5%, 70.8% vs 22.9%, respectively). There were significant differences between these 2 technologies(P<0.001, P<0.001, respectively). However, there was no difference between Panel-FISH and conventional cytogenetics in MDS group (30.4 vs 27.2%, P=0.625). Panel-FISH can increase the detection rate in CLL and MM patients while it did not in MDS patients. However, it can increase the detection rate of aberration clones in MDS cases with normal karyotypes or without enough karyotypes to be analysis.

IL-32 mRNA Expression of Bone Marrow Stromal Cells and Its Correlation with Cell Apoptosis in Patients with Myelodysplastic Syndrome

To investigate the IL-32 mRNA expression of bone marrow stromal cells and its correlation with apoptosis of bone marrow mononuclear cells in patients with myelodysplastic syndrome (MDS). Bone marrow samples from 26 MDS patients and 10 iron deficiency anemia (IDA, as control) patients were collected, RT-PCR was used to detect the IL-32 mRNA expression of bone marrow stromal cells, and the apoptosis of bone marrow mononuclear cells was detected by flow cytometry with Annexin V-FITC/PI dowble staining. The born marrow lymphocytes and NK cells were detected by means of direct immunofluorescence labeling whole blood hemolysis and flow cytometry. IL-32 mRNA expression of bone marrow stromal cells in the MDS patients was significantly higher than that of control group, the IL-32 mRNA expression of bone marrow stromal cells in patients with RA, RAS and RCMD was significantly higher than that in patients with RAEB. There was no obvious difference between RAEB and the control groups. The apoptosis of bone marrow mononuclear cells in MDS group was significantly higher than that in the control group, the apoptosis of bone marrow mononuclear cells in patients with RA, RAS and RCMD was significantly higher than that in RAEB. There was no significant difference between RAEB group and control group. The IL-32 mRNA expression in bone marrow stromal cells significantly correlated with the apoptosis of bone marrow mononuclear cells in MDS patients. The NK cell number in born marrow of MDS patients and the control group had no significant difference. The expression of IL-32 mRNA in bone marrow stromal cells significantly relates with the apoptosis of MDS cells, and the secretion of IL-32 by bone marrow stromal cells may be one of the reasons for the apoptosis of MDS bone marrow cells. It is speculated that the abnormal MDS bone marrow microenvironment is involved in the apoptosis of bone marrow cells.

Effects of decitabine on megakaryocyte maturation in patients with myelodysplastic syndromes.

Thrombocytopenia is a common, often fatal complication experienced by patients with myelodysplastic syndromes (MDS). 5-aza-2′-deoxycytidine (decitabine) has been used to treat MDS patients with thrombocytopenia with a response rate of 45–50%. However, the mechanism of its effects on megakaryocytes remains unclear. In the present study, the effect of decitabine on megakaryocyte maturation was investigated. A total of 20 MDS patients diagnosed with thrombocytopenia were enrolled, including 16 refractory anemia with excess blasts (RAEB)-1 patients and 4 RAEB-2 patients], in addition to 20 leukemia patients that had achieved complete remission and 20 healthy donors. Overall, 65% of MDS patients exhibited a response to decitabine, with an increase in platelet count identified in 80% of patients. In the MDS group, the mean platelet count was significantly increased following one cycle of decitabine chemotherapy (36.85±24.54 vs. 84.90±61; P=0.001); however, no significant difference in megakaryocyte number was identified prior to and following treatment. Additionally, bone marrow mononuclear cells of the MDS patients were cultured in vitro with various concentrations of decitabine (0.0, 2.0, 2.5, 3.0 µM), and cluster of differentiation (CD)41 levels were examined via flow cytometry. The MDS and normal control groups exhibited the highest levels of CD41 expression following treatment with 2.0 µM decitabine (mean fluorescence intensity, 294.07±47.34 and 258.95±28.05, respectively). In conclusion, these results indicate that the DNA-hypomethylating agent, decitabine, may induce the differentiation and maturation of myelodysplastic megakaryocytes in MDS patients, even at low concentrations. Thus, the repeated administration of decitabine at lower doses in MDS patients may be useful in clinical practice, and may lead to the development of alternative treatments for other diseases of abnormal megakaryocyte differentiation, such as idiopathic thrombocytopenic purpura, however, future studies are required to investigate this.

A Study of Human Leukocyte Antigen Mismatched Cellular Therapy (Stem Cell Microtransplantation) in High-Risk Myelodysplastic Syndrome or Transformed Acute Myelogenous Leukemia

The treatment outcomes of myelodysplastic syndrome (MDS) and transformed acute myelogenous leukemia (tAML) remain very unsatisfactory. We designed a combination of human leukocyte antigen (HLA)-mismatched hematopoietic stem cell microtransplantation (MST) with chemotherapy for patients with MDS and tAML and evaluated its effects and toxicity. Patients were between 13 and 79 years old. Patients with MDS (n=21) were given HLA-mismatched MST combined with decitabine and cytarabine; patients with tAML (n=22) were given HLA-mismatched MST combined with decitabine and cytarabine, and also mitoxantrone. Patients in complete remission (CR) also received MST plus decitabine and medium-dose cytarabine chemotherapy without graft-versus-host disease (GVHD) prophylaxis. The overall response rate of the patients with MDS was significantly higher than that of those with tAML (81% vs. 50%; p=.03). The CR rates were 52.4% and 36.4% in the two groups, respectively. There was no difference in the cytogenetic CR rate between the MDS and tAML groups (85.7% vs. 70%, respectively; p=.7). The 24-month overall survival of the patients with MDS was significantly higher than that of the patients with tAML (84.7% and 34.1%, respectively; p=.003). The median recovery times of neutrophils and platelets were, respectively, 14 and 17 days in the patients with MDS, and 16 and 19 days in those with tAML. The treatment-related mortality rates were 4.8% and 18.2%, respectively, in the MDS and tAML groups (p=.34). No GVHD was observed in any patient. Microtransplantation combined with decitabine and chemotherapy may provide a novel, effective, and safe treatment for high-risk MDS and tAML. Microtransplantation (MST) refers to regular chemotherapy combined with granulocyte colony-stimulating factor-mobilized peripheral blood stem cell infusion of human leukocyte antigen-mismatched donor cells without using immunosuppressive agents. It aims to support hematopoietic recovery and perform graft-versus-leukemia (GVL) effects but differs from traditional allogeneic stem cell transplantation because the rate of donor cell chimerism is low and there is and no graft-versus-host disease (GVHD) risk. Thus, a trial was designed to evaluate the safety and efficacy of MST in patients with myelodysplastic syndrome and those with transformed acute myelogenous leukemia. Higher complete remission and cytogenetic complete response rates were observed, and the treatment improved disease progress-free survival, sped hematopoietic recovery, and avoided GVHD.

Significance of Morphological Examination, Cytochemical Staining Combined with Bone Marrow Biopsy in Differential Diagnosis of Myelodysplastic Syndrome with Low Blasts and Hemolytic Anemia

To explore the value of morphological examination, cytochemical staining combined with bone marrow biopsy in the differential diagnosis between myelodysplastic syndrome (MDS) with low blasts and hemolytic anemia (HA). The clinical data of 85 cases of myelodysplastic syndrome with low blasts (< 5%) and 61 patients with hemolytic anemia in Chinese PLA's Gerneral hospital from September 2009 to March 2015 were retrospectively analysed. The clinical characteristics, cytogenetic and molecular features, bone marrow cell count and morphology features, cytochemical staining results and bone marrow biopsy features of above-methioned patients were compared. There was no significant difference (P > 0.05) in clinical data between MDS group and HA group. Megakaryocytic dysplasia-positive rate, and ring sideroblasts positive rate, and PAS positive rate were significantly higher in MDS group than those that in HA group (P < 0.05). Abnormal localization of immature precursors (ALIP) and megakaryocytic dysplasia positive rate in bone marrow biopsy were significantly higher in MDS group than those that in HA group (P < 0.05), 90.6% of MDS with low blasts patients were identifiable by combined detections. Combining detection of morphology, cytochemistry staining and bone marrow biopsy has been confirmed to be more useful for differential diagnosis between MDS with low blasts and HA.

Chronic Iron Overload Damage the Erythropoiesis in MDS Mouse By Increasing GDF11 and ROS Levels

Iron overload (IO) is a kind of disease with an excess of iron depositing in the body's organs and tissues, resulting in the dysfunction of these organs and tissues, which contains hereditary hemochromatosis and secondary IO such as ineffective hematopoiesis in aplastic anemia and myelodysplastic syndrome (MDS) and blood transfusion. Numerous researches and our previous study found that IO can damage the hematopoietic function in bone marrow. MDS is one of the most common IO diseases and IO can further injure the hematopoietic function especially on erythropoiesis in MDS.However, it is indeterminacy on the mechanism behind this stiuation. This study is to explore the possible mechanism of IO on the injury of MDS erythropoiesis though establishing an IO MDS mice model.In this study, we first established an IO mice model by administering iron dextran(25 mg/ml) by intraperitoneal injection every 3 days for 2 months in wild and MDS mouse(NUP98-HOXD13 transgenic mouse). The mouse were devided into four grups: the wild mouse without IO(Ctrl group), the wild mouse with IO (IO group), MDS mouse without IO(MDS group), the MDS mouse with IO(MDS+IO group). After 6 months, the establishment of IO mice models were confirmed by both the iron deposits staining of bone marrow(BM), liver, spleen and the labile iron pool level of bone marrow mononuclear cells( BMMNCs).We next found that IO damage the erythropoiesis in MDS mice. We detected the proportion of different stages BM erythroblasts by flow cytometry plots of Ter119+ CD71+ cells (R1, basophilic erythroblasts, poly red cells, immature red blood cells) and Ter119+ CD71- cells (R2, late erythroblast and reticulocyte ). It was found that the hematopoietic colony-forming unit (CFU-E, BFU-E) in MDS+IO group were less than that in other groups. The proportion of R1(10.39±1.43) and R2(1.58±0.54) in MDS+IO group were the least compared with Ctrl group(R1 17.36±3.45,R2 4.59±1.57),IO group(R1 14.53±2.65,R2 2.56±0.76) and MDS group(R1 13.64±3.27,R2 2.35±0.78). And the reticulocytes proportion in MDS+IO group(11.36±3.65) was the highest compared with Ctrl group(5.159±1.537), IO group(7.61±1.36)) and MDS group(7.35±1.39).Then we further explored the mechanism of IO damage on MDS erythropoiesis. It was reported that TGF-β family and oxidative stress (ROS) played an important role on erythropoiesis. Our previous studies also had shown that the ROS and GDF11(a member of TGF-β family) levels were raised in BMMNCs on C57BL/6 mice with IO. Similarly, we detected them on the BM erythroblast in this study. Firstly, we sorted the R1 and R2 erythroblasts,then we performed RT-PCR to analyze mRNA levels of the TGF-β family. We found that the mRNA levels of GDF11, GDF15, Activin B, Acvr2b and ALK5 in MDS+IO group were the highest compared with that of other groups, while the mRNA levels of Activin A, ALK4 and BMP8 had no significant change in these groups. As we found that the change of GDF11 level was the most significant in MDS+IO group compared with other groups, we focused on the study of GDF11 in the following experiment. The concentration of GDF11 in peripheral serum was detected by ELISA method. It was found that the concentration of GDF11 was the highest in MDS+IO group compared with other groups. And also, the GDF11 related signal pathway proteins (Smad 2, Smad 3, p-Smad 2 and p-Smad 3) were significantly elevated in R1 and R2 erythroblast in MDS+IO group compared with other groups,which indicated that GDF11 related signal pathway was involved in the deficient of BM erythropoiesis in MDS+IO mice. Furthermore, We found that the ROS levels in Ter119+ cells in MDS+IO group, MDS group and IO group were increased 4.15 folds, 2.16 folds , 2.63 folds compared with Ctrl group. RT-PCR was used to detect the mRNA expression levels of the gene NOX4 related to ROS generation and GPX1 related with ROS clearance. It was found that the NOX4 expression levels were the highest in MDS+IO group compared with other groups,while the GPX1 mRNA level was the least in MDS+IO group.In conclusion, our study indicated that chronic IO damage the erythropoiesis in MDS mouse by increasing GDF11 and ROS levels which would provide an theoretical basis to find new targets for the treatment of IO in MDS patients with erythropoiesis dysfunction. DisclosuresNo relevant conflicts of interest to declare.

Eltrombopag Use in Chronic Myelomonocytic Leukemia (CMML) Patients: A Cautionary Tale

▪IntroductionThrombocytopenia is a management challenge for Myelodysplastic syndromes (MDS) patients (pts). The outcome of MDS Pts after hypomethylating agents (HMA) failure is poor. Eltrombopag is an oral, small non-peptide thrombopoietin (TPO) receptor agonist. It has biologically distinct effects in part to its binding site on the TPO receptor that is distinct from that for native TPO and other synthetic agonists. We report our experience among CMML pts treated with eltrombopag in the context of a phase I MDS study.MethodsThis is a phase I study. Pts are allocated to dose cohorts of 50, 100, 150, 200 mg/day. Key eligibility criteria include confirmed diagnosis of MDS or acute myeloid leukemia (AML) with 20-30% myeloblasts. All pts had HMA failure. The mean platelet count within a month of enrollment must be ≤ 50 X 109/L. Key exclusions include splenic enlargement > 8 cm, bone marrow fibrosis > grade 3. We identified CMML pts and compared the responses and adverse events to MDS patients.ResultsAmong 33 pts enrolled on the phase I study, 7 were CMML pts. Table-1 summarizes baseline characteristics. The median age of CMML pts was 76 years, all were white males. Five pts were CMML-2. Majority were classified as higher risk by IPSS, revised IPSS and MD Anderson model. Four pts were proliferative CMML. Among CMML pts, one received eltrombopag at 50 mg, 2 pts at100 mg, 2 pts at 150 mg, and 2 pts at 200 mg. Three pts completed 8 weeks to be evaluable for response and toxicity.Hematological improvement or better by IWG 2006 criteria (HI+) was observed in 6/26 (23%) MDS pts and 1/7 (14%) CMML pts (p 0.16). The responding CMML pt had bilineage response. Five non CMML pts had a platelet response. Two CMML pts became platelet transfusion independent compared to 4 non CMML pts. The rate of AML transformation was 39% and 29% respectively in MDS and CMML pts (p 0.14). The median OS was 7 months for CMML pts compared to 5 months for non-CMML (p 0.2).Five (71%) CMML pts developed leukocytosis on treatment compared to 3 MDS pts (12%). (p 0.001). (3 out of 5 pts had proliferative type CMML). Leukocytosis developed within first cycle in all pts. In 2 CMML pts, leukocytosis resolved after stopping treatment. Four CMML pts (57%) showed peripheral myeloblasts on treatment compared to 11 (42%) in the MDS group (p 0.2). One CMML pt (14%) developed grade 3 fibrosis from grade 0-1 at baseline compared to 3 MDS pts (12%). Data will be presented on ongoing Next generation sequencing for somatic mutations comparing CMML pts to MDS pts and those who developed leukocytosis/circulating myeloblasts to pts who did not.ConclusionsEltrombopag yielded lower responses in CMML pts after HMA failure compared to MDS pts but namely due to stopping treatment in majority of pts due to leukocytosis or circulating myeloblasts. At the time being, eltrombopag should only be offered to CMML pts in the context of clinical trials.Table 1Baseline CharacteristicsCMML (n=7)MDS (n= 26)P valueAgeMean77.5750.20GenderMale7 [100%]18 [69%]0.09RaceWhite7 [100%]24 [92%]0.40ECOG PS0 11 [14%] 6 [86%]6 [23%] 20 [77%]0.70MyeloblastsPlateletsHgbWBCMean % Mean Mean Mean9 30 10.5 1413 22 9.5 30.08 0.20 0.20 0.03Cytogenetic RiskGood Intermediate Poor4 [57%] 2 [29%] 1 [14%]15 [56%] 3 [11%] 8 [33%]0.40WHO classificationRCMD RAEB-1 RAEB-2 RAEB-t (AML) CMML-1 CMMl-2 MDS/MPN-U Del 5q0 0 0 0 2 [29%] 5 [71%] 0 06 [23%] 7 [27%] 11 [42%] 1 [4%] 0 0 0 1 [4%]IPSSLow Int-1 Int-2 High1 [14%] 0 4 [57%] 2 [29%]0 11 [42%] 9 [35%] 6 [23%]0.05R-IPSSVery low Low Intermediate High Very High0 1 [14%] 0 5 [72%] 1 [14%]0 2 [8%] 5 [19%] 9 [35%] 10 [38%]0.20 DisclosuresPadron:Novartis: Speakers Bureau; Incyte: Research Funding. Lancet:Celgene: Consultancy, Research Funding; Amgen: Consultancy; Kalo-Bios: Consultancy; Seattle Genetics: Consultancy; Pfizer: Consultancy; Boehringer-Ingelheim: Consultancy. List:Celgene Corporation: Honoraria, Research Funding. Komrokji:Pharmacyclics: Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding. Off Label Use: use of eltrombopag in MDS and CMML.

Long-Term Outcome of Myelodysplastic Syndromes (MDS) Patients Treated with Erythropoiesis Stimulating Agents (ESAs)

Background: The majority of MDS patients (pts) have anemia and are treated initially with ESAs. Particularly for lower-risk MDS pts (International Prognostic Scoring System (IPSS) Low and Int-1), once ESAs are no longer effective, treatment options are limited to drugs commonly used for higher-risk MDS, such as hypomethylating agents, or off-label use of immunomodulatory drugs. As a result, most pts receive only transfusion support post-ESA, representing a pt group with an unmet medical need frequently targeted for drug development, for whom long-term outcome is unknown.Methods: We studied pts diagnosed with lower-risk MDS from 1997-2014 at MDS CRC institutions and treated with ESAs (epoetin alpha (epo) or darbepoetin (darb)). The best response to treatment was categorized per International Working Group 2006 response criteria (hematological improvement (HI), complete response (CR), or partial response (PR)). The primary endpoint was overall survival (OS) at the time of ESA failure, defined as cessation of treatment due to relapse or refractoriness; a secondary endpoint was time to AML transformation or death, from time of response (for responders) or failure (for nonresponders) determination. Descriptive statistics were used for baseline characteristics. The Kaplan Meier method was used to estimate OS and a log rank analysis was used to compare response categories. Cox regression analysis was performed for multivariable analysis.Results: Of 206 patients included in analyses, median age was 71.6 years (range: 25.3-88.1), 36% were female, 5% were African-American, and 11% had t-MDS. WHO categories included RA (14%), RARS (16%), RCMD (42%), MDS-u (6%), del (5q) (4%), RAEB-1 (9%), RAEB-2 (2%), RARS-T (2%), MDS/MPN-u (3%), and CMML-1 (2%), with pts classified as IPSS Low (39%), Int-1 (61%), or IPSS-R Very Low (16%), Low (55%), Intermediate (26%), and High (4%). IPSS cytogenetic risk groups were Good (72%), Intermediate (22%), and Poor (6%). Baseline median hemoglobin was 9.4 g/dl (range: 5.5-14.2), serum epo level was 97.2 (range: 14.2-3899.0), and 11% were transfusion-dependent. Treatment included darb (59%) and epo (41%) at median doses of 300 mcg (range: 100-500) and 40,000 units (range: 5,000-80,000), respectively. Pts remained on therapy for a median of 30.4 weeks (range: 0.0-447.7) and had a median follow-up of 28.4 months (95% confidence interval (CI): 24.5, 45.4). First treatments following ESA failure included azacitidine (41.7%), decitabine (10.2%), lenalidomide (16.6%), experimental drugs (3.1%), other growth factors (13.6%), ATG and/or other immunosuppressants (8%), chemotherapy (0.1%) , transplant (0.1%) and others (6.6%). The overall response rate (ORR) to ESAs was 18.8%, with 0% achieving CR; 0.1% PR; and 18.7% HI. Responses for epo were 17.3% and for darb were 19.8% (p=.67 for difference). For both ESAs, 81.2% of patients had disease refractory to treatment: 69.4% with stable disease and 12% with progressive disease with no significant differences between epo and darb by responder status. Median response duration for epo and darb were 21.9 weeks (range: 3.0 - 447.7) and 39.1 weeks (range: 0.0 - 350.7) respectively (p=0.045). Median survival from the date of diagnosis was 28.4 months (95% CI: 24.5, 45.4), and from ESA failure was 23.9 months (95% CI: 19.9, 33.0): 21.6 months (95% CI: 15.6, 39.2) for epo and 28.8 months (95% CI: 21.2, 39.7) for darb (p=0.99) (Figure). Median time to AML transformation or death was 17.4 Months (95% CI: 14.1, 22.9): 25.4 months for responders and 16.8 months for non-responders (p=.069). For patients who received ESAs for a minimum of 4 months (39% of pts for epo and 61% for darb), ORR was 16.5%, and median survival from ESA failure was 23.0 months (95% CI: 14.7, 33.0): 22.3 months (95% CI: 13.1, NA) for epo and 24.7 months (95% CI: 14.3, 39.7) for darb (p=0.87).Conclusion: In this large, but uncontrolled cohort, response rates were similar for lower-risk MDS patients treated with epo and darb, though duration was longer for darb. There was a trend for improved outcomes in patients who responded to ESAs. Lower-risk MDS patients treated with ESAs have an OS of less than 2 years from the time of failure, and can thus be considered a high-risk MDS group for whom subsequent therapies are not standardized, representing an unmet medical need. [Display omitted] DisclosuresSekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Steensma:Incyte: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Onconova: Consultancy. Komrokji:Incyte: Consultancy, Honoraria, Research Funding; Novartis: Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics: Speakers Bureau.

Altered Expression of the Repair Genes in CD34+ Cells May be Responsible for Formation and Accumulation of Mutations in MDS Patients

Abstract Background and Aims Recent studies have demonstrated that in most cases of myelodysplastic syndrome (MDS) at least one mutation has been detected, suggesting that abnormal DNA repair may represent both cause and consequence of malignant transformation. In this study we investigated a possible role of different alterations in DNA repair genes in pathogenesis of MDS. Methods Gene expression of CD34+ cells was measured by RT² Profiler PCR Arrays (Qiagen). Polymorphisms and mutations were studied by targeting next generation sequencing (SeqCap EZ System, NimbleGen). Expression analysis of 84 DNA repair genes was performed in 18 MDS patients and subsequent analysis of selected genes was performed on a cohort of 80 patients. The enrichment resequencing of 84 genes was done in 16 patients. Results Five differentially expressed genes between CD34+ cells of patient and control samples were identified (p&lt;0.05). The increased expression was detected in MPG and XPC genes and decreased expression in RAD51, RPA3 and XRCC2 genes. RAD51 gene showed significantly higher expression in the patients with low-risk MDS forms (RA, RARS, del5q) compared to control group (p=0.0005) and to contrast down-regulated expression was detected (p=0.0002) in high-risk MDS patients (RAEB-1 and -2, AML with myelodysplasia). The group of patients with RCMD showed the average expression at the level of the control group. The expression profile shown a gravity-related decreasing trend after dividing the patients according to IPSS-R, IPSS-R cytogenetic groups and blasts count. Expression profile of XRCC2 gene has similar characteristics as RAD51 (p&lt;0.0001). The expression of RPA3 gene was generally decreased (p&lt;0.0001) with a decreasing trend depending on the disease severity according to MDS forms, IPSS-R and cytogenetics. The RAD51, XRCC2 and RPA-3 genes are related to homologous recombination mechanism, with XRCC2 and RPA3 as a supporting factors for RAD51. The survival curves for all the three genes shown significant differences between groups with over-, intermediate- and down-expressed gene (p=0.0001 for RAD51; 0.0022 for XRCC-2 and 0.0054 for RPA3). MPG gene expression was up-regulated by all MDS types with no significant difference between each other (p=0.0015). Deeper testing of XPC gene expression demonstrated the up-regulation only in low-risk MDS group (p=0.0084). Gene-based analysis showed seven candidate SNPs significantly associated with the disease susceptibility using the HapMAP-CEU population from NCBI PubMed database as control group. With the presence of MDS, these polymorphisms are significantly connected: rs4135113 (p=0.03), rs12917 (p=0.003), rs2230641 (p=0.01), rs2228529 and rs2228526 (p=0.04, respectively p=0.03) and rs1799977 (p=0.04). Within the distribution of tested patient groups according to cytogenetics, we observed significant increase of dependency of these polymorphisms (OR 4.1-9.7, p&lt;0.0001) and the presence of MDS in the group of patients with very-high risk cytogenetics. Conclusions DNA repair mechanisms are responsible for correcting DNA damage and preserving genomic integrity. Our study demonstrates, that altered expression of the repair genes in CD34+ cells may be responsible for the formation and accumulation of mutations in hematological malignancies. Furthermore, we have identified genetic variants that might contribute to the pathogenesis of MDS by modifying individual risk for the disease. Supported by grant (NT/13899, NT/14377, and NT/14539) and the project for conceptual development of research organization (00023736) from the Ministry of Health of the Czech Republic. Disclosures No relevant conflicts of interest to declare.

Myelodysplastic syndromes: pathophysiology, clinical and biological features

Myelodysplastic syndromes (MDS) are hemopathies very common in geriatric practice. They are characterized by qualitative morphological abnormalities of one or more myeloid lineages responsible for an ineffective hematopoiesis, and therefore cytopenias of central origin contrasting with a usually rich bone marrow wealth. The MDS are asymptomatic in half of the cases and their discovery is a result of systematic blood analysis or tests to explore another disease. The evolution is marked by worsening cytopenias, and the risk of acute myeloid leukemia transformation with poor prognosis because frequently chemoresistant. The diagnosis of MDS is pronounced after a clinico-biological confrontation to discuss the differential diagnosis taking into account all clinical and cytological data, results of conventional cytogenetics and evolution after vitamin therapy. Knowledge more depth on MDS refine MDS classification criteria by developing successive classifications (FAB 1982, WHO 2001 and 2008) which aim the identification of MDS groups with clinical, biological and common prognostic. The treatment of MDS is essentially symptomatic. The development of new targeted therapeutic strategies enables high hopes in a context where treatment options are a difficult choice, because the advanced age of most patients. Finally, detailed knowledge of risk factors and prognostic scores are very useful to make the best treatment decisions.