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

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.

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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.

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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.

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

Blood ◽

10.1182/blood.v106.11.4926.4926 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4926-4926

Author(s):

Paula Campos ◽

Fabiola Traina ◽

Adriana Duarte ◽

Bruno Benites ◽

Marcelo Brandao ◽

...

Keyword(s):

Bone Marrow ◽

High Risk ◽

Myelodysplastic Syndrome ◽

Mrna Expression ◽

Who Classification ◽

Fas Ligand ◽

Low Risk ◽

World Health ◽

Caspase 8 ◽

Marrow Cells

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.

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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.

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

Blood ◽

10.1182/blood.v126.23.1703.1703 ◽

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.

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Hedgehog Ligands and Receptors Are Deregulated in Myelodysplastic Syndromes

Blood ◽

10.1182/blood.v120.21.3817.3817 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3817-3817

Author(s):

Juliana M. Xavier ◽

Amanda I Dias ◽

Paulo Latuf-Filho ◽

Fabiola Traina ◽

José Vassallo ◽

...

Keyword(s):

Bone Marrow ◽

High Risk ◽

Myelodysplastic Syndromes ◽

Target Genes ◽

Conflicts Of Interest ◽

Megaloblastic Anemia ◽

Low Risk ◽

Hedgehog Pathway ◽

Hematopoietic Stem ◽

Healthy Donors

Abstract Abstract 3817 Background: The Hedgehog pathway has an important role in self-renew of normal and leukemic stem cells and is upregulated in myeloid leukemias, however there are no studies regarding the Hedgehog pathway in myelodysplastic syndromes (MDS). Hedgehog ligands (Sonic hedgehog [SHh], Indian hedgehog [IHh], and Desert hedgehog [DHh]) are produced by stromal cells and bind to the receptor Patched (PTCH). This binding causes activation of Smoothened (SMO) receptor, resulting in downstream transcription of target genes. Aim: To evaluate Hedgehod pathway components in MDS. Methods: Bone marrow (BM) samples were collected from 39 MDS (25 low risk, 14 high risk-WHO 2008) patients and 26 healthy donors (HD). Relative expressions of PTCH and SMO were obtained by Real Time PCR. For immunohistochemistry, BM biopsies were collected from 21 MDS (17 low risk and 4 high risk) patients and 7 megaloblastic anemia (MA) were used as control. The BM sections were stained with antibodies for DHh, SHh and c-Kit and the percentage of stained nucleated cells was based on an average of 4 high-powered fields. Results: Hedeghog receptors PTCH and SMO were overexpressed in MDS BM cells compared to normal BM as follows; PTCH: [median (min-max)] healthy donors= 2.23 (0.42–9.22); low risk= 6.09 (0.41–25.28); high risk= 3.97 (0.42– 21.01); HD vs low risk and HD vs high risk p= 0.02; SMO: healthy donors= 2.33 (0.00–16.11); low risk= 14.79 (1.89– 41.93); high risk= 34.44 (8.04– 164.28)]; HD vs low risk and HD vs high risk p<0.001. Immunohistochemistry assays showed a higher expression of Hedgehog ligands in MDS bone marrow compared to control. For DHh, the number of stained cells in low risk MDS were higher than in MA [MA = 102(84 –183), low risk= 196(69 – 317) high risk 159(131 – 236)]; MA vs low risk p=0.018 and MA vs high risk p= 0.11. Furthermore, SHh staining was 2 and 2.5 fold higher in low and high risk MDS, respectively, compared to MA [MA= 47(18 – 74), low risk 98.25(53 – 129), high risk 116.25 (93 – 125)] MA vs low risk p= 0.001, MA vs high risk p=0.01. Similar to SHh, c-Kit staining was higher in MDS cases as follows: MA= 95.3(61 – 114), low risk= 106(57–136), high risk= 95.3(61 – 114). Interestingly, a high correlation was found between c-kit and DHh (p=0.002, r=0.63) or c-kit and SHh (p=0.001, r=0.7). Conclusion: To our knowledge, this is the first study of the Hedgehog pathway in MDS. We observed overexpression of Hedgehog receptors and ligands. Furthermore, there is a correlation between these ligands and a marker for hematopoietic stem/progenitors cells. According to these data, we propose that deregulation of Hedgehog in MDS may occur in abnormal progenitors found in MDS bone marrow. Disclosures: No relevant conflicts of interest to declare.

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Decitabine Induces Ferroptosis in Myelodysplastic Syndrome

Blood ◽

10.1182/blood-2019-122000 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2995-2995 ◽

Cited By ~ 1

Author(s):

Qi Lv ◽

Huaquan Wang ◽

Zonghong Shao ◽

Limin Xing ◽

Lanzhu Yue ◽

...

Keyword(s):

High Risk ◽

Myelodysplastic Syndrome ◽

Iron Overload ◽

Cell Viability ◽

Cell Lines ◽

Conflicts Of Interest ◽

Inhibitory Effect ◽

Low Risk ◽

Ferritin Concentration ◽

The Difference

Decitabine is one of the classical demethylation drugs in the treatment of myelodysplastic syndrome (MDS); however, the exact mechanism of decitabine has not been fully understood. Such knowledge is essential to develop mechanism-based, targeted approaches in the treatment of MDS. Here, we show that decitabine-induced ROS raise leads to ferroptosis in myelodysplastic syndrome cells. To investigate whether decitabine could induce ferroptosis in MDS cells and its mechanism, cell lines SKM-1 and MUTZ-1 were co-cultured with decitabine and ferroptosis inhibitor (ferrostatin-1), respectively. CCK-8 assay was used to detect the effects of drugs on cell viability. At the same time, we observed whether necroptosis inhibitor (necrostatin-1), apoptosis inhibitor (z-vad-fmk) and iron chelating agent (DFO) could reverse the inhibitory effect of decitabine on MDS cells. The results showed that, necrostatin-1 could increase the cell viability significantly. The growth-inhibitory effect of decitabine on SKM-1 and MUTZ-1 could be partially reversed by ferrostatin-1, DFO and necrostatin-1. The effect of ferrostatin-1 is the most significant. Ferroptosis inducer (erastin) could increase the cytotoxicity of decitabine at different concentrations. Flow cytometry was used to detect the ROS level. Biochemical method was used to detect the intracellular glutathione (GSH) level and glutathione peroxidase (GPXs) activity. The results showed that, the level of GSH and the activity of GPXs decreased while the ROS level increased in SKM-1 and MUTZ-1 cell lines when treated with decitabine, which could all be inhibited by ferrostatin-1. The iron overload model of C57BL/6 mice was next constructed to observe whether iron overload could induce ferroptosis. The results showed that, the concentration of hemoglobin in peripheral blood of mice was negatively correlated with intracellular Fe2+level and ferritin concentration. Iron overload led to decreased viability of bone marrow mononuclear cells (BMMNCs), which was negatively correlated with intracellular Fe2+level. Ferrostatin-1 and necrostatin-1 partially reversed the decline of cell viability in iron overload groups, and erastin promoted the proliferation of BMMNCs in iron overload mice. The level of GSH and the activity of GPXs decreased while the ROS level increased in BMMNCs of iron overload mice compared with the control. DFO could increase the level of GSH in iron overload mice. Ferrostatin-1, z-vad-fmk and DFO could increase the GPXs activity of BMMNCs in iron overload mice. Finally, to explore the role of ferroptosis in the pathogenesis of low-risk and high-risk MDS patients respectively, the BMMNCs were obtained from low-risk MDS, high-risk MDS and lymphoma patients respectively and co-cultured with decitabine and above-mentioned inhibitors. The results showed that, ferrostatin-1, necrostatin-1, z-vad-fmk could significantly reverse the inhibitory effect of decitabine of low-risk MDS patients. Necrostatin-1 and Fer-1 could also reverse the inhibitory effect of decitabine of high-risk MDS patients, although the difference was not significant. Decitabine could significantly increase the ROS level in both MDS groups, which could both be inhibited by ferrostatin-1 or promoted by erastin. Ferrostatin-1, necrostatin-1 and z-vad-fmk could significantly reverse the inhibitory effect of decitabine on GSH level in low-risk MDS patients. Ferrostatin-1 and necrostatin-1 could significantly reverse the inhibitory effect of decitabine on GSH level in high-risk MDS patients. Erastin combined with decitabine could further reduce the GSH level, and the difference was significant in high-risk MDS group. For low-risk MDS group, GPXs activity of ferrostatin-1 combined with decitabine and z-vad-fmk combined with decitabine groups were significantly higher than that of decitabine group. For high-risk MDS group, the activity of GPXs of ferrostatin-1 combined with decitabine and necrostatin-1 combined with decitabine groups were significantly higher than that of decitabine group. Erastin could further decrease the activity of GPXs when compared with decitabine group. Our findings reveal a novel therapeutic mechanism of decitabine and may open a new window for therapeutic targeting in the treatment of MDS. Figure Disclosures No relevant conflicts of interest to declare.

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Profile of Checkpoint Molecules Expression on Bone Marrow Cell Populations in Patients with High-Risk Myelodysplastic Syndrome

Blood ◽

10.1182/blood-2020-141997 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 43-44

Author(s):

Nikolai Yu. Tcvetkov ◽

Elena V. Morozova ◽

Olga S. Epifanovskaya ◽

Elena V. Babenko ◽

Maria V. Barabanshikova ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

High Risk ◽

Myelodysplastic Syndrome ◽

Nk Cells ◽

Conflicts Of Interest ◽

T Cell Response ◽

Cell Populations ◽

Minor Role ◽

Study Cohort

Background Immune checkpoint (IC) inhibitors (ICI) is a promising group of agents with potential effect in myelodysplastic syndrome (MDS). However, there is not enough data on the pattern of IC expression in MDS bone marrow, though this can guide future therapies with ICI. Methods We prospectively included 57 patients with high risk MDS during 2019-2020 years after signing informed consent for participation in research. We used 7 eight-colored panels in each patient to define IC expression on T- and NK-cells (CD3, CD8, CD4, CD56, CD16), myeloid precursors (CD117, CD34), T-regulators (CD25, CD4), myeloid-derived suppressor cells (CD15, CD11b, CD14, HLA-DR, CD33). In each population we assessed expression of CD279, CD152, CD223, TIM3, CD273, CD274, CD275, CD80. Results In lymphoid populations we observed an extremely high percentage of PD-1 positive lymphocytes (10.5 ± 8.0% of all nucleated cells), including PD-1 positive CD4 lymphocytes (6.3 ± 3.7%) and CD8 lymphocytes (4.7 ± 3.5%). Interestingly, on average 63 ± 34% of all lymphocytes were PD-1 positive (Figure 1A), which indicates an extreme degree of T-cell exhaustion and significant exploitation of the PD-1 system by a malignant tumor. Among the PD-1 ligands PD-1L predominated, however it was present only on a small percentage of myeloid precursors (2.2 ± 0.39% of nucleated cells), but most of the PD-1L ligand expression was observed on granulocytes in the process of their differentiation from myeloid precursors (5.8 ± 4.1% of nucleated cells), and in some patients the proportion of such granulocytes reached more than 20%. Moreover, on the cells of the monocytic series, PD-1L is practically not expressed, but CD80 is expressed, a ligand for CTLA4 (1.6 ± 0.7% of nucleated cells). Interestingly, despite the presence of expression of CD80 on monocytic and dendritic cells, the receptor for this ligand on T-lymphocytes was practically absent. The proportion of such cells was only 0.02 ± 0.01%, i.e. the expression of CD80 facilitates the activation of lymphocytes through interaction with CD28 rather than the suppression of the immune response through CD152. Thus, it can be suggested that CTLA4 does not have a clinically significant role in MDS. At the same time, a certain number of TIM3 positive lymphocytes were observed, the proportion of which was 0.42 ± 0.16%, and the percentage of lymphocytes carrying this receptor was 3.6 ± 1.2% (Figure 1A). However, significantly greater TIM3 expression was detected on NK cells. The proportion of such cells was 1.3 ± 0.6% of all nucleated cells, while 30% of all NK cells carried the TIM3 receptor (Figure 1A). Only 0.1% of cells from early myeloid progenitors that expressed TIM3 receptor were detected during flow cytometry, while the proportion of TIM3 positive myeloid cells increased during maturation just in the same way as the PD-1L. Expression of Gal9 was negligible, as it is a protein secreted into the intercellular space. Only a small percentage is associated with cell membranes. The analysis of the correlation matrix showed synchronous changes in the number of most bone marrow T-cell populations. A negative correlation of the level of myeloid tumor precursors actively expressing IC ligands and activated T cells, expressing CD223, NK cells, NKT was observed. The total number of myeloid tumor precursors positively correlated with the number of PD-1 positive lymphocytes (Figure 1B). Conclusion It was found that IC system plays a role in high-risk MDS. Moreover, the expression of ligands is mainly realized during maturation of blast cells into granulocytes, which are the main population expressing these inhibitory molecules. The leading role in the suppression of the T-cell response in MDS is played by the PD-1-PD-1L system, the CTLA4 system plays a minor role in our study cohort. The second most important system is GAL9-TIM3, effecting predominantly NK cells, while T cell are less affected. Thus, the data obtained justify the trials of dual blockade with anti-PD-1 and anti-TIM3 agents in high-risk MDS. Figure 1 Disclosures No relevant conflicts of interest to declare.

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The International Prognostic Scoring System for Waldestrom’s Macroglobulinemia (ISSWM) Is Applicable in Patients Treated with Rituximab-Based Regimens.

Blood ◽

10.1182/blood.v110.11.3615.3615 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3615-3615

Author(s):

Meletios Athanasios Dimopoulos ◽

Efstathios Kastritis ◽

Dimitra Gika ◽

Sossana Delimpassi ◽

Konstantinos Zervas ◽

...

Keyword(s):

High Risk ◽

Alkylating Agents ◽

Single Agent ◽

Primary Treatment ◽

High Risk Group ◽

Low Risk ◽

International Prognostic Scoring System ◽

Intermediate Risk ◽

Number Of Patients ◽

The Impact

Abstract Introduction: An ISSWM was recently proposed (Morel et al, ASH 2006), which was based on a large number of patients treated primarily with alkylating agents and /or nucleoside analogues. The ISSWM based on 5 adverse covariates wich defined 3 risk groups: low, intermediate and high risk with 5-years survival rates of 87%, 68% and 36% respectively. In our current analysis, we assessed the impact of this system in patients with WM who received primary treatment with rituximab-based regimens. Patients and methods: Ninety-three previously untreated, symptomatic patients who received treatment either with single agent rituximab (21 patients) or with the combination of dexamethasone, rituximab, and cyclophosphamide (72 patients) were classified according to the ISSWM, which is based on 5 adverse covariates: age> 65 years, hemoglobin ≤11.5 g/dl, platelet count ≤ 100 x 109/L, β2- microglobulin <3mg/L, serum monoclonal protein concentration >70g/L. Low risk is defined by the presence of ≤ 1 adverse characteristics except age, high risk by the presence of >2 adverse characteristics and intermediate risk by the presence of 2 adverse characteristics or age >65 years. Results: The disease features of the 93 patients were typical of symptomatic WM: age > 65 years in 63%, males 65%, B-symptoms in 22%, splenomegaly in 29%, lymphadenopathy in 34%. 15% of patients were rated as low risk, 65% as intermediate risk and 20% as high risk. Criteria for initiation of therapy included cytopenia, hyperviscosity, constitutional symptoms, organomegaly or IgM-related disorders. Overall, 62% of patients were alive at 6 years. Median survival was not reached for low and intermediate risk and was 38 months for high risk patients (p=0.006). There was a clear separation of the survival curves in the three groups. At the time of last follow-up the percentage of patients alive was 100%, 82% and 58% for patients classified as low, intermediate and high-risk group respectively. Conclusions: The recently proposed ISSWM is applicable in patients with WM who receive primary treatment with rituximab-based regimens and may serve as a basis to compare outcomes in different studies.

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Clinical Characterization, Prognostic Implications, and Response to Therapy In Patients with Myelodysplastic Syndrome (MDS) and Chromsome 17 Abnormality: A Single Institutional Experience

Blood ◽

10.1182/blood.v116.21.2930.2930 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2930-2930

Author(s):

Tapan Kadia ◽

Gautam Borthakur ◽

Stefan Faderl ◽

Farhad Ravandi ◽

Jorge E. Cortes ◽

...

Keyword(s):

Bone Marrow ◽

High Risk ◽

Conflicts Of Interest ◽

Response To Therapy ◽

Chromosome 17 ◽

International Prognostic Scoring System ◽

Cancer Center ◽

Complex Karyotype ◽

Chromosome 5 ◽

Clinical Prognosis

Abstract Abstract 2930 MDS is characterized by recurrent chromosomal abnormalities that reflect the underlying genomic instability of the disease and help to predict clinical prognosis. Abnormalities of chromosome 17 have been described previously in MDS and AML and may have some association to prior exposure to cytotoxic therapy. Abnormalities of the p53 gene, which is located in the short arm of chromosome 17 (17p), have been discovered in the non-deleted allele in a large proportion these cases – suggesting an association and likely contributing to the pathogenesis of this subgroup. Here we present the clinical characteristics, prognosis, and response to therapy of patients (pts) with MDS with and without chromosome 17 abnormalities in our single center experience. We analyzed our records for cases of MDS referred to MD Anderson Cancer Center from 2000–2009. Of the 1638 cases of MDS examined, 88 (5.4%) had evidence of any chromosome 17 abnormality (abnl). The specific chromosome 17 abnormalities were: 17p- in 8 (9%); der 17 in 1 (1%); -17 in 78 (89%); and 17p-, -17 in 1 (1%). In terms of the complete karyotype, the pts could be grouped as: 17 abnl with complex (95%), 17 abnl with -5 or -7 (94%), and 17 abnl non-complex (5%). There were no cases with an isolated 17 abnl. The median age of these pts with chromosome 17 abnormality (cohort A) was 67 yrs (range 16–87), compared to 67 (13-94) in the pts without chromosome 17 abnormality (cohort B, n=1550). The MDS diagnoses by WHO in cohort A were RA, MDS-U in 23 (26%), RAEB in 64 (73%), and 5q- in 1 (1%) compared with 47%, 52%, and 1% in cohort B respectively (p=0.001). Stratification by IPSS (International Prognostic Scoring System) in cohort A revealed 0 (0%) with low risk, 8 (9%) with intermediate-1 (INT-1), 56 (64%) with INT-2, and 20 (23%) with high risk compared with 19%, 40%, 29%, and 9% in the respective IPSS groups in cohort B (p<0.0001). The median bone marrow blast percent in cohort A was 7 (0-19) vs. 4 (0-19) in cohort B (p=0.003). In cohort A, 29 pts (33%) had [0-4% marrow blasts], 38 (43%) had [5-10% blasts], and 21 (24%) had [11-20% blasts] compared to 51%, 26%, and 20% in the respective groups in cohort B (p=0.001). Abnormality in chromosome 17 was highly associated with a chromosome 5 or 7 abnormality, with 83 (94%) of cases having both, compared to only 27% of cases in cohort B having -5 or -7 (p<0.0001). Chromosome 17 abnormality was also strongly associated with a concurrent complex karyotype (defined as ≥ 3 abnormalities) with 84 pts (95%) in cohort A having both compared to only 21% in cohort B having a complex karyotype (p<0.0001). Pts in cohort A were more likely to have a history of prior malignancy 55% vs. 34% in cohort B (p<0.0001), whereas 50% of pts in cohort A were classified as treatment-related MDS compared in only 25% in cohort B. Pts were treated with hypomethylating agents, intense chemotherapy, or other and limited response data were available. The complete response rates in cohort A for the respective treatments were 58% (7/12), 57% (8/14), and 67% (2/3) compared with 50%, 44%, and 29% in cohort B. The median overall survivals for the following cytogenetic groups: diploid, complex without 17 abnormality, -5/-7 without 17 abnormality, and any 17 abnormality were 24 months, 13 months, 10 months, and 7.7 months, respectively (p<0.0001). Pts with MDS with a chromosome 17 abnormality represent a small percentage of the total cases. However, they are characterized by a high risk disease with elevated bone marrow blasts, complex/adverse cytogenetics, association with prior therapy, and a relatively poor survival. There is an association with chromosome 5 and 7 abnormality and may suggest cooperation in the pathogenesis. Given the high incidence of biallelic p53 loss in this group of pts, alternative antineoplastic therapies which circumvent the requirement for p53 function need to be developed. Disclosures: No relevant conflicts of interest to declare.

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