The Relationship Between Erythropoetic Activity and Iron Burden In Patients With Myelodysplastic Syndrome

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5225-5225
Author(s):  
Rui Cui ◽  
Guoqing Zhu ◽  
Zefeng Xu ◽  
Yue Zhang ◽  
Gang Huang ◽  
...  

Abstract Background Although prolonged red blood cells (RBC) transfusion therapy appears to be the main contributor to iron overload, many patients have developed it at an early stage of the disease, before the onset of transfusions in MDS. Growth differentiation factor 15 (GDF-15), a protein produced by erythroid precursors, has been proposed to be a major hepcidin suppressor in ß-thalassemia, but data in the expression of hepcidin and GDF15 levels in MDS are less conclusive. To determine whether the erythropoetic activity affect the iron burden though GDF15 in patients with MDS, we determined the GDF15 levels as well as other markers of erythropoiesis and iron overload (soluble transferrin receptor [sTfR], erythropoietin [EPO], ferritin and hepcidin) in MDS without transfution. Patients and Methods One hundred and seven consecutive patients (mean age 50 years; 62% males) with MDS diagnosed between April, 2011 and March, 2013 at the Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences (CAMS) were included. To be enrolled in this study, patients had to be previously treated without transfusion. To do a comparison with respect to serum hepcidin levels, the hepcidin/ferritin ratio and GDF15 levels, a group of forty healthy individuals (45% males) with rigorous definition of normal iron status were used as controls. Results MDS patients had significantly higher levels of serum SF, GDF-15, and EPO as compared to the controls. GDF15 concentrations significant positively correlated with percent of bone marrow erythroblasts (P <0.001), sTfR (P = 0.018), ISAT (P =0.038) and negatively correlated with TRF (P=0.008). The hepcidin to ferritin ratio was strongly decreased in MDS patients as compared to the controls (P<0.001). The hepcidin to ferritin ratio also showed a significant variability across different MDS subtypes (P = 0.011), with the lowest values in patients with refractory anemia with ringed sideroblasts (RARS). GDF15 levels consistently heterogeneous across different MDS subtypes (P=0.005), with the highest levels in patients with RARS and the lowest levels in the RAEB and RCMD cohorts. A negative correlation between the hepcidin/SF ratio and GDF15 was found(r=-0.279, P=0.014). Both hepcidin and hepcidin/SF ratio were negatively correlated with EPO in MDS patients (r=-0.250, P=0.022 and r=-0.449, P<0.001 respectively). Furthermore, a negative correlation between hepcidin levels and HIF-1α mRNA expression was found in 28 MDS patients(r=-0.377, P=0.048), but there was no relationship in terms of hepcidin and HIF-2α mRNA expression. The hepcidin to ferritin ratio was independently associated with GDF15 concentration and WHO subtype in multivariable analysis (β=-0.292,P =0.029 and β=-0.390,P =0.006). Conclusions Iron overload occurs in MDS patients even without transfusion. Hepcidin concentrations are inappropriately low considering the severe iron overload. High level of GDF15 is a feature of ineffective erythropiesis in MDS. GDF15 is among the erythroid factors down-regulating hepcidin and contributes to iron overload in conditions of dyserythropoiesis in MDS. Disclosures: No relevant conflicts of interest to declare.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4250-4250
Author(s):  
Valeria Santini ◽  
Domenico Girelli ◽  
Alessandro Sanna ◽  
Nicola Martinelli ◽  
Lorena Duca ◽  
...  

Abstract Abstract 4250 Background and Aims. Iron overload is frequently occurring in patients with myelodysplastic syndromes (MDS), with recent data suggesting an impact on both overall and leukemia-free survival1,2. Though prolonged RBC transfusion therapy appears the main contributor, many patients develop iron overload at an early stage of the disease, before the onset of transfusion dependency. It has been postulated that an altered production of hepcidin, the key hormone regulating iron homeostasis, may play a role at this regard. Until recently, studies have been hampered by problems in the development of reliable hepcidin assays, so that only scanty and conflicting data based on semi-quantitative measurement of urinary hepcidin have been reported3,4. This study mainly focused on analyzing serum hepcidin levels in MDS patients by means of a recently validated and improved Mass-Spectrometry based method5. Patients and Methods. One hundred and thirteen consecutive patients (mean age 72.8 ± 9.2 years; 68.1% males) with different types of MDS according to the WHO classification were included in this study. To be enrolled, patients had to be previously untreated or treated only with transfusions. Besides hepcidin, in all subjects we determined serum ferritin, transferrin saturation (TS), non-transferrin-bound-iron (NTBI), along with some putative determinants of hepcidin, like GDF-156 known to be associated with ineffective erythropoiesis, and C-Reactive Protein (CRP) as a surrogate of systemic IL-6 production. Fifty-four healthy individuals (61.1% males) with rigorous definition of normal iron status were used as controls. Main Results. Biochemical markers of iron overload (ferritin and TS), but also CRP and GDF-15 were significantly higher in MDS patients than in controls, even when considering only non-transfused patients. Patients with RARS and the 5q- syndrome appeared as the most iron overloaded, having the highest levels of ferritin, TS, and NTBI. In the whole MDS population, serum hepcidin levels showed a considerable variability, with overall mean values not significantly different from controls [geometric means (gm) with 95% CIs: 5.31 (3.98-7.08) versus 4.2 (3.53-5.0) nM, P=0.28], while the hepcidin/ferritin ratio was significantly lower than in controls [10.1 (7.53-13.53) versus 52.9 (43.6-64.3), P<0.001]. After stratification according to WHO subtypes, hepcidin levels showed significant differences, with the lowest levels in patients with RARS (gm 1.43 nM) and the highest levels in patients with RAEB 1–2 (gm 11.3 nM) and with CMML (gm 10.04 nM) (P=0.003 by ANOVA). The latter groups had substantial elevation of CRP as compared to other MDS subtypes (P=0.008 by ANOVA), while GDF-15 was consistently but uniformly elevated in all MDS subtypes (P=0.97 by ANOVA). Multivariate linear regression models adjusted also for age, sex, and history of RBC transfusions, showed ferritin (β-coefficient 0.45, P=0.002), CRP (β-coefficient 0.21, P=0.02), and different MDS subtypes as the main independent predictors of hepcidin levels. The different degree of correlation between hepcidin and ferritin among the MDS subtypes were analyzed in a general linear model using the F test for slopes. Hepcidin regulation by iron appeared conserved, though relatively blunted in RA, RARS, and 5q- patients, while it was lost in RAEB 1–2 and CMML. Conclusions. Hepcidin levels are consistently heterogeneous in MDS according to different subtypes, likely as the result of the relative strength of competing stimuli. Relative inhibition by ineffective erythropoiesis (but not mediated by GDF-15) seems to prevail particularly in RARS and 5q- syndrome, and is likely to increase the risk of iron overload in these subgroups. On the other hand, patients with RAEB 1–2 and CMML appears to have hepcidin induction that could be driven by cytokines. If confirmed, these results may be relevant not only for a better understanding of iron pathophysiology in MDS, but also for possible future approach with hepcidin modulators7. References: 1) Sanz G, et al. Blood 2008;112: abs 640. 2) Alessandrino EP, et al. Haematologica 2010;95:476-84. 3) Winder A, et al. Br J Haematol 2008;142:669-71. 4) Murphy PT, et al. Br J Haematol 2009;144:451-2. 5) Campostrini N, et al. J Biomed Biotechnol 2010;2010:329646. 6) Tanno T, et al. Nat Med 2007;13:1096-101. 7) Sasu BJ, et al. Blood 2010;115:3616-24. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2676-2676 ◽  
Author(s):  
Luca Malcovati ◽  
Matteo G Della Porta ◽  
Coby M Laarakkers ◽  
Anna Gallì ◽  
Riccardo Albertini ◽  
...  

Abstract The vast majority of patients with myelodysplastic syndrome (MDS) present with anemia, and many of them become transfusion dependent in the long term. Although transfusion iron is primarily taken up by the reticuloendothelial cells, the metal is later redistributed to parenchymal cells, and a portion of MDS patients develop parenchymal iron overload, which may have a negative impact on survival (N Engl J Med2005;352:536–8). The redistribution of transfusion iron from macrophages to parenchymal cells is likely to be modulated by hepcidin levels and erythroid activity. In fact, hepcidin prevents the release of iron from macrophages, and its synthesis is partly down-regulated by erythroid activity, which varies considerably within MDS patients. Expanded but ineffective erythropoiesis is the major mechanism responsible for anemia in low-risk MDS patients, particularly in those with refractory anemia with ringed sideroblasts, while erythroid marrow hypoproliferation is generally found in high-risk patients, typically in those with excess of blasts. We studied 76 patients with MDS followed at the Department of Hematology Oncology, University of Pavia & Fondazione IRCCS Policlinico San Matteo, Pavia, Italy. The WHO classification criteria were employed for diagnosis of MDS. Median time since diagnosis was 12 months (range 0–241). Twenty-five patients were RBC transfusion-dependent (median number of RBC units received was 15, range 4–160). Erythroid activity was evaluated through measurement of soluble transferrin receptor (sTfR) and serum Epo, while body iron status was assessed through serum iron, TIBC and serum ferritin (sFtn). Serum hepcidin was quantified by laser desorption ionization time-of-flight mass spectrometry preceded by weak cation-exchange chromatography exploiting des-Asp hepcidin (hepcidin-24) as an internal standard (www.hepcidinanalysis.com or PLoS ONE2008;16;3:e2706). sTfR levels were found to be independently associated with hemoglobin (Hb) (the higher Hb, the lower sTfR; P&lt;.001), serum Epo (the higher Epo, the lower sTfR; P&lt;.001), and WHO category (patients with purely erythroid disorders having higher values than those with multilineage dysplasia and excess blasts; P&lt;.001), as well as with sFtn (the higher sFtn, the lower sTfR; P=.02). A wide variability in hepcidin level was found in MDS patients (median 6.98 nM, range 0.18–92.05 nM). Patients with pure erythroid disorders had significantly lower hepcidin levels compared with those with multilineage dysplasia or excess of blasts (median values 4.41, 7.62, and 15.31 nM, respectively; P&lt;.001). Transfusion-dependent patients had significantly higher hepcidin levels compared with transfusion-independent subjects (15.31 vs 4.73 nM, P&lt;.001). Significant linear correlations were found between serum hepcidin and Hb (r=−.28, P=0.02), serum Epo (r=.44, P&lt;.001), sFtn (r=.68, P&lt;.001) and sTfR (r=−.64, P&lt;.001). Considering all MDS patients, multivariable analysis showed that serum hepcidin levels were independently determined by sTfR levels (the higher sTfR, the lower serum hepcidin; P&lt;.001) and sFtn (the higher sFtn, the higher serum hepcidin; P&lt;.001). Restricting multivariable analysis to MDS patients receiving regular blood transfusion, the presence of ring sideroblasts and their number were also found to have an independent, negative impact of serum hepcidin levels (P=.005). These findings suggest that in MDS patients the redistribution of transfusion iron from reticuloendothelial cells to parenchymal cells is influenced by erythroid activity through its effect on serum hepcidin levels. MDS patients with expanded but ineffective erythropoiesis have low levels of hepcidin and enhanced iron release from macrophages, and therefore a higher likelihood of parenchymal iron loading. In particular, patients with refractory anemia with ring sideroblasts show low hepcidin levels in spite of iron overload, and therefore appear to be at high risk of parenchymal organ damage. Since these this condition has a benign clinical course, preventing organ damage through iron chelation therapy appears clinically important.


Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1069-1069
Author(s):  
Lining Ju ◽  
Cheng Zhu ◽  
Miguel A. Cruz ◽  
Yunfeng Chen

Abstract Abstract 1069 Binding of GPIbα to VWF tethers platelets to disrupted vascular surface during the haemostatic process. The GPIbα –VWF interaction can also trigger outside-in signaling cascade, resulting in platelet activation, characterized by morphological transformation from discoid to a more spiky shape as well as activation of integrin α IIbβ3. Using the adhesion frequency assay with a biomembrane force probe (BFP), we studied signal initiation by repeated brief contacts of a single platelet with a glass bead coated with VWF-A1 domain and/or fibronectin III 7–10 domain (FNIII7–10) in a precisely controlled fashion (Fig. A). Contacting platelets with beads coated VWF-A1 only resulted in adhesion kinetics mediated by GPIbα –VWF interaction kinetics independent of the activation stage of the platelet. Contacting platelets with beads coated FNIII7–10 only resulted in adhesion kinetics that correlated with the activation stage of the platelet. Discoid-shaped platelets yielded low level adhesions mediated by FN interaction with inactive α IIbβ3 (Fig. B, blue). By comparison, spiky-shaped platelets produced high level adhesions mediated by FN interaction with activated α IIbβ3 (Fig. B, red)that was four times stronger than the interaction with inactive α IIbβ3. Contacting platelets with beads coated both VWF-A1 and FNIII7–10 resulted in two-stage adhesion kinetics. The first stage was mediated by GPIbα –VWF binding, which triggered a second stage consisting of an increase in adhesion after a sub-second delay. The second-stage binding coincided with morphological changes characteristic of platelet activation and matched that mediated by FN interaction with activated α IIbβ3. On the other hand, the concurrent calcium imaging showed as the platelet target was brought to the A1 bead in a repeating manner, the recorded calcium fluorescence intensity climbed up as the repeated touches continue (Fig. C). The peak temporally correlates with the morphological change. Our data indicates that binding of VWF-A1 to platelet GPIbα initiates outside-in signaling, leading to rapid irreversible platelet shape changes and calcium mobilization within a few seconds. Disclosures: No relevant conflicts of interest to declare.


2010 ◽  
Vol 00 (04) ◽  
pp. 34 ◽  
Author(s):  
Rosangela Invernizzi ◽  

A high level of apoptosis may be responsible for the ineffective haematopoiesis in myelodysplastic syndromes (MDS). Recently, it has been demonstrated that the erythroid apoptosis of low-risk MDS is initiated at a very early stage in stem cells and is associated with mitochondrial dysfunction. However, the underlying pathogenetic mechanisms causing malfunctioning of mitochondria and initiation of the intrinsic apoptotic cascade are not completely clear. Recent studies suggest a close relationship between impaired iron metabolism and pathogenesis of myelodysplasia. In fact, iron overload, which is apparent in refractory anaemia with and without ring sideroblasts, may lead to the generation of intracellular free radicals, thereby causing oxidative damage and inducing apoptosis in haematopoietic progenitors. This review summarises current knowledge supporting the role of iron-related oxidative damage in the pathogenesis of MDS. The relationship between mitochondrial iron homeostasis impairment and ineffective erythropoiesis in refractory anaemia with ring sideroblasts as well as the various functions of the cytosolic and mitochondrial ferritins are also discussed.


Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5267-5267
Author(s):  
Giorgio Corinaldesi

Abstract 5267 Insulin-like growth factor (IGF-1) is an activator of the PI3K/AKT (serine/threonine protein kinase) signalling pathway. It can also stimulate cell growth (PDGF, PDAF, VEGF, TGF-alfa and beta), and cellular proliferation, and is a powerful stimulator of directed migration of VSMC (vascular smooth muscle cells). The deregulated action of IGF-1 influences coronary artery disease (CAD) by promoting neo-intimal hyperplasia and leukocyte recruitment, pro-mytotic activity (MAPK: mitogen-activated protein kinases, serine/threonine kinase), and extracellular matrix synthesis in atherosclerotic lesions; it also increases platelet activity, and the expression of P-selectin, IL-6 and platelet CD62. This growth factors may also promote chemotaxis, LDL-cholesterol uptake, and the release of pro-inflammatory cytokines, finally it is a strong inhibitor of apoptosis (BAX, Fas/FasL); in other words, a high level of IGF-1 is a strong risk marker for ischemic heart disease. We have studied a group of 140 patients (92 males and 48 females, mean age was 58 years, range 46–68 years); 72 patients with CAD in early stage of restenosis and 68 control. Patients with arterial restenosis have significantly high levels of IGF-1 (142.8 ng/ml +/− 24.2 ng/ml) that appear closely related to recurrence, than control subjects (mean level of IGF-1: 98.6 ng/ml +/− 14.4 ng/ml). Additional investigations are needed to determine the exact weight of IGF-1 from a slightly clinical point of view, as this marker showed interestingly to have a key role in the pathogenesis of atherosclerosis. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 971-971
Author(s):  
Mrinal M. Patnaik ◽  
Curtis A. Hanson ◽  
Janice M Hodnefield ◽  
Terra L Lasho ◽  
Christy Finke ◽  
...  

Abstract Abstract 971 Unlike the case in acute myeloid leukemia, there is limited information on the prognostic impact of IDH mutations in myelodysplastic syndromes (MDS). In the current study of 277 patients with MDS, IDH mutations were detected in 34 (12%) cases: 26 IDH2 (all R140Q) and 8 IDH1 (six R132S and two R132C). Mutational frequency was 4% (2 of 56) in refractory anemia with ring sideroblasts (RARS), 12% (16 of 130) in refractory cytopenia with multilineage dysplasia, 14% (2 of 14) in MDS-unclassifiable, 14% (6 of 42) in refractory anemia with excess blasts (RAEB)-1, and 23% (8 of 35) in RAEB-2. Normal karyotype was noted in all but one IDH1-mutated cases and 13 IDH2-mutated cases. Multivariable analysis identified presence of mutant IDH1 (p=0.0004; HR 4.0, 95% CI 1.9–8.8), revised International Prognostic Scoring System (IPSS-R) risk category (p<0.0001), and red cell transfusion need (p=0.002) as independent predictors of inferior survival (Figure 1). In a similar multivariable analysis, mutant IDH1 was the only variable associated with shortened leukemia-free survival (p=0.001; HR 7.0, 95% CI 2.3–20.8). The presence of IDH2 R140Q did not affect overall (p=0.54) or leukemia-free (p=0.81) survival (Figure 2). The current study suggests a powerful adverse prognostic effect for mutant IDH1 in MDS. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2555-2555
Author(s):  
Paulo Bahia ◽  
Ana Leite ◽  
Orlando Veloso ◽  
Clarisse LOPES Lobo ◽  
Jorge Marcondes

Abstract Abstract 2555 Poster Board II-532 The purposes of this study were to evaluate the intracranial lesions in adults patients with sickle cell disease (SCD) using magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA). The patients were selected from the main hematological hospital of Rio de Janeiro state (HEMORIO). About 187 patients underwent neurological examination and Transcranial Doppler image (TCDi) with microbubbles. Eighty patients were chosen by an aleatory way to perform MRI and MRA, including those symptomatic and asymptomatic ones. MRI and the MRA were evaluated by two different neuroradiologists, in a blind way about the clinical aspects and TCD results. The middle age was 29 years and 81.3% of the patients had the homozygous form of the SCD. In the MRI, 50% of the patients showed intraparenchymatous lesions. Leukoencephalopathy was the most frequent abnormality in 47.5%, encephalomalacia in 16%, lacunar infarctation in 12.5% and atrophy in 12.5%. Of the encephalomalacia cases, the frontal and parietal lobes were the most affected in 85%. In the MRA study, 34% of the patients presented abnormalities, 26% with vascular stenosis and 19% with vascular occlusions. The anterior circulation was the most compromised in the stenotic cases and the carotid was the main affected artery in the occlusions cases. The silent lesions were observed in 45% of the MRI and 24% of the MRA. The prevalence of parenchymatous lesions and vascular stenosis in cases with genotype SS was significant. There was significant statistic relation between the leukoencefalophalopathy and the vascular stenosis with the medium basal hematocrit. CONCLUSIONS: The use of both methods (MRI and TCD image) demonstrated a powerful tool to study vascular lesions in SCD adults patients. The prevalence of silent infarcts in SCD adults patients was higher than previously estimated in literature (55%). This finding reinforces the prophylactic intervention study with TCD Imagining and prophylactic transfusion therapy in early stage of life. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4704-4704
Author(s):  
Antonios Kattamis ◽  
Polyxeni Delaporta ◽  
Ioannis Papassotiriou ◽  
Dimitra Kyriakopoulou ◽  
Natalia Tourkantoni ◽  
...  

Systematic transfusions are lifesaving for patients with severe congenital anemias, but they eventually lead to iron overload and the indispensable necessity of iron chelation therapy. Current official guidelines for the starting time of chelation therapy derives from data obtained with the use of desferrioxamine, which has been shown to have significant toxicity in very young patients, especially when used in low iron burden. No data exist on the use of the oral iron chelators in this setting. The purpose of this study was to evaluate the changes of iron parameters at the initial period of transfusion therapy in newly diagnosed patients with congenital anemias Methods Nine patients participated in this study. One patient was diagnosed with Diamond-Blackfan anemia, one patient with severe alpha-thalassemia, while 7 had beta-thalassemia. Three of the beta-thalassemia started transfusions at 1.5, 2 and 5 years, respectively. All others started transfusions between 2-4 months of age. Iron, transferrin saturation, ferritin levels. serum transferrin receptors (sTfR), were estimated by standard methods, while labile plasma iron (LPI) by the FeROS LPI kit (Aferrix, Ltd, Israel). The main results of the study show that: 1) transferrin saturation increases rapidly with transfusions (mean levels after 4 transfusions 49.2% (range: 23.8-90.5%), mean after 6 transfusions 69.1% (range: 39.5-112%)), though it has significant diversity in between patients, as indicated also by one patient that continued to have transferrin saturation at 65% even after 12 sessions. Transferrin saturation significantly correlates with ferritin levels (r=0.763, p<0.0001), with the number of previous transfusions (r=0.486, p=0.002) and with the levels of sTfR, which is also an index of the degree of erythropoiesis (r=0.550, p<0.001). The increase of ferritin correlates also with the sTfR levels (r=0.697, p<0.0001), while the rate of increasing transferrin saturation per transfusion correlates to sTfR levels (r=0.486, p=0.002). LPI levels appears early in the transfusion history and correlates with transfusion saturation. Discussion The results of the study indicate that iron overload starts early in the transfusion history of young patients with transfusion-dependent anemias. These findings dispute current guidelines suggesting starting chelation therapy, when the patients have already received 10 transfusions or when ferritin levels reach more than 1000ug/dl. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2845-2845
Author(s):  
Akiko Nagamachi ◽  
Yuko Ozaki ◽  
Hirotaka Matsui ◽  
Akinori Kanai ◽  
Toshiya Inaba

Abstract Polynuclear cells (PNCs) are routinely observed in the bone marrow of MDS patients. They are binuclear, trinuclear or even multinuclear cells with or without micronuclei, the underlying molecular mechanisms for the production of which are largely unknown. Because loss of the long arm of chromosome 7 (7q-) was reported to be associated with the presence of a higher frequency of PNCs, gene(s) preventing bone marrow cells from carrying such nuclear abnormalities may be located at 7q. We previously identified three candidate anti-myeloid tumor suppressor genes, namely Samd9, Samd9L and Miki, from the microdeletion in the 7q21 band frequently detected in JMML patients. SAMD9L-deficient mice develop MDS resembling human diseases associated with 7q-, most likely through enhancement of cytokine signals (Nagamachi et al., Cancer Cell 2013). Miki (mitotic kinetics regulator) translocates from the Golgi apparatus to mitotic centrosomes coincident with the disappearance of the Golgi body after poly-ADP-ribosylation (PARsylation). Miki is indispensable for centrosome maturation [the rapid increase of pericentriolar materials (PCM) during prophase and prometaphase], which is required for the production of robust mitotic spindles to move chromosomes promptly (Ozaki et al., Mol. Cell 2012). Consequently, as observed by time-lapse imaging of HeLa cells expressing histone H2A-GFP, downregulation of Miki by siRNA markedly prolonged the duration of prometaphase to more than several hours (normally around 15 minutes). Chromosomes were scarcely able to align and cells exited from prometaphase either by cell death or by decondensation of each chromosome. In the latter, cells with decondensed chromosomes then fused with one another within 30 minutes to form cells with relatively large nuclei, resulting in PNCs containing various sizes of nuclei including micronuclei. Indeed, reduction of Miki in HeLa cells by siRNA increased the frequency of PNCs from less than 0.5% to 4.5%. To test whether the chaotic chromosome decondensation in prometaphase causes the accumulation of PNCs observed in MDS, we initially used five cell lines derived from MDS associated with 7q-. PARsylated Miki was barely detectable in these cell lines and we found more cells at prometaphase than at metaphase (the ratio of prometa:meta in the lines ranged from 1.7:1 to 5.7:1). In contrast, in seven cell lines expressing PARsylated Miki at high levels, mitotic cells in prometaphase were found less frequently or at roughly the same frequency as those in metaphase (prometa:meta ratio 0.6:1 to 1.3:1). PNCs in five cell lines harboring 7q-were also more frequent (5.9 - 10.2%) than in the seven cell lines expressing high PARsylated Miki (0.8 - 2.4%). In addition, when we reduced Miki expression levels by shRNA in K562 cells, which express PARsylated Miki at high levels, the prometa:meta ratio increased from 1.1:1 to 3.8:1 and PNCs increased from 0.8% to 8.5%. This suggests that, as in HeLa cells, low expression levels of Miki cause prolongation of prometaphase and increase PNCs in blood cells. Fresh bone marrow preparations from 37 patients with MDS were examined to determine whether Miki mRNA-expression levels influence the prometaphase:metaphase ratio and the frequency of PNCs. We found a strong negative correlation (R=-0.59, p<0.01) between Miki mRNA expression levels in mononuclear cells of bone marrow samples and the prometa:meta ratio. We also found a moderate negative correlation (R=-0.4, p<0.05) between PNC frequencies and Miki mRNA expression levels. In addition, there was a strong positive correlation between prometa:meta ratios and PNC frequencies (R=-0.56, P<0.01). In conclusion, lack of one allele of the Miki gene due to 7q-reduces PARsylated Miki, resulting in the increase of PNCs through decondensation of chromosomes in prolonged prometaphase. This may contribute to poor outcome of MDS associated with 7q-through increased chromosome instability. Disclosures No relevant conflicts of interest to declare.


Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2318-2318
Author(s):  
Vinothkumar Rajan ◽  
Rachel Woodside ◽  
Sergey V Prykhozhij ◽  
Jason Berman

Abstract Myelodysplastic syndrome (MDS) is a pre-leukemic state characterized by the failure of the bone marrow to produce mature and functional blood cells. Nearly one-third of MDS patients progress to acute myeloid leukemia (AML). AML is the most common form of acute leukemia in adults and accounts for a high level of mortality in pediatric leukemia. Loss-of-function mutations in the Ten-11 Translocation 2 (TET2) gene are implicated in MDS and AML, but evidence that TET2 mutations are also found in healthy individuals as a result of clonal hematopoiesis calls into question the contribution of TET2 to leukemogenesis. Many studies have tried to mutate the DNA binding domain in TET2 both in mouse and zebrafish, but the blood phenotypes observed have been inconsistent. We created a tet2 zebrafish mutant using CRISPR-Cas9 technology, where we deleted 2.1 kb from exon 2, confirmed by cDNA analysis. A recent publication suggests the importance of Ser99 in the stability of the TET2 protein (Wu et al., 2018) and this conserved serine residue is contained in the region of deletion in our zebrafish tet2 mutant. These tet2 mutant fish were incrossed to create a maternal-zygotic mutant with no tet2 expression. Importantly, quantitative PCR demonstrated that there was no compensatory effect from tet1 and tet3 in the context of tet2 loss. Whole mount in situ hybridization performed on 24-48h tet2 mutant zebrafish embryos provided evidence of a significant reduction of early and mature myeloid and erythroid cells through staining for spi1/pu.1 (myeloid), gata1 (erythroid) myeloperoxidase (mpx; neutrophils), carboxypeptidase A5 (cpa5; mast cells) and l-plastin (lcp1; macrophages). The stem cell compartment was not impacted, as shown by wild-type runx1/c-myb expression. Further, o-dianisidine staining revealed low hemoglobin content in tet2 mutants compared with control embryos. Overall, the early embryos elucidate a refractory anemia like phenotype. Flow cytometry analysis of kidney marrow from 3-month-old adult tet2 mutant fish similarly demonstrated a decrease in the myeloid and erythroid compartments, in keeping with characteristics typical of low stage MDS. We are currently employing RNAseq and MeDIP analysis on blood cell populations from tet2 mutants to determine the genetic and epigenetic signatures associated with this mutation. This zebrafish model of tet2 loss uniquely provides with a dynamic phenotype that starts with refractory anemia with progression through low stage MDS. Thus this model will be beneficial in elucidating the mechanisms underlying how tet2 contributes to MDS and AML and provide a preclinical platform to screen for therapeutic interventions. Disclosures No relevant conflicts of interest to declare.


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