Methylation Analysis of the Wnt Antagonist in Myelodysplastic Syndrome

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5048-5048
Author(s):  
Hongyan Tong ◽  
Chen Mei ◽  
Kongfei Li ◽  
Jie Jin
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Hematological Malignancies ◽  
Conflicts Of Interest ◽  
Methylation Status ◽  
Wnt Signaling Pathway ◽  
Aberrant Methylation ◽  
Methylation Frequency ◽  
Kaplan Meier ◽  
Close Relationship

Abstract Abstract 5048 Background and Objectives: Myelodysplastic syndrome (MDS) is one of the most threatening hematological malignancies. Recently, the epigenetic changes have been recognized in the MDS, and some research found that the aberrant DNA methylation had close relationship with the MDS. Meanwhile, some studies showed that the activation of the Wnt signaling pathway and abnormal methylation of the Wnt antagonists had close relationship with hematological malignancies, such as AML AALL Aand CLL, but less is known in MDS. In our research, we studied the methylation status of Wnt antagonists (DKK1, DKK3, HDPR1, WIF-1, SFRP1 and SFRP4) in the patients with MDS and evaluated the role of them in the pathogenesis and progression of MDS, with providing a new theory support for clarifying the complicated pathogenesis and progression of MDS. Methods: The methylation status of Wnt antagonists (DKK1, DKK3, HDPR1, WIF-1, SFRP1 and SFRP4) in pretreatment bone marrow samples from 53 patients with MDS was measured by methylation-specific polymerase chain reaction (MSP). On the other hand, we collected the clinical materials of the patients with MDS and follow up the patients. Then the correlation between methylation and clinical features as well as prognosis of MDS patients was analyzed byχ2 test and Kaplan-Meier method. Results: In 53 bone marrow samples, the methylation frequencies of the Wnt antagonists were as follows: SFRP4 for 62.3 % (33/53), DKK1 for 45.3 % (24/53), HDPR1 for 34.0 % (18/53), SFRP1 for 15.1 % (8/53), DKK3 for 9.4 % (5/53), and WIF-1 for 5.7 % (3/53). After analyzing individual tumor suppressor gene, clinical parameters and prognostic information, it was found that the patients with the percentage of BM blast above 5% had higher methylation frequency of HDPR1 than the patients with the percentage of BM blast bellow 5% (44.4%∼a13.3%, P=0.034); besides, the methylation frequency of HDPR1 exhibited significant differences in the MDS subtypes (P=0.019), and was significantly correlated with the WPSS (P=0.037); In addition, Kaplan-Meier survival curve indicated that the mean overall survival of patients with methylation was significantly shorter than that of patients without HDPR1 methylation (457.3 days vs. 919.6 days, P=0.037) (Fig.1). Conclusion: The methylation of the Wnt antagonists (DKK1, DKK3, HDPR1, WIF-1, SFRP1 and SFRP4) were observed in patients with MDS and their methylation frequencies were different. The aberrant methylation of HDPR1 may play an important role in the progression and prognosis of MDS. Disclosures: No relevant conflicts of interest to declare.

p15INK4b Methylation Is Related to Blast Percentage, Thrombocytopenia, the Number of Lineages Involved in Cytopenia and Survival in Myelodysplastic Syndrome at the Time of Diagnosis: a Pyrosequencing Study.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3793-3793
Author(s):  
Miyoung Kim ◽  
Bora Oh ◽  
Song-yee Kim ◽  
Hyun-Kyung Park ◽  
Sang Mee Hwang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Cytosine Methylation ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Bone Marrow Examination ◽  
Specific Pcr ◽  
Hypomethylating Agent ◽  
Kaplan Meier ◽  
System Variables

Abstract Abstract 3793 Poster Board III-729 Introduction The inactivation of tumor suppressor genes by promotor hypermethylation, particularly p15INK4b, is believed to contribute to the initiation and the progression of myelodysplastic syndromes (MDS). p15INK4b methylation was found to be more frequent in high-risk MDS (RAEB) at the time of diagnosis, and to be associated with progression to AML in the studies using methylation-specific PCR (MSPCR). We investigated if the quantity of p15INK4b methylation is related to International Prognosic Scoring System variables (IPSS) and survival in the myelodysplastic syndrome (MDS) patients and if the quantity of p15INK4b methylation reflects the bone marrow status (the percentage of blasts or the degree of dysplasia) of the patients under treatment with or without hypomethylating agents. Materials and Methods The study included 74 de novo MDS patients including 24 with follow-up bone marrow examination: 10 under treatment with hypomethylating agent and 14 under treatment with other than hypomethylating agent including stem cell transplantation (SCT). We pyrosequenced 63 base pairs of p15INK4b including 11 CpGs using PSQ96MA system (Biotage, Uppsala, Sweden), with the extent of CpG cytosine methylation assessed in terms of methylation index (MtI) and methylation level (MtL). Result Patients with >5% bone marrow blasts had higher MtI and MtL (58.8% and 10.1%, respectively) than patients with <5% blasts (44.0% and 6.1%, p = 0.031 and p = 0.030, respectively). Methylation quantity was not associated with chromosomal aberrations. The MtI and MtL of patients with thrombocytopenia were higher than patients without thrombocytopenia (62.1% vs. 44.1%, p = 0.009, and 11.2% vs. 6.2%, p = 0.036, respectively); they were higher in patients with cytopenias in °Ã2 lineages than in patients with either unilineage or no cytopenia (56.5% vs. 39.4%, p = 0.055, and 9.8% vs. 4.1%, p = 0.036, respectively). The survival of patients with >50% MtI or >7% MtL was worse than patients with <50% MtI or <7% MtL (p = 0.023 and p = 0.031, respectively). Methylation quantity of p15INK4b did not correlate with the bone marrow status of the patients under treatment regardless of using hypomethylating agent or not. Conclusion Using pyrosequencing, we showed that the quantity of p15INK4b methylation correlates with thrombocytopenia and blast percentage in MDS patients. Heavy p15INK4b methylation in MDS is associated with adverse survival. Methylation quantity of p15INK4b did not appear to reflect the bone marrow status of the patients under treatment regardless of the use of hypomethylating agent in this study, however, further study with controlled patient group is required to clarify this issue. Fig. 1. Heavy methylation of the p15INK4b promotor region is a prognostic factor for poor survival in MDS patients. (a) Kaplan-Meier curves of MDS patients with MtI >50% and patients with MtI <50% (mean survival time: 1118 days vs. 1864 days, respectively). (b) MDS patients with MtL >7% and patients with MtL <7% (738 days vs. 1804 days, respectively). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mutational Characteristics and Changing Pattern from Idiopathic Cytopenia of Undetermined Significance to High-Risk Myelodysplastic Syndrome Stratified By IPSS-R

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3009-3009
Author(s):  
Eun-Ji Choi ◽  
Young-Uk Cho ◽  
Seongsoo Jang ◽  
Chan-jeoung Park ◽  
Han-Seung Park ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Myelodysplastic Syndrome ◽  
Rna Splicing ◽  
Conflicts Of Interest ◽  
Low Risk ◽  
International Prognostic Scoring System ◽  
Intermediate Risk ◽  
Sequencing Data ◽  
Undetermined Significance

Background: Unexplained cytopenia comprises a spectrum of hematological diseases from idiopathic cytopenia of undetermined significance (ICUS) to myelodysplastic syndrome (MDS). Revised International Prognostic Scoring System (IPSS-R) is the standard tool to assess risk in MDS. Here, we investigated the occurrence, characteristics, and changing pattern of mutations in patients with ICUS and MDS stratified by IPSS-R score. Methods: A total of 211 patients were enrolled: 73 with ICUS and 138 with MDS. We analyzed the sequencing data of a targeted gene panel assay covering 141 genes using the MiSeqDx platform (Illumina). The lower limit of variant allele frequency (VAF) was set to 2.0% of mutant allele reads. Bone marrow components were assessed for the revised diagnosis according to the 2016 WHO classification. Lower-risk (LR) MDS was defined as those cases with very low- or low-risk MDS according to the IPSS-R. Higher-risk (HR) MDS was defined as those cases with high- or very high-risk MDS according to the IPSS-R. Results: Patients with ICUS were classified as very low-risk (39.7%), low-risk (54.8%), and intermediate-risk (5.5%) according to the IPSS-R. Patients with MDS were classified as LR (35.5%), intermediate-risk (30.4%), and HR (34.1%). In the ICUS, 28 (38.4%) patients carried at least one mutation in the recurrently mutated genes in MDS (MDS mutation). The most commonly mutated genes were DNMT3A (11.0%), followed by TET2 (9.6%), BCOR (4.1%), and U2AF1, SRSF2, IDH1 and ETV6 (2.7% for each). IPSS-R classification was not associated with mutational VAF and the number of mutations in ICUS. In the 49 LR MDS, 28 (57.1%) patients carried at least one MDS mutation. The most commonly mutated genes were SF3B1 (20.4%), followed by TET2 (12.2%), U2AF1 (10.2%), DNMT3A (10.2%), ASXL1 (10.2%), and BCOR (6.1%). Higher VAF and number of mutations were observed in LR MDS compared to ICUS patients. In the 42 intermediate-risk MDS, 27 (64.3%) patients carried at least one MDS mutation. The most commonly mutated genes were ASXL1 (23.8%), followed by TET2 (21.4%), RUNX1 (16.7%), U2AF1 (14.3%), DNMT3A (14.3%), SF3B1 (9.5%), and SRSF2, BCOR, STAG2 and CBL (7.1% for each). In the 47 HR MDS, 36 (76.6%) patients carried at least one MDS mutation. The most commonly mutated genes were TET2 (25.5%), followed by DNMT3A (14.9%), TP53 (14.9%), RUNX1 (12.8%), U2AF1 (10.6%), ASXL1 (10.6%), and SRSF2 and KRAS (6.4% for each). As the disease progressed, VAF and number of the MDS mutations gradually increased, and mutations involving RNA splicing, histone modification, transcription factor or p53 pathway had a trend for increasing frequency. Specifically, ASXL1, TP53, and RUNX1 mutations were the most striking features in patients with advanced stage of the disease. Cohesin mutations were not detected in ICUS, whereas these mutations were detected at a relatively high frequency in HR MDS. Our data were summarized in Table 1. Conclusions: We demonstrate that on disease progression, MDS mutations are increased in number as well as are expanded in size. Furthermore, a subset of mutations tends to be enriched for intermediate- to HR MDS. The results of this study can aid both diagnostic and prognostic stratification in patients with unexpected cytopenia. In particular, characterization of MDS mutations can be useful in refining bone marrow diagnosis in challenging situations such as distinguishing LR MDS from ICUS. Disclosures No relevant conflicts of interest to declare.

Impact of TET2 mutations On Responsiveness to Demethylating Agents in MDS.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1606-1606
Author(s):  
Anna M. Jankowska ◽  
Mohammed Shaik ◽  
Heather Cazzolli ◽  
Rebecca Ganetzky ◽  
Mikkael A. Sekeres ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Methylation Status ◽  
Epigenetic Silencing ◽  
Specific Gene ◽  
Aberrant Methylation ◽  
Global Methylation ◽  
Myeloid Malignancies ◽  
Demethylating Agents ◽  
Potential Marker ◽  
Epigenetic Instability

Abstract Abstract 1606 Poster Board I-632 Aberrant epigenetic silencing of tumor suppressor and differentiation genes constitutes an important mechanism in the pathogenesis of MDS and related myeloid malignancies. Demethylationg agents such as azacitidine and decitabine lead to degradation of DNMT1 and may reverse aberrant methylation. While the drugs demonstrate efficacy in MDS, response rates are variable. Thus, many primarily refractory patients are exposed to these therapies unnecessarily. While search for markers of responsiveness included study of methylation status of potential marker promoters or global methylation patterns, to date predictive tests have not been developed. Similarly, mechanisms of epigenetic instability responsible for wide-spread promoter methylation have not been clarified, preventing development of diagnostic markers. TET2 mutations are frequent events in a variety of MDS subtypes, particular chronic myelomonocytic leukemia (CMML) and other MDS/MPN, as well as AML derived from those conditions. It is likely that TET2 alterations are important in the pathogenesis of myeloid malignancies, but little is known regarding the function of TET2. A recent report indicated that a related family member, TET1, converts 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (hmC). Hydroxylation of 5mC prevents DNMT1 from homologous methylation of daughter DNA strands during cell division, thus preventing maintenance methylation (Tahiliani et al. Nature 324, 2009). Consequently, closely related TET2 may play a role in epigenetic regulation. As a consequence, TET2 mutations may lead to accumulation of aberrantly methylated CpG islands. Of utmost importance is whether TET2 mutations or resultant epigenetic silencing of specific gene or gene groups affects response to hypomethylating agents. We hypothesized that TET2 mutations play a role in epigenetic instability and may serve as markers of responsiveness/refractoriness to the therapy with demethylating agents. We have determined TET2 mutational status by sequencing all exons in 32 patients with myeloid malignancies (MDS (N=18) and MDS/MPN (N=14)) who underwent therapy with the demethylating agents azacitidine (N=27) or decitabine (N=5). For definition of response we applied International Working Group Criteria in patients who received a sufficient dose and number of cycles to allow assessment of response. Overall response rate (complete+partial responses (CR+PR) + hematologic improvement (HI)) was achieved for 9/32 patients (28%) after 35 cycles, including 4 patients who achieved CR, 2 PR, and 3 CI. In total, 12 TET2 mutations were identified in 9/32 patients (28%), of whom 5 had MDS/MPN (3 with CMML-1/2) and 4 had sAML. Unique compound heterozygosity was found in 3 patients; consequently biallelic inactivation of TET2 was found in 4 patients. For analyses, patients with partial and complete responses were compared with refractory patients and response was correlated with the presence of TET2 mutations. Among TET2-mutated patients, only 1 patient responded to therapy, whereas 8 additional patients, including 3 patients with biallelic inactivation of the TET2 gene, did not show any improvement (11%). In contrast, among patients with WT TET2 gene, responses were seen in 8/23 patients (35%; p=.19). While the cohort of treated patients was small, our preliminary results indicate that the presence of TET2 mutations may represent a negative predictor of response to demethylating agents. Disclosures No relevant conflicts of interest to declare.

Methylation Status of the Collagen Prolyl-3 Hydroxylases (C-P3H) and C-P4H Genes in Multiple Myeloma: P3H2 Is Selectively Methylated

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4639-4639
Author(s):  
Eleftheria Hatzimichael ◽  
Aggeliki Dasoula ◽  
Konstantinos Lagos ◽  
Georgianna Kartalou ◽  
George Dranitsaris ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Cpg Island ◽  
Methylation Status ◽  
Cpg Islands ◽  
Clinical Parameter ◽  
Small Population ◽  
Cpg Methylation ◽  
Post Translational Modification

Abstract Abstract 4639 Background–Aim: Prolyl hydroxylation is an important and the most common post-translational modification that affects the structure and function of collagen. Enzymes that participate in this process are the collagen prolyl-3 hydroxylases (C-P3H) and the C-P4H. We have previously showed that P3H3 is methylated in multiple solid tumour types, whereas methylation in P3H2 appears to be specific for breast cancer and multiple myeloma (MM). In this study we assessed the CpG methylation status of the P3H genes (P3H1, P3H2, P3H3) in a larger cohort of MM patients and for the first time the CpG methylation status of the P4H genes (P4HA1, P4HA2, P4HA3) and investigated for clinical relevance. Patients and Methods: Bone marrow aspirate samples from 47 MM patients (28 males, median age 66 years) were obtained at diagnosis. Methylation-specific PCR (MSP) was employed to study the CpG methylation in P3H1, P3H2, P3H3, P4HA1, P4HA2 and P4HA3 CpG islands. Genomic DNA was isolated and bisulphite modified using commercially available kits (QIAmp DNA mini kit, Qiagen and EZ DNA methylation kit, Zymo Research respectively). Control methylated and unmethylated genomic DNAs were included in each experiment. For the study of the P3H2 CpG methylation status we used 2 sets of independent primers, which map to different areas of P3H2 CpG island. Ten bone marrow samples from patients with border line thrombocytopenia that were proved to have no neoplasia served as negative controls. Logistic regression analyses were used to measure the association between gene methylation and age, b2 microglobulin, serum albumin, hypercalcemia, ISS stage, extramedullary disease, bone disease, anemia and renal failure (eGFR< 50 ml/min). Results: None of the six genes under study was methylated in the control bone marrow samples. Methylation in the CpG island of P3H2 was detected in 44 % of patients (95% CI 27.8–62.8%) No methylation was detected in the CpG islands of P3H1, P3H3, P4HA1, P4HA2 and P4HA3. Trends noted were that patients with methylated P3H2 were more likely to have ISS stage 3 (OR 2.2, p=0.1). Overall no significant association was found with any clinical parameter although the study might have been underpowered due to the small population size. Conclusions: P3H2 is methylated at high frequency in MM, whereas P3H1, P3H3, P4HA1, P4HA2, P4HA3 were unmethylated, implying a striking specificity for methylation in P3H2 in MM. These findings warrant further evaluation in a larger sample of patients in order to better define the prognostic and clinical utility of P3H2 methylation in MM. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
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.

Mutation Analysis in 200 Cases of Newly Diagnosed Myelodysplastic Syndrome By Next Generation Sequencing: Association with Established Prognostic Variables and IPSS-R

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1703-1703
Author(s):  
Kankana Ghosh ◽  
Parsa Hodjat ◽  
Priyanka Priyanka ◽  
Beenu Thakral ◽  
Keyur P. Patel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Next Generation Sequencing ◽  
Myelodysplastic Syndrome ◽  
Mutation Analysis ◽  
Conflicts Of Interest ◽  
International Prognostic Scoring System ◽  
P Value ◽  
Newly Diagnosed ◽  
Next Generation ◽  
Generation Sequencing

Abstract INTRODUCTION Myelodysplastic syndrome (MDS) is known to have numerous genomic aberrations that predict response to treatment and overall survival. We aimed to assess various mutations in newly diagnosed MDS cases by next generation sequencing (NGS) and their association with various well-established clinicopathologic parameters and the Revised International Prognostic Scoring System (IPSS-R). MATERIALS AND METHODS We performed molecular studies on DNA extracted from bone marrow aspirate specimens in 200 newly diagnosed treatment naïve MDS patients presenting at a single institution from 08/2013 to 03/2015 as part of routine clinical work up in a CLIA certified molecular diagnostics laboratory. Cases met criteria for MDS per WHO 2008 criteria. The entire coding sequences of 28 genes (ABL1, ASXL1, BRAF, DNMT3A, EGFR, EZH2, FLT3, GATA1, GATA2, HRAS, IDH1, IDH2, IKZF2, JAK2, KIT, KRAS, MDM2, MLL, MPL, MYD88, NOTCH1, NPM1, NRAS, PTPN11, RUNX1, TET2, TP53, WT1) were sequenced using a NGS-based custom-designed assay using TruSeq chemistry on Illumina MiSeq platform. FLT3 internal tandem duplications (ITD) and codon 835/836 point mutation were detected by PCR followed by capillary electrophoresis. CEBPA mutation analysis was performed by PCR followed by Sanger sequencing on 186 patients. RESULTS Median age was 67 years. Patients included 139 males (69.5%) and 61 females (30.5%). Hematologic parameters are as follows [median (range)]: Hb 9.6 g/dL (5-16.7), platelets 75 K/μ L (5-652), WBC: 2.8 K/μ L (0.4-20.8), ANC 1.3 K/μ L (0.0 -12.0), AMC 0.2 K/μ L (0.0-3). Bone marrow (BM) blasts [median (range)] were 4% (0-19). Of 192 patients with cytogenetic analysis performed, 65 (33.85%) had diploid karyotype, 53 (27.6%) had one, 21 (10.93%) had two, 13 (6.77%) had three, 40 (20.83%) had > three abnormalities. IPSS-R risk categorization of the 200 cases is as follows: very low (17 cases, 8.5%), low (46, 23%) intermediate (42, 21%), high (47, 23.5%), very high (48, 24%). Mutations identified by NGS are as detailed in Table 1. Of the 4 patients with FLT mutations detected, the breakdown is as follows: FLT3 ITD (3, 75%), FLT3 D835 (1, 25%), FLT3, ITD + D835 (0, 0%). CEBPA mutation was detected in 12 of 186 (6.45%) cases assessed. CEBPA was detected in 12 (6.45%). Sixty three (31.5%) cases had no mutations detected in the genes analyzed by NGS or PCR, 80 (40%) had mutations in one, 42 (21%) had mutations in two, 8 (4%) in three and 7 (3.5%) in > three genes. We found positive associations between mutated genes and various parameters as detailed in Table 2. No association was found between frequency of any particular mutation and the IPSS-R score. CONCLUSIONS: MDS is a heterogeneous group of myeloid neoplasms at the genetic level. Multiple genetic mutations in a large subset of cases likely indicate clonal evolution. A subset of mutations has significant association with well-established clinico-pathologic parameters like WBC and BM blast percentage. With longer follow-up, we could use this data to refine IPSS-R. Table 1. Number of cases % cases TP53 46 23 TET2 33 16.5 RUNX1 27 13.5 ASXL1 25 12.5 DNMT3A 17 8.5 EZH2 12 6 IDH2 8 4 IDH1 7 3.5 NRAS 7 3.5 JAK2 5 2.5 FLT3 4 2 PTPN11 3 1.5 EGFR 2 1 MPL 2 1 WT1 2 1 GATA2 1 0.5 KIT 1 0.5 KRAS 1 0.5 MYD88 1 0.5 NPM1 1 0.5 BRAF 1 0.5 Table 2. Mutated genes p value WBC ASXL1 <0.042 AEC TET2 <0.016 BM blast % RUNX1, CEBPA <0.008, p<0.02 BM myelocyte % TP53, TET2, RUNX1, DNMT3A <0.014, <0.014, <0.015, <0.038 AEC: absolute eosinophil count, BM: bone marrow Disclosures No relevant conflicts of interest to declare.

scholarly journals GDF11 Increased in Patients with Myelodysplastic Syndrome

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5224-5224
Author(s):  
Yu Han ◽  
Huaquan Wang ◽  
Zonghong Shao
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Myelodysplastic Syndrome ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Risk Group ◽  
Low Risk ◽  
Mean Corpuscular Hemoglobin ◽  
Corpuscular Hemoglobin ◽  
Normal Controls

Abstract Objective To analyze the concentration of growth differentiation factor 11(GDF11) in peripheral blood of patients with myelodysplastic syndrome (MDS), so as to evaluate the relationships between these changes and erythropoiesis functions and to explore the role of GDF11 in the pathogenesis of MDS. Methods The concentration of GDF 11 in peripheral blood was detected by enzyme-linked immuno sorbent assay in 44 MDS patients and 10 normal controls from September 2014 to June 2015 at our hospital. The percentage of nucleated erythrocyte (CD235a) in bone marrow was detected by flow cytometry. The correlation between these changes and erythropoiesis functions, including red blood cell count, hemoglobin, reticulocyte (RET%), hematokrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular-hemoglobin concentration (MCHC) and late erythroblast in bone marrow were evaluated. Results (1)The concentration of GDF11(128.67±47.62)in high-risk MDS patients was significantly higher than that of low-risk MDS patients (65.96±36.55,p<0.01)and higher than that of normal controls (29.76±10.10,p<0.01); The concentration of GDF11 in low-risk MDS patients was significantly higher than that of normal controls (p<0.05). (2) The expression of CD235a in high-risk group(38.49±5.42)was not different with that in low-risk group(42.64±7.36, p>0.05). (3)In high-risk MDS patients, the expression of GDF11 was negatively correlated with Hb, RET%, RBC, MCHC, Hct in peripheral blood and late erythroblast, CD235a+ cells in bone marrow(r=-0.437,r=-0.428,r=-0.444,r=-0.553,r=-0.661,r=-0.436,r=-0.52,all p<0.05),and the expression of GDF11 was positively correlated with MCV(r=0.52, p <0.05),but it was not correlated with MCH (p >0.05).(4) In low-risk MDS patients, the expression of GDF11 was negatively correlated with Hb, RET% (r=-0.491Ar=-0.606,both p<0.05),it was not correlated with RBC, MCHC, MCV, MCH, Hct, late erythroblast and CD235a+ cells (all p>0.05). Conclusion GDF11 increased in patients with MDS and it was negatively correlated with late erythropoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Genetic Mutation in Cytopenic Patients: Distinctive Genomic Profile between Preclinical Vs. Clinical Myelodysplastic Syndrome

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5429-5429
Author(s):  
Kritanan Songserm ◽  
Amornchai Suksusut ◽  
Sunisa Kongkiatkamon ◽  
Kitsada Wudhikarn ◽  
Chinnachote Teerapakpinyo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Myelodysplastic Syndrome ◽  
Somatic Mutations ◽  
Conflicts Of Interest ◽  
Gene Mutations ◽  
Genetic Alterations ◽  
Genetic Mutation ◽  
Low Risk ◽  
Genomic Profile

Genetic mutation in cytopenic patients: Distinctive genomic profile between preclinical vs. clinical myelodysplastic syndrome. Introduction Myelodysplastic syndromes (MDS) are heterogeneous groups of clonal hematopoietic disorders. The current diagnosis of MDS is based on morphologic assessments of dysplasia which are subjected to inter-observer variability and cytogenetic abnormalities which are frequently absent. Somatic mutations in myeloid-related genes have been identified in MDS. However, they are also found in idiopathic cytopenia of unknown significance (ICUS) that shows no significant dysplasia. Therefore, we aimed to explore the clinical implications of genetic mutations in ICUS and compared with MDS. The secondary objective was to find association between degree of dysplasia and somatic mutations. Materials and Methods The patients with peripheral cytopenia ≥1 lineage (ANC < 1,800/mm3, hemoglobin < 10 gm/dL, platelet < 100x109/mL) without explainable causes were enrolled. Bone marrow aspirates were evaluated independently by 2 hematologists. Of note, dysplasia are defined by WHO 2008 classification (eg. Erythroid lineage: ring sideroblasts, megaloblastoid change; granulocytic lineage: hypogranularity, pseudopelger-huet anomaly; megakaryocytic lineage: hypolobate, micro-megakaryocyte). The significant dysplasia cut off was 10% in single lineage or more. If there was a discrepancy, the third hematologist would help to reach the final consensus. We extracted DNA from bone marrow and performed next generation sequencing (NGS) that targeted 143 myeloid-related genes. Results Forty-eight patients were enrolled in this study. The median age at diagnosis was 70 years (71-96). Results of bone marrow examinations were categorized by morphology into 3 groups; non-significant dysplasia (dysplasia < 10%) 27%, low risk MDS (IPSS-R ≤3.5) 42% and high-risk MDS/sAML (IPSS-R >3.5/Blast≥20% in BM or peripheral blood) 31%. Most of cases (77%) carried normal cytogenetics while other genetic alterations were complex chromosome (6%), -Y (6%), del(5q) (4%), trisomy 8 (2%), del(20q) (2%), i(17q) (2%). Thirty from 48 cases (62%) harbored more than 1 somatic mutation. Twenty-eight gene mutations were identified. Mutations were detected 1.6 mutation per 1 patient in average. Most frequent somatic mutations were ASXL1:10/80 (12%), TET2:9/80 (11%), MFDS11: 6/80 (7%), TP53:6/80 (7%), and RUNX1:5/80 (6.25%). The proportions of cases with somatic mutations were not different across the groups (no dysplasia 50%, non-significant dysplasia 80% and significant dysplasia 62%). According to mutation types in each group, mutations in epigenetic pathways were the most frequent mutations across all patient subgroups (ICUS 64.7%, low-risk MDS 51.8 %, and high-risk MDS 52.5%). Mutations in transcription factor were predominated in MDS (18.5% and 25.0% in low-risk and high-risk MDS, respectively) compared to ICUS (11.7%). Individual average frequency of gene mutations was significantly different between disease subtype (high risk MDS 2.7 gene/person, low risk MDS 1.1 gene/person, ICUS 1.3 gene/person (P=.038). Higher variant allele frequency (VAF) of mutated genes was significantly observed in high risk MDS (38.3%) compared to low risk MDS (30.8%) and preclinical MDS (29.0%) (P=.03). Conclusion In conclusion, molecular profiling was significantly different between preclinical MDS and MDS groups in terms of types of somatic mutations and VAF. This unique contrast could be used to distinguish between preclinical MDS and clinically significant MDS. In contrast, degree of marrow dysplasia was not associated with number of gene mutations in this study. Prediction for clinical consequent of somatic mutations in CCUS requires long term follow up. Disclosures No relevant conflicts of interest to declare.

Myelodysplastic Syndrome-Derived Stromal Cells Support Leukemia-Initiating Cells and Contradirectly Eliminate Normal CD34+ Clonogenic Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1219-1219
Author(s):  
Hiroto Horiguchi ◽  
Masayoshi Kobune ◽  
Shohei Kikuchi ◽  
Satoshi Iyama ◽  
Kohichi Takada ◽  
...  
Keyword(s):  
Stem Cell ◽  
Myelodysplastic Syndrome ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Methylation Status ◽  
Gene Expressions ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Clonogenic Cells

Abstract Introduction The failure of normal hematopoiesis in myeloid neoplasm could be induced by a variety of mechanism. Regarding myelodysplastic syndrome (MDS)/acute leukemia (AML), aberrant hematopoietic stem/progenitor cells with exhibiting ineffective hematopoiesis and impaired differentiation ability gradually substitute it for normal hematopoietic stem/progenitor cells during a long term as a consequent of replacement of stem cell niche. However, it has not yet been clarified precise mechanism how MDS stem/progenitor cells could replace normal hematopoietic stem/progenitor cells. Methods In an attempt to analyze the supporting activity of bone marrow (BM) stromal cells, we first established the MDS/AML-derived stromal cells and healthy volunteer (HV)-derived-stromal cells. Next, MDS/AML-derived CD34+ cells or normal CD34+ cells were cocultured with established stromal cells using cytokines including stem cell factor, thrombopoietin, flt3-ligand in the presence of notch ligand (for normal CD34+ cells) or IL-3 (for AML/MDS derived cells). Subsequently, we analyzed clonogenic cells after 2 weeks coculture, 5 week cobblestone area-forming cells (CAFC) and repopulating cells in immunedeficient mice (NSG mice). Results The support of clonogenic cells after 2 weeks coculture and 5 weeks CAFCs was observed after coculture with normal CD34+ cells and HV-derived stromal cells. Furthermore, these cocultured cells engrafted into immunedeficient mice. Interestingly, the number of colony-forming units (CFU) mixed cells (MIXs) and CAFC derived from CD34+ cells was drastically reduced after coculture with MDS/AML-derived stromal cells. Nevertheless, MDS/AML-derived stromal cells support the proliferation of leukemia-initiating cells (L-ICs) and L-ICs were detected after third replating. These results indicate that MDS/AML-derived stromal cells preferentially support leukemia stem/progenitor cells, but not normal CD34+ cells. We compared the mRNA expression between (HV)-derived-stromal cells, MDS/AML-derived stromal cells and 5-aza-dC-treated stromal cells. The expression of several factors including hedgehog-interacting protein (HHIP) was reduced in MDS/AML-derived stromal cells. 5-aza-dC treatment restored the expression in some of genes and the stromal supporting activity for normal CD34+ cells partially recovered. Conclusion These results suggest that reduction of several gene expressions was detected in MDS/AML stromal cells by changes of methylation status. The epigenetic alteration of stromal genome may be involved in the progression of myeloid disorders. Disclosures: No relevant conflicts of interest to declare.

p53 Co-Activator Aspp1 Induces Apoptosis in Damaged Hematopoietic Stem Cells and Prevents Malignant Transformation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 603-603 ◽  
Author(s):  
Masayuki Yamashita ◽  
Eriko Nitta ◽  
Toshio Suda
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
Hematopoietic Stem Cells ◽  
Hematological Malignancies ◽  
Aberrant Methylation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Apoptotic Genes

Abstract Accumulation of DNA damage in hematopoietic stem cells (HSCs) is associated with aging, bone marrow failure and development of hematological malignancies. Mutation accumulation in HSCs precedes the development of leukemia and lymphoma, and these “pre-leukemic HSCs” can survive after chemotherapy, contributing to the relapse of the disease. Thus, understanding for the DNA damage response at a HSC level is a matter of critical importance for lifelong hematopoiesis, yet the protection mechanism for HSCs from DNA damage accumulation remains to be elucidated. During our study on the response of HSCs to ionizing radiation (IR), we have detected higher responsiveness of HSCs to DNA damage compared with committed progenitor cells: higher p53 activation was observed in HSC-enriched LSK (Lin-Sca1+cKit+) cells and LT-HSCs (CD150+CD41-CD48-LSK) than in myeloid progenitor-enriched LKS- cells. Of note, when treated with 4 Gy IR, LSK cells exhibited stronger upregulation of pro-apoptotic genes Bax, Noxa and Puma compared with LKS- cells, whereas upregulation of survival-contributing p21 and Mdm2 genes was comparable between the two populations. Corresponding to such characteristic behavior, we have identified apoptosis-stimulating protein of p53 1 (Aspp1) as a novel specific regulator of HSCs that provides HSCs with high sensitivity to apoptosis. We found that mRNA and protein of Aspp1 were specifically detected in LSK cells and LT-HSCs. To uncover the roles of Aspp1 in the regulation of HSCs, we evaluated HSCs of adult Aspp1 knockout (KO) mice. These mutant mice exhibited a major increase in the absolute number of LSK cells (1.5-fold; P<0.05) and LT-HSCs (2-fold; P<0.0005). Furthermore, self-renewal capacity of Aspp1-null HSCs was significantly enhanced as measured by serial competitive bone marrow (BM) transplantation assays (P<0.01). To assess the cause of enhanced self-renewal of Aspp1-null HSCs, we examined gene expression profile of Aspp1-null LSK cells before and after BM transplantation using multiplex quantitative RT-PCR array. Aspp1-null LSK cells showed higher expression of multiple quiescence-related genes including Tek, Mpl and Ndn. In line with this, Ki67 staining revealed that Aspp1-null LSK cells showed resistance to the loss of quiescence after serial BM transplantation (P<0.01), and Aspp1 KO mice showed accelerated recovery of peripheral blood and BM when treated with a single dose of 5-FU (P<0.05). Moreover, when serially transplanted or subjected to 4 Gy IR in vivo, Aspp1-null LSK cells exhibited higher resistance to apoptosis which was detected as decreased proportion of Annexin V-positive cells (P<0.05). Gene expression analysis consistently revealed that the induction of pro-apoptotic genes Bax, Noxa and Puma was impaired in irradiated Aspp1-null LSK cells. As a result of the reduced apoptosis, Aspp1-null LSK cells exhibited the tendency to retain persistent DNA damage after genotoxic stress as assessed by γH2AX and 53BP1 foci (chi-square test, P<0.05). Importantly, by breeding Aspp1 KO mice with Mx1-Cre mice and p53flox/flox mice, we verified that Aspp1 synergized with p53 to regulate self-renewal and genomic integrity of HSCs beyond its canonical p53-dependent function. Aspp1 loss further enhanced self-renewal capacity of HSCs in a p53-null background when assayed by serial BM transplantation (P<0.05). Likewise, Aspp1 deficiency further accentuated the accumulation of DNA damage after IR exposure in the absence of p53 (P<0.05). Consequently, whereas approximately half of the recipients receiving p53-null LSK cells died of thymic lymphoma, the recipient mice transplanted with LSK cells deficient for both Aspp1 and p53 were 100% lethal within 6 months after BM transplantation (log-rank test, P<0.01). These mice succumbed to hematological malignancies, mostly T-cell acute lymphoblastic lymphoma and leukemia (ALL) (88%) but also B-cell (6%) and myeloid (6%) malignancies. Taken together, our study demonstrates that Aspp1 attenuates HSC quiescence and induces apoptosis in damaged HSCs, in both p53-dependent and -independent manners, thereby inhibiting the development of leukemia and lymphoma in conjunction with p53 in HSCs. As loss of Aspp1 expression due to aberrant methylation of its promoter has already been proven to be an independent poor prognosis factor in ALL patients, Aspp1 may be a potential target for stem cell-directed therapy of leukemia and lymphoma. Disclosures No relevant conflicts of interest to declare.

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