FLIP (Long) and FLIP (Short) Expression in Bone Marrow Cells in Patients with Myelodysplastic Syndrome: Correlation with FAB and WHO Classification.

Abstract The paradox of peripheral cytopenias despite of normo/hypercellular marrow in myelodysplastic syndrome (MDS) has been ascribed to excessive intramedullary hematopoietic cell apoptosis. Several apoptosis-inducing systems, including Fas/Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL) and its receptors, are upregulated in MDS. FLIP (FLICE (FAS-associated death-domain-like IL-1β-converting enzyme)-inhibitory protein) was identified as a FAS and TRAIL signal inhibitor. The largest variant FLIPLong (FLIPL) was originally characterized as a molecule with inhibitory activity on caspase-8. The short splice form termed FLIPShort (FLIPS) has also been characterized as a potent (TRAIL-induced) apoptosis inhibitor. However, whereas FLIPL and FLIPS have been described as death receptor pathway inhibitors, recent data suggest that physiologically, FLIPL may have caspase-8-activating properties. This study aims to characterize the expression of FLIPL and FLIPS based on mRNA, by Real-time quantitative PCR, in marrow cells from MDS patients and to correlate the expression with French-American-British (FAB) and World Health Organization (WHO) classification. For each sample, results were first calculated as a ratio of the total transcript number of FLIPL or FLIPS and the total transcript number of the endogenous reference gene (β-actin) to obtain a normalized target value. Transcript ratios of each sample were normalized against the respective ratio of a pool of 6 normal bone marrow donors (NBM), and the ratio between the two was used as measure for the relative FLIPL or FLIPS level. We hypothesized that FLIPL and FLIPS expression differed between low and high risk of MDS. Marrow aspirates were obtained from 6 NBM and 16 patients with MDS out of treatment (7 males, 9 females; 23–78 (median 64) yo). The National Ethical Committee Board approved this study, informed-written consent was obtained from all patients and donors. According to FAB classification, patients were distributed as: 10 RA, 2 RARS and 4 RAEB. According to WHO classification: 10 RCMD, 2 RCMD-RS, 3 RAEB-1 and 1 RAEB-2. FLIPS mRNA expression were significantly higher in high risk DS according to FAB and WHO classification; RA/RARS compared with AREB (0.08 [0.0–2.3] vs 0.67 [0.36–1.54]; P = 0.03); RCMD and RCMD-RS compared with RAEB-1 and RAEB-2 (0.08 [0.0–2.3] vs 0.67 [0.36–1.54]; P = 0.03). However, FLIPL mRNA expression also tended to be higher in high risk MDS according to FAB and WHO classification, though not significantly different: RA/RARS compared with AREB (1.18 [0.06–3.43] vs 1.65 [0.51–3.63]; P = 0.46); RCMD and RCMD-RS compared with RAEB-1 and RAEB-2 (1.18 [0.06–3.43] vs 1.65 [0.51–3.63]; P = 0.46). Lower FLIPS level in low risk MDS marrows, in addition to the well described upregulation of extracellular proapoptotic signals, would explain the increased susceptibility of hematopoietic cells in low risk MDS marrow to death-inducing stimuli. The fact that FLIPL expression did not differ according to FAB and WHO classification could be related to the hypothesis that FLIPL may have caspase-8-activating properties rather than anti-apoptotic activity. Differential regulation of FLIPL and FLIPS according to risk groups in MDS patients might result in different rates of apoptosis. Further studies are needed to elucidate the mechanisms controlling and regulating FLIP expression in normal and malignant hemopoietic cells.

Download Full-text

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

Blood ◽

10.1182/blood-2019-125236 ◽

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.

Download Full-text

Flow Cytometric Detection of Human Telomerase Reverse Transcriptase Expression in CD34 Positive Precursor Fraction in Myelodysplastic Syndrome Bone Marrow.

Blood ◽

10.1182/blood.v108.11.4300.4300 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4300-4300

Author(s):

Hiroshi Handa ◽

Takafumi Matsushima ◽

Norifumi Tsukamoto ◽

Masamitsu Karasawa ◽

Hiroyuki Irisawa ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

High Risk ◽

Myelodysplastic Syndrome ◽

Disease Progression ◽

Risk Group ◽

Telomerase Activity ◽

Low Risk ◽

Htert Expression ◽

Trap Assay

Abstract Telomerase activity has been found in most common cancers indicating that telomerase detection may be a useful marker in cancer diagnosis. For detection of telomerase activity and the expression of associated genes in cells, TRAP assay and RT-PCR are customarily used. Immunohistochemical detection of hTERT is useful to detect telomerase-positive cells in a background of non- cancerous cells. We developed a method for the detection of intra-nuclear hTERT protein, in a sub-population of hematopoietic cells, using concurrent staining cell surface antigen and multi color flow cytometry. Human leukemia and myeloma cell lines showed 100% positivity, whereas neutrophils of normal subjects showed 0% positivity, it is consistent with telomerase activity assessed by TRAP assay (r=0.71, p<0.0001) and previous observations. Then we applied this method to analyze hTERT expression in myelodysplastic syndrome (MDS). Forty MDS patients samples were obtained, 36 patients were diagnosed as low risk MDS (RA), 14 patients were diagnosed as high risk MDS (RAEB or RAEB-t) according to FAB classification. All samples were acquired after informed consent was obtained from the patients. Expression of hTERT protein was higher in CD34-positive blast-gated cells than CD34-negative blast-gated cells. The percentage of the CD34+ cells expressing hTERT ranged from 9.66% to 90.91% in low risk MDS patients, whereas from 50.46% to 97.68% in high risk MDS. The expression level was higher in the high risk group compared to that in the low risk group in MDS (p=0.0054, p=0.0084). This observation implied that telomerase up-regulation and hTERT expression were important for disease progression and could be a marker of more advanced disease. In subsets of MDS and AML bone marrow specimens obtained from these patients, we examined the hTERT expression in CD34+/CD38 high cells and CD34+/CD38 low cells containing stem cell fraction. Of interest, some of the patients showed higher expression of hTERT in CD34+/CD38 low cells than in CD34+/CD38 high cells. This observation is inconsistent with previous reports describing normal bone marrow hematopoietic cell findings. We speculated that this phenomenon could be a marker of MDS abnormality and that telomerase up-regulation may be initiated in the more primitive precursor fraction containing hematopoietic stem cells during the disease progression. Telomerase studies may be useful for definition of the risks associated with disease severity. Multi-parameter nature of flow cytometry and its ability to identify cellular sub-populations will facilitate a fuller understanding of the mechanisms of activation of telomerase.

Download Full-text

Let-7a, Mir-17 and Mir-20a Expression Levels in CD34+ Bone Marrow Cells of Patients with Myelodysplastic Syndromes (MDS) Are Associated with Established Prognostic Factors, Supporting Their Implication in the Pathogenesis of the Disease,

Blood ◽

10.1182/blood.v118.21.3792.3792 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3792-3792 ◽

Cited By ~ 1

Author(s):

Christos K Kontos ◽

Vassiliki Pappa ◽

Diamantina Vasilatou ◽

Maria-Angeliki S Pavlou ◽

Frida Kontsioti ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Prognostic Factors ◽

High Risk ◽

Mirna Expression ◽

Who Classification ◽

Low Risk ◽

Expression Levels ◽

Cd34 Cells ◽

Bone Marrow Blasts

Abstract Abstract 3792 Introduction: MicroRNAs are single, small non-coding RNA molecules of approximately 21–26 nucleotides, which regulate the expression of numerous genes. miRNAs may act either at the post-transcriptional or the post-translational level to repress gene expression; still, upregulation of gene expression has been noticed in some cases as a direct effect of miRNA function. The importance of miRNAs in carcinogenesis is emphasized by the association of cancers with alterations in miRNA expression. Many miRNAs, including let-7a and those of the miR-17-92 cluster (miR-17, miR-20a, etc.), have been shown or are predicted to affect the activities of targeted mRNAs encoding proteins that have oncogenic or anti-oncogenic functions. let-7a downregulates KRAS, while miR-17 and miR-20a downregulate E2F1. Both these proteins are overexpressed in myelodysplastic syndromes (MDS) and have been shown to be involved in the pathobiology of the disease. Purpose: In the current study, we examined the prognostic value of let-7a, miR-17 and miR-20a levels in MDS and their potential as novel molecular biomarkers. Furthermore, we investigated the protein expression levels of validated targets of these three miRNAs in bone marrow CD34+ cells of MDS patients. Material and Methods: We evaluated 43 patients with MDS (34 men, 9 women) with a median age of 73 years (range 45–87). According to WHO classification, 12 patients (27.9%) were diagnosed with RA, 6 (13.9%) RCMD, 8 (18.6%) with RAEB-I, 7 (16.3%) with RAEB-II, 8 (18.6%) with AML, and 2 (4.7%) with CMML. According to IPSS, 13 patients (32.5%) had low risk, 14 (35.0%) intermediate I risk, 6 (15.0%) intermediate II, and 7 (17.5%) high risk disease. WPSS classification was: 8 (23.5%) very low risk, 5 (14.7%) low risk, 8 (23.5%) intermediate, 9 (26.5%) high risk, and 4 (11.8%) very high risk. We isolated CD34+ cells from bone marrow mononuclear cells from MDS patients, as well as from peripheral blood of donors of CD34+ cells for stem cell transplantation, using magnetic beads. Extraction of small RNA-containing total RNA from CD34+ cells was performed and cDNA of let-7a, miR-17 and miR-20a was synthesized using specific primers. miRNA expression levels were determined using quantitative real-time PCR, the TaqMan® chemistry and the relative quantification (2−ΔΔCT) method. The snoRNA RNU48 was used as reference gene. Furthermore, total protein was extracted from CD34+ cells using a lysis buffer and subsequently quantified using the Bradford assay. Western blot analysis was carried out for MYC, E2F1, Cyclin D1 (CCND1), BCL2 and KRAS, while Actin was used as reference protein. Results: In MDS patients, let-7a expression levels were 0.053–506.1 copies/RNU48 copies, while miR-17 and miR-20a expression levels were 0.005–2694.5 and 0.003–3116.7 copies/103RNU48 copies, respectively. No significant differences were found between patients and controls regarding let-7a, miR-17 and miR-20a expression. let-7a underexpression was associated with high (>10%) bone marrow blasts percentage (P =0.036), presence of WHO classification subtypes with poor prognosis (RAEB-I, RAEB-II and AML) (P =0.020), and high IPSS (P =0.037). Furthermore, miR-17 underexpression was related to high (>10%) bone marrow blasts percentage (P =0.008), intermediate and/or high risk karyotype (P =0.018) and high IPSS (P =0.016). Moreover, miR-20a underexpression was associated with high IPSS (P =0.037) and WPSS (P =0.013). Interestingly, protein expression levels of all targets analyzed in the current study were shown to be lower in samples overexpressing let-7a, miR-17 and/or miR-20a, in comparison with the corresponding protein levels noticed in specimens showing lower expression of these three miRNAs. Conclusion: To the best of our knowledge, this is the first study showing that expression levels of let-7a, miR-17 and miR-20a are associated with established prognostic factors in MDS, including IPSS and WPSS. Furthermore, these three miRNAs seem to be implicated in the pathogenesis of the disease, most probably by finely tuning the expression of target proteins that are involved in highly important molecular pathways, therefore affecting key cellular functions, such as cell cycle control, apoptosis, cell proliferation, and regulation of gene expression. Undoubtedly, further studies are needed to confirm the present findings and clarify their association with the pathogenesis of different MDS subgroups. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Stathmin 1 Is Involved In The Highly Proliferative Phenotype Of High-Risk Myelodysplastic Syndromes and Acute Leukemia Cells

Blood ◽

10.1182/blood.v122.21.861.861 ◽

2013 ◽

Vol 122 (21) ◽

pp. 861-861

Author(s):

João Agostinho Machado-Neto ◽

Paula de Melo Campos ◽

Patricia Favaro ◽

Mariana Lazarini ◽

Irene Lorand-Metze ◽

...

Keyword(s):

Bone Marrow ◽

High Risk ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Leukemia Cell ◽

Low Risk ◽

Leukemia Cells ◽

Healthy Donors ◽

Stathmin 1 ◽

Marrow Cells

Abstract Introduction : Stathmin 1, also known as Oncoprotein 18 (OP18) or Leukemia-associated phosphoprotein p18 (LAP18), is an important cytoplasmic microtubule-destabilizing protein that plays a critical role in the process of mitosis, proliferation and accurate chromosome segregation through regulation of microtubule dynamics. High levels of Stathmin 1 have been reported in solid tumors and have been associated with poor prognosis in various types of cancers. The identification of overactive proteins in leukemia cells, compared to normal hematopoietic cells, as well as understanding the molecular and cellular basis of the disease may provide new therapeutic opportunities. Aims: To evaluate Stathmin 1 expression in proliferating and non-proliferating hematopoietic cells, in bone marrow cells from healthy donors and from patients with myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). In addition, we evaluated the effect of Stathmin 1 silencing on proliferation and apoptosis in the U937 acute myeloid leukemia cell line. Materials and Methods: A panel of human leukemia cell lines that included myeloid (K562, KU812, NB4, HL60, P39, HEL, U937, KG1 and THP1) and lymphoid cells (Jurkat, MOLT4, Daudi, Raji, Namalwa and Karpas 422) in exponential growth was used. Peripheral blood lymphocytes (PBL) were induced, or not, to proliferate upon PHA stimulation for 72 hours. A total of 30 healthy donors and 117 patients at diagnosis (MDS=52 [low-risk=36, high-risk=16], AML=49, and ALL=16) were included in the study. Stathmin 1 gene and protein expression was evaluated by qPCR and Western blot. Stathmin 1 was stably knocked down with specific shRNA-expressing lentiviral vector and cell growth was examined by MTT assay, clonogenicity by colony formation and apoptosis by AnnexinV/PI. Appropriate statistical analyses were performed; results are expressed as median (minimum- maximum). Results: A higher expression of Stathmin 1 was observed in all leukemia cell lines, when compared with normal non-proliferating hematopoietic cells. We also observed a marked increase in Stathmin 1 expression in PBL induced to proliferate with PHA after 72 hours. Stathmin 1 transcripts were significantly increased in total bone marrow cells from patients with AML (2.01 [0.35-8.88]; p=.0009) and ALL (2.94 [1.16-10.82]; p=.0004), compared with healthy donors (1.01 [0.38-4.08]). No difference in Stathmin 1 expression was observed between healthy donors and MDS patients. When the MDS group was stratified by the WHO classification into low and high-risk MDS, Stathmin 1 expression was significantly higher in the high-risk, when compared with low-risk MDS (1.62 [0.42–3.28] vs. 1.13 [0.36–2.61], p=.03). Similar results were found in isolated CD34+ bone marrow cells, Stathmin 1 transcripts were significantly increased in CD34+ AML cells compared with CD34+ normal cells, and in high-risk compared with low-risk MDS (all p≤.02). Interestingly, 3 out of 5 MDS patients showed a significant increase in Stathmin 1 transcripts after disease progression. Also, a significant positive correlation was observed between percentage of bone marrow blasts and Stathmin 1 expression in MDS patients (p=.03; r=.31). In U937 leukemia cells, Stathmin 1 silencing significantly reduced cell proliferation (p=.02) and clonal growth (p<.0001), but did not modulate apoptosis. Conclusions: Stathmin 1 is overexpressed in high-risk MDS and acute leukemia cells, and is upregulated during MDS progression, suggesting that Stathmin 1 plays a role in the highly proliferative phenotype. Our study adds new insights to the role of Stathmin 1 in leukemogenesis. Future studies are necessary to validate whether Stathmin 1 is a predictive marker for MDS progression, and to determinate whether Stathmin 1 is a “driver” or a “passenger” during malignant transformation. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

GDF11 Increased in Patients with Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v126.23.5224.5224 ◽

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.

Download Full-text

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

Blood ◽

10.1182/blood-2019-128037 ◽

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.

Download Full-text

Favourable response to antithymocyte or antilymphocyte globulin in low-risk myelodysplastic syndrome patients with a 'non-clonal' pattern of X-chromosome inactivation in bone marrow cells

European Journal Of Haematology ◽

10.1034/j.1600-0609.2002.01625.x ◽

2002 ◽

Vol 68 (4) ◽

pp. 210-216 ◽

Cited By ~ 29

Author(s):

M. Aivado ◽

A. Rong ◽

M. Stadler ◽

U. Germing ◽

A. Giagounidis ◽

...

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndrome ◽

X Chromosome ◽

Bone Marrow Cells ◽

X Chromosome Inactivation ◽

Low Risk ◽

Chromosome Inactivation ◽

Antilymphocyte Globulin ◽

Favourable Response ◽

Marrow Cells

Download Full-text

Clonal T Cell Expansion in High Risk Myelodysplastic Syndrome (MDS).

Blood ◽

10.1182/blood.v106.11.3435.3435 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3435-3435

Author(s):

Jeffrey S. Painter ◽

Bai Fanqi ◽

Alan Cantor ◽

Alan F. List ◽

P.K. Epling-Burnette

Keyword(s):

Bone Marrow ◽

T Cell ◽

High Risk ◽

Myelodysplastic Syndrome ◽

Peripheral Blood ◽

Cell Expansion ◽

Who Classification ◽

Refractory Anemia ◽

Risk Patients ◽

T Cell Expansion

Abstract Myelodysplastic Syndrome (MDS), a hematologic malignancy, is associated with cytogenetic and molecular abnormalities in maturing hematopoietic cells that occurs in 40–70% of cases. These abnormalities directly contribute to increased apoptosis and ineffective hematopoiesis of erythroid and myeloid progenitors while frequently sparing lymphoid subsets. In addition to these direct mechanisms of hematopoietic failure, failed hematopoiesis mediated indirectly by an autoimmune mechanism has been suggested to have importance in a subset of patients with the disease. Autoimmune-mediated bone marrow suppression is suggested by the positive outcome of several clinical trials using immunosuppressants. Depletion of autoreactive T cells with deleterious effects on bone marrow formation is considered the immunologic foundation for these therapeutic responses and bone marrow hypocellularity has been indicated as the best predictor of response. Antigen-driven expansion of immunodominant T cell clones can lead to overrepresentation of cells expressing individual T Cell Receptors (TCRs), which is known as TCR skewing. Treatment of hypocellular MDS patients with immunosuppressive therapies is associated with normalization of a skewed TCR- phenotype. The overall incidence of immunodominant T cell expansions has not been determined. The goal of our study was to assess the frequency of clonal T cell expansion in peripheral blood of MDS patients. Peripheral blood was analyzed from 52 patients for T cell CDR3-length skewing by genomic multi-plex PCR. All patients met the clinical criteria of MDS as defined by the WHO classification scheme. Patients with Refractory Anemia (RA) with and without Ringed Sideroblasts (RARS) represented 13% (n=7), Refractory Cytopenia with Multilineage Dysplasia (RCMD and RCMD-RS) represented 48% (n=25), and Refractory Anemia with Excess Blasts (RAEB1, RAEB2, and MDS that had progressed to AML) represented 38% (n=20). TCR-skewing occurred in 29 out of 52 patients with MDS (55%) compared to one out of 20 (5%) in age-matched normal controls. There was no difference in the frequency of clonal expansions based on the WHO classification. Based on the International Prognostic Scoring System (IPSS), we found that high risk patients (Int-2 and high) had a significantly higher incidence of clonal expansions than did patients with low risk disease (low and Int-1) (47% vs. 20%, respectively, p<0.05). Indeed, patients with the highest incidence of having clonal T cell expansion were in the Int-2 risk category (86% by TCR-Vβ analysis, n=7). Of 47 patients with known bone marrow cellularity classification, we enrolled only eight (17%) that were classified as hypocellular, ten (21%) normal cellular, and 29 (62%) hypercellular. No patient with a hypocellular bone marrow in our analysis showed evidence of clonal T cell expansion (p<0.05). These results suggest that clonal T cell expansion occurs prominately in high risk patients. We believe that cellular immunity in MDS could have both beneficial and deleterious effects. Antigen-specific cellular immune responses against pre-leukemic cells would be advantageous, while autoimmune destruction of normal bone marrow cells in the environment of an aggressive immune response would be deleterious. More information is needed about the role that clonal T cell expansion plays in high risk MDS.

Download Full-text