Reduced PLCG1 expression Is Associated with Inferior Survival in Myelodysplastic Syndromes

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1700-1700
Author(s):  
Masayuki Shiseki ◽  
Mayuko Ishii ◽  
Mari Ohwashi ◽  
Kentaro Yoshinaga ◽  
Naoki Mori ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Phospholipase C ◽  
Myelodysplastic Syndromes ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Expression Level ◽  
Rt Pcr ◽  
Control Subjects ◽  
Chromosome 20

Abstract The PLCG1 gene encodes phospholipase C g isoform, which is involved in diverse physiological and pathological cellular processes through catalyzing the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce the second messenger molecules inositol 1,4,5-trisphosphate and diacylglycerole. Until now, roles of phospholipase C g 1 in hematopoiesis have been shown in animal models. Plcg1 deficient mouse lacks erythropoiesis and die in early embryonic stage. In zebrafish model, it was demonstrated that phospholipase C g 1 is required in granulocyte maturation. Physiological and/or pathological role of phospholipase C g 1 in human hematopoiesis has not been elucidated. In human, the PLCG1 gene is located on long arm of chromosome 20. Deletion of long arm of chromosome 20 (del(20q)) is commonly observed in myelodysplastic syndromes (MDS). Previously, we determined the common deleted region (CDR) of del(20q) in MDS, and the PLCG1 gene is located within the CDR. Reduced expression of genes located in the CDR due to haploinsufficiency may play role in molecular pathogenesis of MDS. Therefore, we analyzed PLCG1 expression in bone marrow mononuclear cells in MDS patients with or without del(20q), and investigated its clinical significance in the present study. Mononuclear cells separated from bone marrow samples taken at the time of diagnosis with written informed consent from patients were used. To analyze PLCG1 expression, quantitative RT-PCR was performed. Total RNA was extracted from mononuclear cells and subjected to cDNA synthesis. Real-time RT-PCR was carried out using cDNA as template by the TaqMan probe method (Applied Biosystems) with co-amplification of the endogenous control gene, human GAPDH (Applied Biosystems) were performed. The human PLCG1 primer-probe set was from Applied Biosystems. A total of 109 MDS patients, 65 males and 44 females with median age of 69 years (range: 22-91 years), with (n=20) or without (n=89) del(20q), were included in the present study. They were classified as RCUD (n=11), RCMD (n=55), RARS (n=11), RAEB-1 (n=16), and RAEB-2 (n=16) according to WHO classification. They were categorized in four IPSS risk groups, low risk (n=21), intermediate-1 risk (n=52), intermediate-2 risk (n=20), and high risk (n=6). Relative PLCG1 expression level was significantly reduced in MDS patients with del(20q) compared to control subjects (n=20) (P=0.011). Median values of relative PLCG1 expression level in MDS patients with del(20q) and control subjects were 0.94 and 1.96. In addition, relative expression level of PLCG1 in whole MDS cohort significantly lower than that in control subjects (P=0.046). Expression patterns of PLCG1 among, were not different. Median values of relative PLCG1 expression level in five WHO-subtypes, RCUD, RCMD, RARS, RAEB-1, and RAEB-2 were 1.50, 1.55, 1.29, 1.13, and 1.12, respectively, but no statistically difference was observed. WHO-subtypes with high blast counts (RAEB-1 and RAEB-2) showed trend in association with reduced PLCG1 expression compared with those with low blast counts (RCUD, RCMD, and RARS) (median value: 1.01 vs. 1.54, P =0.11). To investigate prognostic implication of PLCG1 expression in MDS, we analyzed impact of PLCG1 expression on overall survival (OS). Based on PLCG1 expression level, 109 patients were divided into four groups, high (Q1), intermediate (Q2, Q3), and low (Q4) quartiles. Kaplan-Meier analysis demonstrated that the lowest quartile (Q4) showed significantly worse survival compared with remaining quartiles (Q1-Q3) (P =0.0015). The estimated 5-year OS rates in Q1-3 group and Q4 group were 63.2% and 29.7%, respectively. Percentage of patients with WHO-subtypes with high blast count (RAEB-1 and RAEB-2) was significantly higher in Q4 than other quartiles (46.9% vs 22.1%, P =0.019 by Fisher's exact test). The present study demonstrated that reduced PLCG1 expression is associated with inferior clinical outcome, indicating that PLCG1 expression could be a useful prognostic marker in MDS. Association between reduced PLCG1 expression and WHO-subtypes with high blasts counts, suggesting that PLCG1 dysfunction play a role in disease progression. Disclosures No relevant conflicts of interest to declare.

Research of Gene Expression in Patients with Myelodysplastic Syndromes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4433-4433
Author(s):  
Bao-An Chen ◽  
Bo Zhang ◽  
Chong Gao ◽  
Guo-Hua Xia ◽  
Ze-ye Shao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Clinical Significance ◽  
Myelodysplastic Syndromes ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Bone Marrow Mononuclear Cells ◽  
Rt Pcr ◽  
Normal People ◽  
Significant Difference

Abstract Abstract 4433 Object This study was aimed to investigate the expression of c-FLIPL, c-FLIPS and DLK1 mRNA in Myelodysplastic Syndromes (MDS) patients, as compared with normal people and AML patients, and to find its clinical significance. Methods The expression of c-FLIPL, c-FLIPS and DLK1 mRNA in bone marrow mononuclear cells (BMNNC) of 16 patients with MDS, 8 patients with AML and 3 controls were detected by RT-PCR. Results The expression of DLK1 mRNA was up-regulated in MDS, including RA and RAEB, as compared with controls(P<0.05). There was no significant difference in expression of DLK1 between RA and RAEB(P>0.05). The expression of DLK1 was significant higher in AML patients, compared with controls(P<0.05). There was no significant difference between MDS and AML patients(P>0.05). The expression of c-FLIPL mRNA was higher than that in controls, both in RA and RAEB(P<0.05). There was no significant difference in expression of c-FLIPL between RA and RAEB(P>0.05). In eight AML patients, c-FLIPL gene's expression was up-regulated, as compared with controls(P<0.05). Between AML and MDS patients, there was no significant difference(P>0.05); The expression of c-FLIPS mRNA had no significant difference between MDS patients and controls(P>0.05), but its expression in RAEB was significant higher as compared with RA patients and controls(P<0.05). And in AML patients, the expression of c-FLIPS was higher than that in controls(P<0.05), but there was no significant difference between AML and MDS patients(P>0.05). Conclusion It is concluded that the expressions of DLK1, c-FLIPL and c-FLIPS mRNA in MDS/AML patients are abnormal as compared with normal people, although there are no significant difference have been found between AML and MDS. These genes may play critical roles in escaping malignant clone of MDS from apoptosis and acquiring the ability to divide unlimitedly, they can become important indexes for evaluating of development in MDS. Disclosures: No relevant conflicts of interest to declare.

JAK2 Inhibition with TG101348 Inhibits Monosomy 7 Myelodysplastic Syndromes (MDS) Bone Marrow Cells In Vitro: a Potential Targeted Therapy for Monosomy 7 MDS

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 973-973 ◽  
Author(s):  
Matthew J. Olnes ◽  
Andrea Poon ◽  
Zachary Tucker ◽  
Neal S. Young ◽  
Elaine M Sloand
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Myelodysplastic Syndromes ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Dependent Manner ◽  
Monosomy 7

Abstract Abstract 973 The myelodysplastic syndromes (MDS) are bone marrow disorders characterized by cytopenias and a variable risk of progression to acute myeloid leukemia (AML). Monosomy 7 is the second most common cytogenetic abnormality in MDS, and the most frequent karyotypic aberration occurring in aplastic anemia patients following immunosuppressive therapy. Monosomy 7 MDS carries a particularly poor prognosis, with patients manifesting severe cytopenias and a high propensity to develop treatment-refractory AML. There are currently no targeted therapies for this disorder. We previously reported that monosomy 7 bone marrow mononuclear cells (BMMNCs) express high levels of a differentiation-defective granulocyte colony stimulating factor (G-CSF) receptor isoform (IV), an alternative splice variant that exhibits constitutive signaling through the JAK-2 and STAT-1 pathway, while levels of STAT-3 and -5 are unchanged (Sloand et al, PNAS, 2006, 103:14483). As a result, the cell's ability to differentiate is limited, while its ability to proliferate remains intact. Here we examine the effects of the highly selective JAK2 inhibitor TG101348 on monosomy 7 aneuploidy in BMMNCs, as well as the activity of this compound on CD34+ stem cells and CD13+ myeloid cells in culture, and on the JAK-2 signaling apparatus. Incubation of BMMNCs with TG101348 for 5 days significantly decreased absolute numbers of monosomy 7 aneuploid cells in a concentration dependent manner versus vehicle- treated controls (0.187 × 106 vs 1.08 × 106, P=0.007), while diploid cell numbers remained stable (0.338 × 106 vs 0.213 × 106, P=0.50). Flow cytometry experiments demonstrated that incubation with increasing concentrations of TG101348 decreased the absolute number of CD34+CD13- stem cells, and increased numbers of more differentiated CD34-CD13+ myeloid cells, with median CD34+/CD13+ ratios of 6.547 and 2.216 for cells treated with vehicle and 100 nM TG101348, respectively. By immunoblot, STAT-1 protein expression in monosomy 7 BMMNCs treated with 1uM TG101348 was decreased relative to vehicle- treated controls, while there was no difference in STAT-3 and STAT-5 levels. Thus TG101348 decreases monosomy 7 MDS blasts in vitro through inhibition of JAK-2/STAT-1 signaling, a finding that warrants further study of this agent in clinical trials for patients with monosomy 7 MDS and AML. Disclosures: No relevant conflicts of interest to declare.

TET2 mRNA Expression in Bone Marrow Mononuclear Cells of the Patients with Myelodysplastic Syndromes and it's Clinical Significances

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5036-5036
Author(s):  
Zonghong Shao ◽  
Wei Wang ◽  
Rong Fu ◽  
Jun Wang ◽  
Lijuan Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mrna Expression ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Lower Proportion ◽  
Bone Marrow Mononuclear Cells ◽  
Rt Pcr ◽  
Important Indicator ◽  
Significant Difference ◽  
Lower Expression

Abstract Abstract 5036 Objective This study was aimed to investigate the expression of TET2 mRNA in the bone marrow mononuclear cells(BMMNC)of patients with myelodysplastic syndrome(MDS)and its clinical significance. Methods The mRNA expression of TET2 in bone marrow mononuclear cells(BMMNC) of 25 patients with MDS and 16 controls were detected by RT-PCR. Results The expression of TET2 mRNA in BMMNC was down-regulated in MDS (0.9509±0.3841)compared with that in controls(1.2515±0.3749)(P<0.05), but was no significant difference of BMMNC expression of TET2 among RA, RCMD and RAEB. Patients with higher expression of TET2(≥0.9) presented significantly lower proportion of bone marrow blasts[(1.04±1.68)%] than that [(6.13±8.17)%] of those with lower expression (<0.9) of TET2 (P<0.05). The expression of TET2 mRNA in BMMNC of MDS patients was inversely correlated with malignant clone burden (r=-0.398,P<0.05) and IPSS (r=-0.480,P<0.05). Conclusions The mRNA expression of TET2 in BMMNC of MDS patients decreased, which might useful as an important indicator for the evaluation of MDS clone burden. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Ribosomal Protein L23 Negatively Regulates Cell Apoptosis Via Its Suppression of Miz1 and Activation c-Myc in Higher-Risk Myelodysplastic Syndromes

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5508-5508
Author(s):  
Yuekun Qi ◽  
Lingyun Wu ◽  
Chunkang Chang ◽  
Feng Xu ◽  
Qi He ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Ribosomal Protein ◽  
Normal Control ◽  
Myelodysplastic Syndromes ◽  
Cell Apoptosis ◽  
Conflicts Of Interest ◽  
Negative Regulation ◽  
Gene Set Enrichment Analysis ◽  
Negative Regulator ◽  
Expression Level

Abstract Ribosomal protein (RP) L23 has been suggested to be a negative regulator of cell apoptosis. Previously, we reported that RPL23 is over-expressed in higher -risk myelodysplastic syndromes (MDS), which is negatively correlated with apoptosis of bone marrow CD34+ cells and predicts poor prognosis. However, the mechanism of the negative regulation of cell apoptosis by RPL23 in higher- risk MDS remains unclear. In the present study, lentiviral transfection of a RPL23-RNAi vector led to generate RPL23-knockdown (KD) in MDS cell line (SKM-1). Gene microarray was performed in RPL23-KD and control (NC) cells. We employed Gene Set Enrichment Analysis (GSEA) to identify functional gene sets and pathways differentially expressed between the two groups. The results showed that RPL23 knockdown induced remarkable proliferation inhibition, distinct pro-apoptosis and cell cycle arrest when compared with NC. Global gene expression profile showed, by comparison to NC samples, 306 genes upregulated while 448 genes downregulated. Among genes varying wildly, increased expression of Miz-1 (FC = 3.79, p=1.19E-20) and lowered expression of c-Myc (FC= -2.613, p=1.49E-05) were identified in RPL23-KD cell, which were verified both by qRT-PCR and western-blot. mRNA and protein levels of p15and p21significantly increased in RPL23-KD cells. In bone marrow sample from 46 patients with higher risk MDS, the mRNA level of Miz1 was remarkably lower than normal controls, which was negatively correlated to RPL23 expression level (r= -0.252, p = 0.048). The mRNA expression level of c-Myc in higher-risk MDS was significantly higher than normal control, which is positively correlated to the RPL23 expression level (r = 0.70, p = 0.004). IHC showed equidirectionally increased c-Myc and RPL23 and lowered expression of Miz1 in patients with higher-risk MDS compared to normal control. In conclusion, the negative regulation of cell apoptosis by RPL23 in higher-risk MDS might be through its suppression of Miz1 and activation of c-Myc. Disclosures No relevant conflicts of interest to declare.

Over-Expression of IGF-IR in Malignant Clonal Cells in Bone Marrow of Myelodysplastic Syndromes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4832-4832
Author(s):  
Qi He ◽  
Xiao Li ◽  
Zheng Zhang ◽  
Qingqiao Zhang ◽  
Feng Xu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Myelodysplastic Syndromes ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Expression Level ◽  
Over Expression ◽  
Significant Difference ◽  
Clone Cells

Abstract Abstract 4832 To explore the expression level of insulin-like growth factor-1 receptor (IGF-IR) in malignant clone cells of myelodysplastic syndromes (MDS). Method Fluorescence in situ hybridization (FISH) and immunochemistry (APAAP, alkaline phosphatase anti-alkaline phosphatase) were used together to detect the expression of IGF-IR in the bone marrow cells of 26 MDS patients with known abnormal karyotypes. Result The average IGF-IR expression level on the surface of clone cells from the 26 MDS cases was markedly elevated compared to the corresponding level in normal cells (78.2±13.7% vs 14.1±14.0%,P <0.0001). The percentages of malignant clone cells in all 26 MDS cases were significantly correlated with the respective percentages of IGF-IR positive nucleated cells (r = 0.909; P <0.0001). No significant difference in the IGF-IR expression level on the clone cells were observed either between high- and low-risk MDS patients or among MDS patients with different abnormal karyotypes. Conclusion IGF-IR might be taken as a marker of clone cells in MDS because of its propensity to cause malignant proliferation. Disclosures No relevant conflicts of interest to declare.

Reduced Response to Stromal Cell-Derived Factor-1(SDF-1) May Lead to Low Expression of p57kip2 in the Bone Marrow of Patients with De Novo Myelodysplastic Syndromes (MDS)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2782-2782
Author(s):  
Youshan Zhao ◽  
Chunkang Chang ◽  
Juan Guo ◽  
Shucheng Gu
Keyword(s):  
Bone Marrow ◽  
Normal Control ◽  
Myelodysplastic Syndromes ◽  
Stromal Cell ◽  
Mononuclear Cells ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Cell Percentage ◽  
Stromal Cell Derived Factor ◽  
Low Expression

Abstract Abstract 2782 Purpose To explore the expression of p57 in the bone marrow(BM) of patients with de novo myelodysplastic syndromes(MDS), and the role in MDS pathogenesis and prognosis, as well as the relationship between the expression of p57 and SDF-1/CXCR4 signal. Patients and Methods The expression of p57 and CXCR4 in bone marrow mononuclear cells (BMMCs) of 67 de novo MDS patients (including 46 low-risk, 21 high-risk according IPSS) were measured by real-time quantitative (RT)-PCR. BM CD34+ cell percentage was measured by flow cytometry. Besides, p57 expression was investigated when stromal cell-derived factor-1(SDF-1) was co-cultured with BMMCs from MDS cases and normal control respectively. Furthermore, comparison of p57 expression level before and after treatment in 25 MDS patients was studied. Results P57 expression was significantly decreased in MDS patients when compared to normal controls (p=0.003). Patients with abnormal karyotype showed lower expression of p57 compared to normal karyotype cases (P=0.028). A positive correlation between p57 and CXCR4 expression was investigated (r=0.609, P<0.001), and P57 expression was negatively correlated to BM CD34+ cell percentage (r=−0.393, P=0.008) in MDS patients. Additionally, P57 expression increased after treatment who received hematological response (P=0.016). P57 expression in BMMCs from normal control increased significantly when co-cultured with SDF-1 in vitro, which could be blocked by AMD3100, whereas SDF-1 induced a mild increase of p57 expression in MDS when compared to normal control (P<0.001). Conclusion Low expression of the p57 is common in MDS, which may play a role in pathogenesis and indicate poor prognosis. SDF-1 induced mild p57 expression increasing in MDS patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Prognostic Significance of Reduced BCAS4 expression in Bone Marrow Cells of Patients with Myelodysplastic Syndromes

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4258-4258
Author(s):  
Masayuki Shiseki ◽  
Mayuko Ishii ◽  
Mari Ohwashi ◽  
Kentaro Yoshinaga ◽  
Naoki Mori ◽  
...  
Keyword(s):  
Risk Factors ◽  
Bone Marrow ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Risk Groups ◽  
Older Age ◽  
Expression Level ◽  
Expression Of Genes ◽  
Kaplan Meier ◽  
Lysosome Related Organelles

Deletion of long arm of chromosome 20 (del(20q)) is commonly observed in myelodysplastic syndromes (MDS). Reduced expression of genes located within the common deleted region (CDR) of del(20q) due to haploinsufficiency may play a role in molecular pathogenesis of MDS. In the previous study, we examined expression of genes located within the CDR which we determined using array-CGH, in bone marrow mononuclear cells in MDS patients with or without del(20q), indicating that BCAS4 expression was significantly reduced in bone marrow cells in MDS patients with or without del(20q). The BCAS4 gene, which was identified as a fusion transcript expressed in MCF7 cells, encodes 23kD protein. Although function of BCAS4 protein remains unclear, it could be a member of "cappuccino" family, which belong to lysosome-related organelles. Abnormality of genes encoding lysosome-related organelles cause variety of congenital disorders, including the Hermansky-Pudlak syndromes, which is characterized by oculocutaneous albinism and bleeding tendency due to platelet dysfunction as a result of lysosome abnormalities. In the present study we investigated clinical implication of BCAS4 expression level in MDS patients. Mononuclear cells separated from bone marrow samples taken at the time of MDS diagnosis were used for analysis. Written informed consent was obtained from patients before study. To analyze BCAS4 expression, quantitative RT-PCR was performed using cDNA from mononuclear cells as template by the TaqMan probe method (Applied Biosystems) with co-amplification of the endogenous control gene, human GAPDH (Applied Biosystems). Samples from 103 MDS patients, 64 males and 39 females with median age of 67 years (range: 20-91 years), with (n=14) or without (n=89) del(20q), were examined in the present study. Patients were classified as RCUD (n=12), RCMD (n=55), RARS (n=9), RAEB-1 (n=10), and RAEB-2 (n=13), according to WHO 2008 classification, and in RAEB-T (n=4) according to FAB classification. They also were categorized in four IPSS risk groups, low risk (n=30), intermediate-1 risk (n=46), intermediate-2 risk (n=18), and high risk (n=9). There was no significant difference in relative BCAS4 expression level between patients with del(20q) and those without del(20q), and among WHO subtypes. Higher IPSS risk groups (INT-2 and High) showed trend in association with reduced BCAS4 expression compared with lower IPSS risk groups (Low and INT-1) (P=0.104). We analyzed impact of BCAS4 expression on overall survival (OS). Based on BCAS4 expression level, 103 patients were divided into four groups, highest (Q1), intermediate (Q2, Q3), and lowest (Q4) quartiles. The Kaplan-Meier analysis demonstrated that Q4 showed significantly worse OS compared with remaining quartiles (Q1-Q3) (log-rank test, P=0.0031). The estimated 2-year OS rates in Q1-3 group and Q4 group were 75.1% and 48.9%, respectively. According to the COX proportional hazards model, univariate analysis showed lower BCAS4 expression (Q4 vs Q1-Q3) was associated with worse OS (hazard ratio 3.43, 95%CI 1.89-6.11, P=0.0001) as well as older age (65 years or older vs less than 65 years), and higher IPSS risk groups (INT-2 and High vs Low and INT-1). Multivariate analysis indicated that lower BCAS4 expression showed trend for association with worse OS (hazard ratio 1.90, 95%CI 0.96-3.64, P=0.0651) by analyzing with two variables (older age and higher IPSS groups). Next, we investigated whether OS is predicted by combination of three variables, BCAS4 expression level, IPSS risk groups, and age at diagnosis. We defined lower BCAS4 expression (Q4), higher IPSS (INT-2 and High), and older age (65 years or older), as risk factors. The Kaplan-Meier analysis showed that survival curves were well separated according to number of risk factors (0, 1, and 2 or more) (P<0.0001). The estimated 1-year, 2-year, and 5-year survival rates were 100%, 100%, and 86.5% in patients without risk factor, 75%, 70.2%, and 51.7% in patients with one risk factor, and 54%, 34.3%, and 11.4% in patients with two or more risk factors. The present study demonstrated that reduced BCAS4 expression is associated with inferior clinical outcome, indicating that BCAS4 expression level could be a useful prognostic marker in MDS, especially by combination with IPSS risk and patients age at diagnosis. Disclosures Tanaka: Bristol-Myers Squibb: Research Funding.

scholarly journals Defective Autophagy in MPN Were Predominantly Detected in Megakaryocytes in Philadelphia Negative (Ph -) Myeloproliferative Neoplasm

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5386-5386
Author(s):  
Jen-Chin Wang ◽  
Guanfang Shi ◽  
Karan Josan ◽  
Preethi Ramachandran ◽  
Vladimir Gotlieb ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Western Blot ◽  
Cell Signaling ◽  
Mononuclear Cells ◽  
Myeloproliferative Neoplasm ◽  
Beclin 1 ◽  
Positive Staining ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear ◽  
Cytoplasmic Staining

We previously reported that autophagy was defective in classical Philadelphia negative (Ph -) Myeloproliferative Neoplasm (MPN) by demonstrating increased p62, decreased Beclin-1 by RT-PCR and Western Blot (WB) methods and decreased LC3-II by WB (ASH annual meeting poster 2018) in peripheral blood mononuclear cells. Now, we further performed immunohistochemical staining of these autophagy markers on the bone marrow biopsy specimens. Methods: Formalin-fixed and paraffin-embedded bone marrow samples were stained with primary antibodies against Beclin-1 (mouse monoclonal, Millipore), LC3B (rabbit monoclonal, Cell Signaling Technology), and p62 (mouse monoclonal, Cell Signaling Technology). The tissue sections were analyzed using VENTANA BenchMark Ultra System (Ventana Medical Systems, Inc.) according to the manufacturer's protocol. Patients who were studied included 12 essential thrombocythemia (ET), 10 polycythemia (PV), and 5 myelofibrosis (MF) (including 1 post-ET MF and 1 post-PV MF). Scoring was given based on the degree of cytoplasmic staining. Score 1 included weak cytoplasmic stain, score 2 included moderate cytoplasmic stain and score 3 was given for strong cytoplasmic staining . Results: 1) As shown in Fig1, the predominant cells which stained positive including p62, Beclin-1 , or LC3 B were mostly found on the megakaryoctes, although very few of other cell types were found to have positive staining as well 2) As shown in Table 1, the immunostaining results were in general correlated to the RT-PCR , or western blot findings that a defective autophagy was demonstrated in MPN patients with combined finding of a stronger staining in p62,and weak staining in Beclin-1 and LC3B. Conclusion: We have demonstrated defective autophagy in these Ph (-) MPN, by immunostaining of the bone marrow specimens, and demonstrating positivity for defective autophagy predominantly in megakaryocytes. Since megakaryocytes are the most important cells involved in the pathogenesis of MPN, we propose that defective autophagy in megakaryocytes play an important role in the pathogenesis of these diseases. Disclosures Wang: Incyt: Research Funding.

scholarly journals DNA methylation identifies genetically and prognostically distinct subtypes of myelodysplastic syndromes

2019 ◽  
Vol 3 (19) ◽  
pp. 2845-2858 ◽  
Author(s):  
Brian Reilly ◽  
Tiffany N. Tanaka ◽  
Dinh Diep ◽  
Huwate Yeerna ◽  
Pablo Tamayo ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Myelodysplastic Syndromes ◽  
Mononuclear Cells ◽  
Scoring Systems ◽  
Bone Marrow Mononuclear Cells ◽  
Key Points ◽  
Prognostic Scoring Systems

Key Points Targeted DNAm profiling of MDS patient bone marrow mononuclear cells identifies several distinct DNAm clusters. Clusters enrich for specific genetic lesions and show differences in survival independent of clinical prognostic scoring systems..

Heterogeneous Interactions of Leukemic and Control Bone Marrow Stroma Cells with an AML Cell Line and AML Leukemic Blasts.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1047-1047
Author(s):  
Ulrike Buttkereit ◽  
Sana Mohamad ◽  
Monika Lindemann ◽  
Joachim R. Goethert ◽  
Bertram Opalka ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Immune Escape ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Tumor Stroma ◽  
Malignant Cell ◽  
Cell Counting ◽  
Set Up

Abstract Abstract 1047 Tumor-stroma interaction plays a pivotal role for malignant cell survival, proliferation, immune escape, and drug resistance. We had previously shown that bone marrow (BM) stroma from leukemia patients had different gene expression patterns and support of normal CD34+ cells compared to controls (Ctr). Aim of this study was to evaluate growth, survival, and colony-forming potential issues of M-07e AML cells and AML blasts as indicator cells (IC) upon contact with non-leukemic and leukemic stroma. Plastic-adherent BM cells from leukemic or Ctr non-leukemic donors were cultured for several weeks. Polyclonal as well as isolated single fibroblast colony (F-CFU) cultures were set up. Short-term and long-term co-cultures of BM stroma used M-07e AML cells and AML blasts as IC. After long-term co-culture colony forming units (CFU) were determined in the adherent and non-adherent fraction of the IC. Proliferation of IC was determined by cell counting and 3H-TdR incorporation assays. The human HS-5 stroma cell line was used in selected experiments. The F-CFU frequency was determined from 39 Ctr samples, 6 MDS samples, 59 leukemic samples, and 12 lymphoma samples with BM infiltration. Succession of F-CFU numbers in respective samples was AML / MDS < Ctr < MM / FL II << 2°AML. Polyclonal stroma cells from AML BM supported M-07e IC survival slightly but not significantly better than Ctr stroma. No noteworthy support for primary AML blasts was observed with either stroma. Stroma from both AML and Ctr donors stimulated colony formation of M-07e IC without marked differences in the frequency of compact and diffuse colonies. In 3H-TdR assays M-07e IC were stimulated by IL-3 and certain but not all stroma samples tested. Notably, IL-3-induced proliferation of M-07e IC was decreased in the presence of stroma and almost completely suppressed by HS-5 cells. When single F-CFU were isolated, expanded, and tested for their capacity to support M-07e IC stimulating as well as non-stimulating F-CFU were found. Support competence of stroma cells decreased with passage number. Primary BM polyclonal stroma cells and single F-CFU from leukemic and non-leukemic donors showed a broad heterogeneity with respect to support of cell growth or colony-forming potential in favour of the AML M-07e IC line. Thus, the interaction of stroma and normal or leukemic hematopoietic cells seems to be a complex system awaiting further investigations. Disclosures: No relevant conflicts of interest to declare.

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