The Somatic Mutation of PIG-A Gene and the Expressions of EGR-1 and WT1 Genes in Bone Marrow Cells of the Patients with Paroxysmal Nocturnal Hemoglobinuria

Abstract Abstract 4379 Objective To investigate the somatic mutations of PIG-A gene and the expressions of EGR-1 and WT1 genes in the bone marrow cells of the patients with Paroxysmal nocturnal hemoglobinuria, then explore the characteristics of PIG-A gene mutation in Chinese patients with PNH and the possible mechanism of PNH clonal expansion related to EGR-1 and WT1 genes. Methods The quantities of CD55− cells and CD59− cells in both peripheral blood and bone marrow were examined with flow cytometric assay(FCM), and then sorting CD59− cells from bone marrow mononuclears (BMMNCs). The mutant segments of PIG-A gene were identified by DNA sequencing; The expressions of EGR-1 and WT1 mRNA in CD59− and CD59+ BMMNC were measured with semiquantitive RT-PCR. Results Nine in sixteen patients with PNH and one in four patients with AA-PNH were measured for their PIG-A gene mutation. Five single base substitution were found, including A→G, G→T, A→T, T→G and G→C, resulting in one consense and seven missense mutations. Each of 3 PNH patients had more than two point mutations of PIG-A gene. Insert or deletion mutation of PIG-A gene were not found. The relative mRNA expressions of EGR-1and WT1 genes in CD59− BMMNC, CD59+ BMMNC of PNH or AA-PNH patient and BMMNC of normal controls were (1.00±0.11), (0.86±0.14), (0.85±0.06) and (1.06±0.16), (0.90±0.14), (0.88±0.06), the expression in CD59− BMMNC was significantly higher than that in CD59+ BMMNC or normal controls (p<0.05). There was no significant difference between the expression in CD59+ BMMNC and normal controls(p>0.05). The relative mRNA expressions of EGR-1 and WT1 in BMMNC of PNH and AA-PNH cases were positively correlated with the proportion of CD59− cells(coefficient correlation was 0.509 and 0.481 respectively, p<0.05), but not related to the proportion of CD59+ cells. Conclusions The PIG-A gene mutation in some Chinese patients mainly manifested as single base substitution, occurred at random sites and without hot spot, the overexpression of EGR-1 and WT1 genes in PNH clone could promote the abnormal clone expansion. Disclosures: No relevant conflicts of interest to declare.

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The Expressions of CD114, CD117 and STAT5 in CD34+CD59− and CD34+CD59+ Bone Marrow Cells of the Patients with Paroxysmal Nocturnal Hemoglobinuria

Blood ◽

10.1182/blood.v120.21.4404.4404 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4404-4404

Author(s):

Rong Fu ◽

Shaoxue Ding ◽

Zonghong Shao ◽

Lijuan Li ◽

Hui Liu ◽

...

Keyword(s):

Bone Marrow ◽

Signaling Pathway ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Granulocyte Colony Stimulating Factor ◽

Clone Cells ◽

Stimulating Factor ◽

Normal Controls ◽

Marrow Cells

Abstract Abstract 4404 Objective: To measure the expressions of granulocyte colony-stimulating factor receptor (G-CSFR, CD114) and stem cell factor receptor (C-KIT, CD117) on the membrane of CD34+CD59− and CD34+CD59+ bone marrow cells of the patients with paroxysmal nocturnal hemoglobinuria(PNH), and the signaling pathway protein STAT5 within the cytoplasm of those cells. Methods: The expressions of CD114 and CD117 on the cell membrane and STAT5 protein within the cytoplasm of bone marrow CD34+CD59+ and CD34+CD59− cells from 26 PNH patients and 14 normal controls were examined by flow cytometry (FCM). Results: The percentage of CD114 positive cells in CD34+CD59− cells of PNH patients was (43.23±19.77)%,which was significantly lower than that in CD34+CD59+ cells of PNH patients (73.72±17.42) (P<0.01) or normal controls (65.91±13.70)% (P<0.01). There was no statistic difference between the two latter (P>0.05). The percentage of CD117 positive cells in CD34+CD59− cells of PNH patients was (49.20±26.80)%,which was significantly lower than that in CD34+CD59+ cells of PNH patients (67.62±17.41) (P<0.01) or normal controls (70.21±12.68)% (P<0.01). There was no statistic difference between the two latter (P>0.05). The STAT5 MFI in CD34+CD59− and CD34+CD59+ cells of PNH patients and CD34+CD59+ cells of normal controls was (270.01±181.26), (205.05±146.16), (227.39±156.65) respectively. There was no statistic difference among the three groups (P>0.05). Conclusions: In PNH, CD114 and CD117 expressed lower on bone marrow PNH clone cells than normal clone cells, but the expressions of signaling pathway protein STAT5 within the cytoplasm was normal. Disclosures: No relevant conflicts of interest to declare.

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High Incidence of TET2 Mutation in Chinese Patients with MDS and AML with Previous History of MDS

Blood ◽

10.1182/blood.v120.21.4943.4943 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4943-4943

Author(s):

Lin Li ◽

Zhu Xuejun ◽

Jiang Pengjun ◽

Zhang Wenxi ◽

Yu Hui ◽

...

Keyword(s):

Bone Marrow ◽

Gene Mutation ◽

Bone Marrow Cells ◽

Previous History ◽

Chinese Patients ◽

Hypomethylating Agents ◽

Tet2 Mutation ◽

History Of ◽

Vitro Effect ◽

Marrow Cells

Abstract Abstract 4943 Myelodysplastic syndromes(MDS) are a heterogeneous group of myeloid neoplasms characterized by cytopenia, dysplasia in one or more cell lines, ineffective haematopoiesis, and increased risk of development of acute myeloid leukemias(AML). The precise mechanism of onset and evolution of MDS has not been clarified. Recently, the ten-eleven translocation 2 (TET2) gene has been found mutated in 15–26% of MDS and AML. The TET2 paralog TET1 catalyzes the conversion of 5-methylcytosine to 5-hydroxymethylcytosine; TET2 shares a homologous domain thought to catalyze this conversion, and is hypothesized to act as a tumor suppressor gene by regulating DNA methylation and epigenetic control of gene expression at critical loci important for myelopoiesis and leukemogenesis. Moreover, the hypomethylating agents 5-azacytidine and decitabine, have demonstrated good perspective in the treatment of high-risk MDS, but their effects might be limited to some patients. Given the effect of TET2 in epigenetic regulation, it is supposed that the status of TET2 gene mutation affect the therapeutic effect of hypomethylating agents related above. Hence, the aim of this study was to determine the clinical characteristics of Chinese patients with TET2 mutations and the vitro effect of 5-azacytidine and decitabine on CD34+Lin−bone marrow cells from patients with or without TET2 gene mutation in MDS and AML with previous history of MDS(AML-MDS). In our study, the entire coding sequence of the TET2 gene (exons 3 to 11) were sequenced. DNA for detection of mutations were from PB or BM samples of 17 MDS(RA 1, RARS 1, RCMD 4, RAEB-1 7 and RAEB-2 4) and 8 AML-MDS patients in our hospital. Among the patients, there were 19 males and 6 females. The median age was 59(46∼83) year old. Cytogenetic analysis was proceeded in 16 patients and abnormal karyotypes were detected in 7 patients. According to IPSS, cytogenetics was favorable in 9, intermediate in 3, unfavorable in 4. All patients were found with one or more TET2 mutation, including 20 frameshift and 57 point mutations. Except 2 mutations in exon 9 and 1 in exon7, others were all in exon 3 or 11. Only 9 mutations were homozygous and others were heterozygous. Both homozygous and heterozygous TET2 mutations were found, suggesting that the presence of wild type allele or residual activity is not protective. A large proportion of frameshift mutations caused stop codons resulting in loss of function while missense mutations may lead to decreased function. In addition, CD34+Lin-bone marrow cells from 4 AML-MDS patients were sorted with immune magnetic beads from MACS and cultured with StemSpan SFEM supplemented of StemSpan CC100. Decitabine and 5-azacytidine were respectively employed with concentration of 0. 1, 0. 25, 0. 50μM for 12h, 24h, 48h, and 72h. Cell proliferation was inhibited and apoptosis was induced by both drugs. Three AML-MDS patients had previously been treated with decitabine and LD AraC for 1 to 3 cycles. More observation is needed to evaluate if presence of TET2 mutation represent a negative predictor of response to demethylating agents. In conclusion, our preliminary data showed that high proportion of Chinese MDS and AML-MDS patients carried TET2 mutation, of which most showed heterozygous status and involved exon 3 or 11. Decitabine and 5-azacytidine might have effects on CD34+Lin− bone marrow cells from AML-MDS patients in vitro. Disclosures: No relevant conflicts of interest to declare.

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NKG2D-Mediated Marrow Injury in Paroxysmal Nocturnal Hemoglobinuria and Its Related Disorders.

Blood ◽

10.1182/blood.v110.11.3674.3674 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3674-3674

Author(s):

Nobuyoshi Hanaoka ◽

Tatsuya Kawaguchi ◽

Kentaro Horikawa ◽

Shoichi Nagakura ◽

Sonoko Ishihara ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Bone Marrow Cells ◽

Close Association ◽

Bone Marrow Failure ◽

Γδ T Cells ◽

Refractory Anemia ◽

Nkg2d Ligands ◽

Marrow Cells

Abstract Immune mechanism is considered to exert in the pathogenesis of marrow failure in paroxysmal nocturnal hemoglobinuria (PNH), idiopathic aplastic anemia (AA) and myelodysplastic syndromes (MDS); however, the molecular events are unknown. We have currently reported the appearance of NKG2D ligands such as cytomegalovirus glycoprotein UL16 binding proteins (ULBPs) and MHC class I-related chains A and B (MICA/B) on granulocytes and CD34+ marrow cells of some patients with PNH and its related diseases (Hanaoka N, et al. Blood. 2006;107:1184–1191). ULBP and MICA/B are stress-inducible membrane proteins that appear in infection and transformation. The ligands share NKG2D receptor on lymphocytes such as NK, CD8+ T, and γδ T-cells and promote activation of the lymphocytes. Cells expressing the ligands are then deadly injured by NKG2D+ lymphocytes (Groh, PNAS 1996; Cosman, Immunity 2001). Indeed, cells expressing NKG2D ligands were killed in vitro by autologous NKG2D+ lymphocytes of our patients (Hanaoka N, et al. Blood. 2005;106:304a; Blood. 2006;108:295a). In further analysis, ligands were detected on granulocytes in 47 (53%) of 88 patients: 11 (58%) of 19 PNH, 28 (60%) of 47 AA, and 8 (36%) of 22 refractory anemia. Ligands were also detected on immature bone marrow cells in all 11 patients (3 PNH, 5 AA, and 3 refractory anemia) who permitted analysis of their marrow cells. In the patients, it is conceivable that blood cells were exposed to a certain stress to induce NKG2D ligands, leading to NKG2D-mediated marrow injury. We also observed a close association of the ligand expression with pancytopenia and favorable response to immunosuppressive therapy by prospective analysis of 5 patients (3 AA-PNH syndrome and 2 AA) for more than one year up to 5 years. Thus, we here propose that NKG2D-mediated immunity, which drives both NK and T-cells, is critically implicated in the pathogenesis of bone marrow failure of PNH and its related disorders.

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Expression of dlk1 in the Bone Marrow Cells of Patients with Myelodysplastic Syndrome and Its Clinical Significance

Blood ◽

10.1182/blood.v118.21.5042.5042 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5042-5042

Author(s):

Zonghong Shao ◽

Lanzhu Yue ◽

Rong Fu ◽

Lijuan Li ◽

Erbao Ruan ◽

...

Keyword(s):

Bone Marrow ◽

Early Diagnosis ◽

Myelodysplastic Syndrome ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Rt Pcr ◽

Normal Controls ◽

Lower Expression ◽

Bone Marrow Blasts ◽

Marrow Cells

Abstract Abstract 5042 Objective To investigate the expression of dlk1 gene (delta-like 1) in the bone marrow cells of patients with Myelodysplastic syndrome (MDS), and explore the molecular marker for early diagnosis of MDS. Methods The expression of dlk1 mRNA in the bone marrow cells of cases with MDS, AML and normal controls were measured by RT-PCR, aiming to search for the cytogenetic marker of MDS malignant clone. Results The expression of dlk1 mRNA in bone marrow cells of MDS patients (0.7342±0.3652) was significantly higher than that of normal controls (0.4801±0.1759) (P<0.05), and was significantly positively correlated with the proportion of bone marrow blasts(r=0.467,P<0.05). The expression of dlk1 mRNA significantly increased as the subtype of MDS advanced (P<0.05). Patients with abnormal karyotypes displayed significantly higher expression of dlk1 mRNA (0.9007±0.4334) than those with normal karyotypes (0.6411±0.2630) (P<0.05). Patients with higher expression of dlk1(≥0.8) presented significantly higher malignant clone burden (0.4134±0.3999) than those with lower expression (<0.8) of dlk1 (0.1517±0.3109) (P<0.05). Conclusion dlk1 gene was highly expressed in MDS patients, which increased as the subtype of MDS advanced. The expression of dlk1 mRNA was significantly positively correlated with the proportion of bone marrow blasts. High expression of dlk1 gene suggests high malignant clone burden of MDS. Disclosures: No relevant conflicts of interest to declare.

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Two abnormal clones in the bone marrow cells of a patient with paroxysmal nocturnal hemoglobinuria

Clinical Genetics ◽

10.1111/j.1399-0004.1979.tb00988.x ◽

2008 ◽

Vol 16 (3) ◽

pp. 178-182 ◽

Cited By ~ 5

Author(s):

A. M. Cohen ◽

F. Shabtai ◽

U. Lewinski ◽

B. Klein ◽

M. Djaldetti

Keyword(s):

Bone Marrow ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Bone Marrow Cells ◽

Marrow Cells

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A systematic analysis of bone marrow cells by flow cytometry defines a specific phenotypic profile beyond GPI deficiency in paroxysmal nocturnal hemoglobinuria

Cytometry Part B Clinical Cytometry ◽

10.1002/cyto.b.21064 ◽

2012 ◽

Vol 84B (2) ◽

pp. 71-81 ◽

Cited By ~ 6

Author(s):

Francesco Mannelli ◽

Sara Bencini ◽

Benedetta Peruzzi ◽

Ilaria Cutini ◽

Alessandro Sanna ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Bone Marrow Cells ◽

Systematic Analysis ◽

Phenotypic Profile ◽

Marrow Cells

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Defective Capacity of Bone Marrow From Nude Mice to Restore Lethally Irradiated Recipients

Blood ◽

10.1182/blood.v42.5.671.671 ◽

1973 ◽

Vol 42 (5) ◽

pp. 671-678 ◽

Cited By ~ 26

Author(s):

Dov Zipori ◽

Nathan Trainin

Keyword(s):

Bone Marrow ◽

Immune Responses ◽

Nude Mice ◽

Bone Marrow Cells ◽

Intrinsic Defect ◽

Proliferative Capacity ◽

Colony Forming Capacity ◽

Normal Controls ◽

Infective Agent ◽

Marrow Cells

Abstract The proliferative capacity of bone marrow cells from thymus-deprived nude mice was investigated in lethally irradiated recipients. Although the colony-forming capacity of these cells was found to be similar to that of normal littermates, a reduction in the number of nucleated cells was observed in the bone marrow of nude mice. Moreover when the radioprotective effect of such cells was studied, it was found that 5 x 105 or 2 x 106 bone marrow cells from nude mice were less effective in restoring hemopoiesis and establishing permanent chimerism than similar amounts of bone marrow cells of normal controls. In addition, irradiated animals surviving after injection of bone marrow cells from nude mice were found to have lower immune responses to SRBC than normal chimeras. The possibility that mortality of irradiated recipients injected with bone marrow of nude mice is due to the presence of a latent infective agent or of some inhibitory factor of hematopoiesis in the bone marrow of such nude mice is shown to be improbable. Alternatively it is suggested that nude mice suffer from an intrinsic defect in the proliferative capacity of their bone marrow colony-forming cells (CFUs).

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Study on the Burden of Abnormal Hematopoietic Clone of the Patients with Myelodysplastic Syndromes.

Blood ◽

10.1182/blood.v106.11.4899.4899 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4899-4899

Author(s):

Zonghong Shao ◽

Huaquan Wang ◽

Jun Shi ◽

Yanran Cao ◽

Hong Liu ◽

...

Keyword(s):

Bone Marrow ◽

Serum Level ◽

Normal Control ◽

Myelodysplastic Syndromes ◽

Bone Marrow Cells ◽

Normal Control Group ◽

Haematopoietic Stem Cell ◽

Control Group ◽

Normal Controls ◽

Marrow Cells

Abstract Background Myelodysplastic syndromes (MDS) is a group of clone haematopoietic stem cell diseases. The burden of abnormal hematopoietic clone plays key roles in the development of this disease and needs to be further studied quantitively. Methods The ratio of the counted bone marrow cells with abnormal chromosomes to the total counted bone marrow cells was regarded as the index of MDS clone burden. The disease severity related parameters including white cell count, hemoglobin, platelet count, lactate dehydrogenase level, bone marrow blast, myeloid differentiation index, the ratio of cFU-GM to CFU-GM, micromegakaryocyte, transfusion, IL-2, TNF, CD4+ and CD8+ T cells of MDS patients were assayed and the correlations between those parameters and MDS clone burden were also analysed. Results The clone burden of MDS patients was (67.4±36.2)%. MDS clone burden correlated positively with bone marrow blasts(r =0.483, P=0.012), negatively with hemoglobin level(r = −0.445, P= 0.023); The number of blasts, hemoglobin and erythocytes in high clone burden(>50%) and low clone burden(≤50%) groups were (7.78±5.51)% and (3.45±3.34)%(P=0.035), (56.06±14.28)g/L and (76.40±24.44)g/L(P=0.013), (1.82±0.12)×1012/L and (2.32±0.21)×1012/L(P=0.034)respectively. CD4+ T lymphcytes of MDS patients and normal controls were (274.18±71.85)×106/L and (454.82±205.88)×106/L(P=0.012)respectively. CD8+ T lymphcytes of MDS patients and normal controls were (240.45±150.01)×106/L an (305.27±145.14)×106/L(P=0.317)respectively. The serum level of interleukin 2 of MDS patients and normal control group were (6.29±3.58)ng/ml and (3.11±1.40)ng/ml (P=0.002) respectively. The serum level of tumor necrosis factor of MDS patients and normal control group were (2.42±1.79)ng/ml and (1.68±0.69)ng/ml(P=0.124)respectively. The ratio of CD4 to CD8 in high clone burden MDS patients was (1.90±0.52), and that in low clone burden patients was (0.97±0.44)(P=0.022). Conclusion The quantitive clonal karyotype abnormalities and deficient T cell immunity are important parameters for evaluating MDS severeity and prediciting it’s progression.

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Expression of CD123 and CD114 on the Bone Marrow Cells of Patients with Myelodysplastic Syndromes

Blood ◽

10.1182/blood.v116.21.4966.4966 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4966-4966

Author(s):

Zonghong Shao ◽

Lanzhu Yue ◽

Rong Fu ◽

Huaquan Wang ◽

Lijuan Li ◽

...

Keyword(s):

Bone Marrow ◽

Molecular Marker ◽

Myelodysplastic Syndromes ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Normal Controls ◽

Bone Marrow Blasts ◽

Marrow Cells

Abstract Abstract 4966 Background Recent studies have shown that interleukin-3 receptor α(CD123) is highly expressed on leukemia stem cells of patients with acute myeloid leukemia, and is correlated with tumor load and poor prognosis. The expression of CD123 may also be high in patients with myelodysplastic syndromes (MDS). In this study, the expression of CD123 as well as granulocyte colony stimulating factor (G-CSF) receptor (CD114) on the bone marrow cells of patients with MDS was investigated in order to explore the molecular marker of the malignant clone of MDS. Methods Forty-two patients with MDS, who were diagnosed in the hematological department of General Hospital of Tianjin Medical University from 2008 to 2009, and twelve normal controls were enrolled in this study. FACS was used to measure the expression of CD123 on CD34+CD38- cells and CD114 on CD34+ cells of the bone marrow of these patients and controls and the clinical significance was analyzed. The expression of CD114 on CD123+CD34+CD38- cells was further measured to explore the molecular marker of the malignant clone in MDS. Results The ratio of CD34+CD38-/CD34+ in the bone marrow cells of MDS patients was [(14.03±5.27)%], significantly higher than that of normal controls [(7.70±4.36)%] (P<0.01); The ratio of CD123+CD34+CD38-/CD34+CD38- in the bone marrow cells of MDS patients[(48.39±28.15)%]was significantly higher than that of normal controls [(8.75±11.71)%] (P<0.01), and was significantly positively correlated with the proportion of bone marrow blasts(r=0.457, P<0.05). The ratio of CD114+CD34+/CD34+ in the bone marrow cells of MDS patients [(33.05±21.71)%] was lower than that of normal controls [(38.99±19.07)%], but with no significance(P>0.05). The expression of CD114 on CD123+CD34+CD38-cells [(34.82±29.58)%] was significantly lower than that on CD123-CD34+CD38-cells [(53.48±27.41)%] of MDS patients (P<0.05). Conclusions MDS patients displayed higher proportion of CD34+CD38-/CD34+ than normal controls. CD123 was highly expressed in the bone marrow of patients with MDS, significantly correlated with the proportion of bone marrow blasts, thus might be the marker of MDS malignant clone. CD123+CD34+CD38-cells exhibited lower expression of G-CSF receptors, which might partly explain why MDS clone responsed worse to G-CSF in vitro and in vivo. Disclosures: No relevant conflicts of interest to declare.

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