Abnormalities In Myelodysplastic Syndromes Research Articles

Incidence of Cytogenetic Abnormalities in Myelodysplastic Syndrome in the African American Population: A Decade-Long Single-Center Real-World Experience

Incidence of Cytogenetic Abnormalities in Myelodysplastic Syndrome in the African American Population: A Decade-Long Single-Center Real-World Experience

The der(1;7)(q10;p10) defining a distinct profile from -7/del(7q) in myelodysplastic syndromes: Asystematic review and meta-analysis.

Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by ineffective hematopoiesis due to stem cell abnormalities. Monosomy 7q aberrations are a common cytogenetic abnormality in MDS. Specifically, an unbalanced translocation der(1;7)(q10;p10) [der(1;7)] has been identified in MDS patients, which is a monosomy 7q aberration variant like -7/del(7q). However, knowledge of der(1;7)'s features remains limited. Existing studies have compared the clinical and genetic characteristics of der(1;7) to those of -7/del(7q) but yielded inconsistent findings. Accordingly, we conducted meta-analyses comparing der(1;7) to -7/del(7q). Publications were searched from the following databases up to January 10, 2023: Pubmed, Web of Science, Embase, Cochrane, and ClinicalTrials.gov. Eligible studies were assessed for risks of bias. Relevant data were extracted from included studies and analyzed using random-effects models. Publication bias was evaluated and sensitivity analyses were performed. The comparative meta-analyses included 405 MDS patients with der(1;7) from nine studies. The analysis revealed that der(1;7) was associated with a greater male preponderance (86.1% vs. 68.3%, Odds Ratios (ORs) 2.007, p < 0.01) than -7/del(7q), lower platelets counts compared to del(7q), higher hemoglobin levels than -7, lower absolute neutrophil counts, and higher percentage of patients with non-excess blasts (66.9% vs. 41.3%, ORs 2.374, p = 0.01) in comparison with -7/del(7q). The der(1;7) existed more as a sole karyotype aberration (55.6% vs. 37.0%, ORs 2.902, p = 0.02), co-occurred more often with +8 (22.7% vs. 4.2%, ORs 5.714, p = 0.04) whereas less -5/del(5q) (1.5% vs. 41.3%, ORs 0.040, p < 0.01) and complex karyotype (7.3% vs. 54.8%, OR 0.085, p < 0.01). The der(1;7) was associated with higher frequencies of RUNX1 (40.8% vs. 12.3%, ORs 4.764, p < 0.01), ETNK1 (28.1% vs. 2.5%, ORs 42.106, p < 0.01) and EZH2 (24.8% vs. 6.9%, ORs 3.767, p = 0.02) mutations, but less TP53 mutation (2.4% vs. 45.3%, ORs 0.043, p < 0.01). Moreover, der(1;7) patients had longer time to progression (Hazard Ratios (HRs) 0.331, p = 0.02), better overall survival (OS) than -7 patients (HRs 0.557, p < 0.01), but similar OS with del(7q) patients (HRs 0.837, p = 0.37). The findings revealed distinct clinical, cytogenetic, and molecular characteristics distinguishing der(1;7) from -7/del(7q), indicating der(1;7) defines a unique subtype within MDS with monosomy 7q. These findings support classifying der(1;7) as a separate MDS entity in future.

MicroRNA dysregulation in myelodysplastic syndromes: implications for diagnosis, prognosis, and therapeutic response.

Myelodysplastic syndromes (MDS) are a group of malignant clonal hematological disorders with heterogeneous clinical course and risk of transformation to acute myeloid leukemia. Genetic and epigenetic dysregulation, including alterations in microRNA (miRNA) expression, plays a pivotal role in MDS pathogenesis influencing disease development and progression. MiRNAs, known for their regulatory roles in gene expression, have emerged as promising biomarkers in various malignant diseases. This review aims to explore the diagnostic and prognostic roles of miRNAs in MDS. We discuss research efforts aimed at understanding the clinical utility of miRNAs in MDS management. MiRNA dysregulation is linked to specific chromosomal abnormalities in MDS, providing insights into the molecular landscape of the disease. Circulating miRNAs in plasma offer a less invasive avenue for diagnostic and prognostic assessment, with distinct miRNA profiles identified in MDS patients. Additionally, we discuss investigations concerning the role of miRNAs as markers for treatment response to hypomethylating and immunomodulating agents, which could lead to improved treatment decision-making and monitoring. Despite significant progress, further research in larger patient cohorts is needed to fully elucidate the role of miRNAs in MDS pathogenesis and refine personalized approaches to patient care.

Inactivation of p53 provides a competitive advantage to del(5q) myelodysplastic syndrome hematopoietic stem cells during inflammation.

Inflammation is associated with the pathogenesis of myelodysplastic syndromes (MDS) and emerging evidence suggests that MDS hematopoietic stem and progenitor cells (HSPC) exhibit an altered response to inflammation. Deletion of chromosome 5 (del(5q)) is the most common chromosomal abnormality in MDS. Although this MDS subtype contains several haploinsufficient genes that impact innate immune signaling, the effects of inflammation on del(5q) MDS HSPC remains undefined. Utilizing a model of del(5q)-like MDS, inhibiting the IRAK1/4-TRAF6 axis improved cytopenias, suggesting that activation of innate immune pathways contributes to certain clinical features underlying the pathogenesis of low-risk MDS. However, low-grade inflammation in the del(5q)-like MDS model did not contribute to more severe disease but instead impaired the del(5q)-like HSPC as indicated by their diminished numbers, premature attrition and increased p53 expression. Del(5q)-like HSPC exposed to inflammation became less quiescent, but without affecting cell viability. Unexpectedly, the reduced cellular quiescence of del(5q) HSPC exposed to inflammation was restored by p53 deletion. These findings uncovered that inflammation confers a competitive advantage of functionally defective del(5q) HSPC upon loss of p53. Since TP53 mutations are enriched in del(5q) AML following an MDS diagnosis, increased p53 activation in del(5q) MDS HSPC due to inflammation may create a selective pressure for genetic inactivation of p53 or expansion of a pre-existing TP53-mutant clone.

Rare cytogenetic abnormalities in MDS evolving from fanconi anemia-A case report

Fanconi anemia (FA) is a genetically heterogenous rare autosomal recessive disorder. Mutations in FANCA gene are the most frequent among FA patients accounting for 60-65%. FA is characterised by congenital malformations, progressive bone marrow failure (BMF) and increased risk of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The risk of developing hematological abnormalities in FA patients is around 98% by 40 years of age. The risk of clonal cytogenetic abnormalities during BMF is around 67% by 30 years of age and risk of developing MDS or AML is 52% by 40 years of age. The frequent chromosomal abnormalities are 1q+, monosomy 7 and gains of 3q. Partial duplications/triplications of chromosome 1q are known to represent a nonrandom chromosomal anomaly in myeloid disorders.

Impairment of FOXM1 expression in mesenchymal cells from patients with myeloid neoplasms, de novo and therapy-related, may compromise their ability to support hematopoiesis.

Bone marrow mesenchymal stem cells (BM-MSCs) exhibit multiple abnormalities in myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), including reduced proliferative and clonogenic capacity, altered morphology, impaired immunoregulatory properties and capacity to support hematopoiesis. Here, we investigated expression of the FOXM1 gene, a transcription factor driving G2/M gene expression, in BM-MSCs isolated from patients with MDS and AML, de novo and therapy-related, compared to BM-MSCs isolated from healthy donors (HD). We observed a statistically significant downregulation of FOXM1 expression in BM-MSCs isolated from MDS and AML patients, as compared to controls. In parallel, expression of FOXM1 mitotic targets (CCNB1, CDC20, PLK1 and NDC80) was suppressed in patients' BM-MSCs, as compared to HD. No differences in the expression of FOXM1 and its mitotic targets were observed in BM-mononuclear cells from the different sources. From a functional standpoint, silencing of FOXM1 mRNA in healthy MSC induced a significant decrease in the expression of its targets. In this line, healthy MSC silenced for FOXM1 showed an impaired ability to support hematopoiesis in vitro. These findings suggest that deregulation of FOXM1 may be involved in the senescent phenotype observed in MSC derived from myeloid neoplasms.

U2AF1 Splicing Factor Mutations Affect RNA Subcellular Compartmentalization in Myeloid Malignancies

Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic disorders characterized by morphological dysplasia, cytopenias and high risk of progression to acute myeloid leukemia (AML). Despite recent improvements in treatment strategies, the 5-year relative survival rate in MDS remains only 31%, dropping to 24% in AML. This highlights the need for a deeper characterization of the pathogenic mechanisms driving disease development and progression. Splicing factor (SF) mutations are the most common genetic abnormality in MDS and secondary AML. These mutations, in particular U2AF1 mutations, are associated with increased risk of AML transformation and poor survival. U2AF1 S34F and Q157R mutations determine RNA splicing alterations that eventually result in ineffective hematopoiesis. However, molecular mechanisms and biological consequences underlying the clonal advantage of SF-mutant over wildtype cells remain to be fully elucidated. To address this aim, we recently combined high-resolution transcriptomics and imaging techniques in preclinical models of U2AF1-mutant and wildtype MDS/AML (Biancon et al., Mol Cell 2022). Our results showed that U2AF1 mutations cause widespread RNA binding and splicing alterations that directly affect the biology of RNA granules, i.e. dynamic condensates of RNAs and RNA binding proteins implicated in RNA metabolism, signaling and gene regulation. In particular, in U2AF1-mutant vs WT cell lines and primary cells we observed increased formation of stress granules (SGs), cytoplasmic RNA granules mediating cellular adaptation to stress conditions. Crucially, the enhanced stress response in U2AF1-mutant cells was associated with improved cell fitness, that was reverted by SG inhibition. Dysregulation of SGs, and potentially of other subcellular RNA condensates, may therefore represent a novel therapeutic vulnerability in MDS/AML patients carrying U2AF1 mutations. We here integrated single-cell (sc)RNA-seq data to further support the direct link between the U2AF1-mutant genotype and the increased SG phenotype in myeloid malignancies. We first isolated CD34+ blasts from bone marrow samples of MDS patients with (n=3) or without (n=3) U2AF1 S34F mutation. We then performed 10x Genomics scRNA-seq obtaining an average of 3,478 sequenced cells per sample for downstream analyses. Cells from different patients were integrated with Seurat v4.0.1 and annotated with the recently released Cell Marker Accordion (https://github.com/TebaldiLab/TheCellMarkerAccordion), confirming the expected skewing towards granulocyte-monocyte progenitor cells and erythroid lineage in S34F-mutant patients. Single cells were subsequently classified according to the detection of the S34F mutation (VarTrix v1.1.19), into: "WT" (WT cells in U2AF1-WT patients), "S34F_WT" (WT cells in U2AF1-S34F patients), "S34F" (S34F-mutant cells in U2AF1-S34F patients). Each cell was also associated to an "SG expression score", measuring the collective abundance of 149 transcripts enriched in SGs and aberrantly bound-spliced by mutant U2AF1 (Biancon et al., Mol Cell 2022). Importantly, S34F-mutant cells represented the category with the highest SG expression score (Figure 1A), consistently indicating an increased stress adaptation that might explain the clonal advantage of splicing factor-mutant cells in MDS. To further understand the effect of U2AF1-mutant binding and splicing alterations on other RNA condensates, we focused on Cajal bodies (CBs), nuclear granules involved in snRNA transcription and assembly of spliceosomal RNPs. Immunofluorescence (IF) staining for coilin, a known CB marker, followed by confocal imaging revealed a decrease in the number of CBs in U2AF1 S34F vs WT myeloid cell lines (Figure 1B). Of note, IF data were supported by RNA-seq data performed in the same cellular system (Biancon et al., Mol Cell2022), showing a significantly lower expression of snRNAs in U2AF1 mutant vs WT cells. The role of CBs in cancer is currently unknown, but the disruption of CBs may be associated to altered RNA splicing kinetics and increased genomic instability. Collectively, our results suggest that U2AF1 mutations affect the compartmentalization and aggregation properties of targeted RNAs, providing the rational to further investigate RNA granule perturbations in splicing factor-mutant myeloid malignancies for therapeutic potential. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clone Size By Conventional Cytogenetics in Myelodysplastic Syndromes Correlates with Clinical Features and Outcomes

Introduction Chromosomal abnormalities are detected in 30-40% of myelodysplastic syndromes (MDS) patients (pts) using conventional karyotyping of 20 metaphases. Conventional cytogenetics remain crucial for risk stratification and certain cytogenetic abnormalities are MDS defining. The Revised international prognostic scoring system (IPSS-R) and its new molecular version (IPSS-M) account for conventional cytogenetic abnormalities regardless of the number of metaphases those abnormalities are detected among (dichotomous assignment). We aimed to assess correlation of "clone size by conventional cytogenetics” defined as percentage of metaphases where chromosome abnormalities are detected with certain clinical features and outcomes in MDS. Methods We retrieved cases from Moffitt MDS database with cytogenetics of interest (i.e. del5q, del20q, del7q/-7, chromosome 3 (ch3), trisomy 8 (+8) and complex karyotype). Inclusion for this study required presence of at least 10 metaphase analysis of chromosome abnormalities by conventional cytogenetics at time of diagnosis or prior to any therapy. We assessed clinical features of interest and outcomes based on % of cells/metaphases harboring the chromosome abnormality using G-banding "conventional cytogenetics clone size". Results This study included 1096 pts, 151 with isolated del5q, 134 pts with del20q, 113 pts with del7q/-7, 38 pts with isolated ch3, 163 pts with +8, and 497 pts had complex karyotype. Detailed disease baseline characteristics, molecular/cytogenetic data, risk stratification and outcomes of all those cohorts were available and reviewed. Among 151 isolated del 5q, pts with >75% metaphases harboring del5q had more severe anemia (hgb < 8.0 g/dl), 29% compared to 10%,12%, and 18% for pts with <25%, 25-50, and 51-75% metaphases, respectively (p .03). Among pts who were treated with lenalidomide (n=98), there was a trend for higher response rate among pts with >75% del5q metaphases (61% vs 46%; p=.09). There was no difference in presence of TP53-m based on % of metaphases harboring del5q. We identified 134 pts with del20q, pts with >75% del20q metaphases had more thrombocytopenia (< 100 u/l), 65% compared to 42%, 26%, and 42% for pts with <25%, 25-50, and 51-75% metaphases, respectively (p .005). For 163 pts with +8, the median OS was 59 months (mo) for pts with <25% +8 detected metaphases compared to 36 mo for those >25%. (p=.05) For 113 pts with del7q/-7, the median OS was worse for -7 (n= 81) compared to del7q (n=32); (18.5 vs 29 mo, p =.024). The median OS was 98 mo for del7q/-7 with <25% metaphases compared to 18 mo for those > 25% metaphases, p=.009. There was no difference in response to hypomethylating agents based on 25% cutoff. We had 38 pts with isolated ch3 abnormality, the median OS was 122 mo for 2 pts with <25% metaphases compared to 24 mo for those >25%, p=.09. Finally, among 497 pts with complex karyotype, the median OS was 20 mo for those with <25% metaphases compared to 12 mo for >25% (p=.01). There was no difference in presence of TP53-m based on % of metaphases. Conclusions The number of cells harboring chromosome abnormalities in MDS using regular karyotyping "clone size by conventional cytogenetics” strongly correlated with clinical features and outcomes. A 25% cut off should be examined in context of IPSS-R and IPSS-M scoring for cytogenetic risk rather than dichotomous assignment to risk group. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Correlation of Mutational Profiles and Cytogenetics with Morphologic Dysplasia in Myelodysplastic Syndromes

Introduction: Myelodysplastic syndromes (MDS) are a spectrum of clonal bone marrow failure disorders demonstrating cytopenias, dysplasia, and risk of progression. Although ~50% of MDS patients have normal karyotype (NK), a subset of MDS exhibits genetic alterations that result in an abnormal karyotype. However, the relationship between the molecular landscape and histologic features in MDS have not been well explored. Using next generation sequencing (NGS) and cytogenetics, we sought to correlate genetic abnormalities in MDS with morphologic dysplasia. Methods: After IRB approval, we retrospectively identified NGS studies performed with a 164 gene panel in patients with MDS at Stanford (2018-2021). Molecular and cytogenetic data were collected for 225 MDS patients. Peripheral blood and bone marrow (BM) slides were collected from 61 patients for histological review. As controls, 32 staging BMs were obtained from untreated lymphoma patients. A hematopathologist performed a blinded intense morphologic review. A subset of cases was reviewed by 2 other hematopathologists and the intraclass correlation coefficient (ICC) was calculated to evaluate concordance. The degree of dysplastic features and overall dysplasia was scored. The standardized mean difference (SMD) was calculated to quantify the degree of dysplasia between comparison groups. Statistical analysis was performed using R (3.6.3); p value < 0.05 was considered statistically significant. Results: MDS cases had higher degrees of dysplasia than control cases in megakaryocytic (median 37.5% vs 0%), granulocytic (median 20% vs 0%), and erythroid (median 10% vs 0%) lineages. To assess specificity, using a cutoff of 10%, 1/31 control cases (3.1%) showed megakaryocytic dysplasia, 2/32 (6.3%) had erythroid dysplasia, while none showed granulocytic dysplasia. Dysplasia in > 2 lineages in >10% of cells was specific for MDS, detected in 49/61 (80.3%) of MDS cases vs 0/32 control cases (Table 1). Within the MDS group, lack of multilineage dysplasia was more common in NK (8/25; 32%) vs non-NK (3/36; 8.3%), suggesting MDS-NK is morphologically subtle. Using a cutoff of 10%, 13/25 (52%) NK lacked granulocytic dysplasia vs 10/36 (27.8%) in non-NK, 8/25 (32%) of NK lacked erythroid dysplasia vs 10/36 (27.8%) in non-NK, and 5/25 (20%) NK lacked megakaryocytic dysplasia vs 3/35 (8.3%) in non-NK. The most strongly associated dysplastic features for MDS included erythroid megaloblastic change (SMD=1.25), erythroid nuclear features (SMD=1.041), atypical megakaryocyte nuclei (SMD=1.189), separated megakaryocyte nuclei (SMD=1.131), and hypogranulated granulocytes (SMD=1.096). Based on 10 MDS and 5 control cases, rough concordance on overall dysplasia was demonstrated among 3 hematopathologists. Granulocytic dysplasia showed the highest reproducibility (ICC for granulocytic = 0.948, erythroid = 0.547, megakaryocytic = 0.531). The percent of cells with dysplasia in the MDS cohort did not correlate with number of mutations. The overall number of mutations between MDS with NK and non-NK were relatively similar (Figure 1). The non-NK group exhibited a greater frequency of pathogenic TP53 variants than NK (31.5% vs 2.6%, p < 0.0001). Conversely, SF3B1 and TET2variants occurred more frequently in the NK cohort than non-NK (SF3B1: 32.5% vs 12.6%; TET2: 29.8% vs 12.6%, both p values < 0.003). In MDS cases with a TET2 variant, NK was associated with a significantly higher total number of mutations than non-NK cases (median [IQR]: 5[5-7] vs 4[3-6], p = 0.031). Using Random Forest models, exploratory multivariate analysis identified the presence of mutations in TP53, SF3B1, TET2, and overall megakaryocytic, granulocytic, and erythroid dysplasia as the prominent parameters that distinguished NK from non-NK MDS cases. There was no significant difference in age at diagnosis between NK and non-NK MDS in all NGS cases (median age 74 vs 70, p = 0.10) and morphologically scored cases (median age 70 in both groups). Conclusion: Our study demonstrates that granulocytic dysplasia was the most specific morphologic finding for MDS with high reproducibility among 3 hematopathologists. Detection of dysplasia in > 2 lineages in >10% cells was also specific for MDS, while MDS-NK appear morphologically subtle. Mutations in TET2 were more frequent in MDS-NK. Our findings highlight the importance of NGS and histopathology in helping establish a diagnosis of MDS-NK. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evaluation of miR-145 and miR-146a as potential biomarkers for diagnosis of Myelodysplastic Syndrome

Objective: Myelodysplastic syndromes (MDS) are a group of heterogeneous hematopoietic stem cell disorders characterized by ineffective hematopoiesis, bone marrow dysplasia, and peripheral cytopenias. microRNAs (miRNAs) are small non-coding RNAs that play key roles in post-transcriptional regulation of gene expression and have been determined potential in disease diagnostics and therapeutics owing to their stability. Recent evidence suggests that haploinsufficiency of the miR-145 and miR-146a, encoded from 5q Common Deleted Region (CDR) may contribute to the phenotype in MDS. Although, interstitial del(5q) is the most common chromosomal abnormality in MDS, these findings are inconsistent in Turkish patients. Therefore, we aimed to investigate assess the diagnostic value of miR-145/miR-146a and their relation with del(5q) or monosomy 5 in MDS. Methods: In order to determine the association between del(5q) and expression miR-145/miR-146a, conventional cytogenetics (CC), FISH, and qRT-PCR methods were performed for 24 patients with MDS and 20 healthy individuals. Additionally, ROC curves were generated to evaluate putative diagnostic value of miRNAs. Results: Cytogenetic examination revealed clonal cytogenetic abnormalities in 43.4% of cases. miR-146a decreased in 23 of 24 patients regardless of chromosome 5 abnormalities (p

PB1912: MYELODYSPLASTIC SYNDROME WITH ISOLATED 5Q DELETION AND ASSOCIATED 5Q DELETION TO 13Q DELETION

Background: The 5q deletion is the classic chromosomal abnormality in myelodysplastic syndrome (MDS). Critical regions are 5q31 and 5q33, leading to gene haplodeficiency and other pathogeneic mechanisms. 13q deletion occurs in about 2% of MDS cases. This deletion involved band 13q14. Aims: Here we describe cytogenetic abnormalities in a Tunisian cohort of MDS patients and we shed the light on cases with 5q deletion. Methods: Cytogenetic analyses of bone marrow cells were performed according to standard procedures for 12 Tunisian patients presenting with MDS. Cytogenetic analysis was performed on lymphocytes and bone marrow cells. Cultured cells were treated with colchicine and hypotonic KCl solution. The pellet was fixed and washed in methanol-acetic acid (3:1). The cells were resuspended in fixative and dropped onto slides. Chromosome-banding analysis was performed using RHG and GTG banding techniques. Three karyotypes and 25 metaphases were analyzed according to the International System for Human Cytogenetic Nomenclature. Results: Cytogenetic abnormalities were detected in 9 cases and del5q was recorded in four patients (3 patients with refractory anemia (RA) and a fourth with RA and excess blasts-T (RAEB-T)). The del5q was isolated in two RA cases as a del (5) (q13 q33) in a new diagnosed and a treated patients. It was associated in two patients: a RA patient explored during the evolution, harboring a del (5) (q12 q33) associated to a del (13) (q12 q14) and the RAEB-T patient harbouring a del (5) (q21 q31) associated to a clonal hyperploidy. Summary/Conclusion: The diagnosis of MDS was based on cytomorphology and cytogenetics. However, nowadays, an increasing number of molecular aberrations have been discovered and included in the WHO revised new classification of myeloid neoplasms. The del13q described in MDS seems to be a marker of the evolution of the clonal cells harboring 5q deletion. However, the molecular basis and the implicated genes of the deletion of the long arm of chromosome 13 associated to the 5q deletion clone remain until now not well explored.

Molecular and cytogenetic characterization of myelodysplastic syndromes in cell-free DNA

AbstractMolecular and cytogenetic studies are essential for diagnosis and prognosis in patients with myelodysplastic syndromes (MDSs). Cell-free DNA (cfDNA) analysis has been reported to be a reliable noninvasive approach for detecting molecular abnormalities in MDS; however, there is limited information about cytogenetic alterations and monitoring in cfDNA. We assessed the molecular and cytogenetic profile of a cohort of 70 patients with MDS by next-generation sequencing (NGS) of cfDNA and compared the results to sequencing of paired bone marrow (BM) DNA. Sequencing of BM DNA and cfDNA showed a comparable mutational profile (92.1% concordance), and variant allele frequencies (VAFs) strongly correlated between both sample types. Of note, SF3B1 mutations were detected with significantly higher VAFs in cfDNA than in BM DNA. NGS and microarrays were highly concordant in detecting chromosomal alterations although with lower sensitivity than karyotype and fluorescence in situ hybridization. Nevertheless, all cytogenetic aberrations detected by NGS in BM DNA were also detected in cfDNA. In addition, we monitored molecular and cytogenetic alterations and observed an excellent correlation between the VAFs of mutations in BM DNA and cfDNA across multiple matched time points. A decrease in the cfDNA VAFs was detected in patients responding to therapy, but not in nonresponding patients. Of note, cfDNA analysis also showed cytogenetic evolution in 2 nonresponsive cases. In summary, although further studies with larger cohorts are needed, our results support the analysis of cfDNA as a promising strategy for performing molecular characterization, detection of chromosomal aberrations and monitoring of patients with MDS.

Non-Invasive Genetic Profiling and Monitoring in Myelodysplastic Syndromes

Introduction. Myelodysplastic syndromes (MDS) are clonal heterogeneous disorders in which molecular studies and cytogenetics are essential for diagnosis, classification and prognosis. Cell-free DNA (cfDNA) analysis has been reported as a reliable non-invasive approach for detecting molecular abnormalities in MDS. However, there is limited information about cytogenetic alterations and monitoring in cfDNA from MDS patients.Objective. To assess and monitor the molecular and cytogenetic abnormalities by next generation sequencing (NGS) using cfDNA from patients with MDS.Patients and methods. Bone marrow (BM) aspirates and peripheral blood (PB) samples were collected from 70 newly diagnosed or treatment-naïve patients with MDS (Table). PB samples from 21 healthy controls were also studied. Molecular characterization was performed in paired samples of BM DNA and cfDNA by NGS in all patients. Libraries were prepared using a custom panel including 48 myeloid-associated genes and genomic regions localized at the most frequently altered chromosomes in MDS (QIAseq Custom DNA Panels, Qiagen) and sequenced using Illumina. 20/70 (28.6%) showed cytogenetic/FISH alterations at diagnosis, 2 of them with infrequent alterations in MDS, not covered by the panel (+14, del(9q)). 6 cases presented chromosome Y loss as a single alteration, not covered by the NGS panel. Copy number variant (CNV) analysis was performed by NGS to detect cytogenetic alterations in both cfDNA and BM, and confirmed by chromosomal microarrays (CMA) in BM DNA (CytoScan/OncoScan, ThermoFisher).Results. We obtained a median amount of cfDNA of 58.4 ng/ml in MDS patients that was significantly higher than that obtained in healthy controls (median 32.4 ng/ml, P=0.023). Sequencing of BM DNA and cfDNA showed a comparable mutational profile (187/201 mutations, 93.0% concordance). The most frequently mutated genes were TET2 (44.3%), SF3B1 (37.1%), ASXL1 (20.0%), DNMT3A (20.0%), SRSF2 (15.7%), ZRSR2 (12.9%) and U2AF1 (12.9%). A strong correlation was observed between the VAFs of BM DNA and cfDNA (r s=0.797, P<0.001). VAFs of SF3B1 mutations detected in cfDNA were significantly higher than VAFs in BM DNA (P=0.016).The analysis of cytogenetic alterations by NGS showed abnormalities in 10/70 MDS patients in both BM DNA and cfDNA. Interestingly, in a patient without analyzable metaphases in karyotype, del(20q) was found by NGS and confirmed by CMA. Overall, CMA and NGS were highly concordant to detect chromosomal aberrations although they do not reach the sensitivity achieved by karyotype/FISH (Figure 1). However, all cytogenetic aberrations detected by NGS in BM DNA were also detected in cfDNA.Molecular and cytogenetic alterations were monitored in sequential samples from 7 cases (median follow up: 13 months, range 10-30). We observed an excellent correlation between the VAF of mutations in BM DNA and ctDNA across multiple matched time points. A decrease in the VAF was detected in patients responding to therapy (either hypomethylating agents or chemotherapy), but not in non-responding patients (Figure 2). Of note, cfDNA analysis also showed cytogenetic evolution in 2 cases not responding to azacitidine (del(12p) and +21). From the two untreated patients, one acquired a subclonal del(7q) not detected by NGS and observed only in few metaphases, and the second patient showed a clonal expansion of the NF1 mutation at the time of AML transformation.Conclusions. Analysis of cfDNA allows the characterization and monitoring of molecular abnormalities in patients with MDS. Cytogenetic alterations were detectable in most cases by NGS in both BM DNA and cfDNA although with a lower detection rate than karyotype/FISH.Acknowledgements. ISCIII-FEDER, PI16/0153, PI19/0005, 2017SGR205, PT20/00023 and XBTC.Figure/Table legends:Table. MDS patients included, classified by disease subtype. (SLD: single lineage dysplasia; MLD: multilineage dysplasia; RS: ring sideroblasts; EB: excess blasts; MDS-U: MDS-unclassifiable)Figure 1. Detection of cytogenetic alterations by conventional karyotype, FISH, CMA and NGS.Figure 2. Monitoring of molecular and cytogenetic alterations in 7 patients with MDS. 5 patients receiving treatment (3 azacitidine, 1 FLAG-IDA + HCT, 1 inhibitor of hypoxia-inducible factor (HIF)) and 2 untreated cases were included. BM VAF dynamics are shown with a dotted line and cfDNA dynamics are shown with a solid line. [Display omitted] DisclosuresBesses: Gilead: Research Funding. Salar: Janssen: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Gilead: Research Funding; Celgene: Consultancy, Speakers Bureau. Bellosillo: Thermofisher Scientific: Consultancy, Speakers Bureau; Roche: Research Funding, Speakers Bureau; Qiagen: Consultancy, Speakers Bureau.

Acquisition Patterns and Interaction of Molecular, Obvious and Cryptic Cytogenetic Abnormalities in Myelodysplastic Syndromes

Abstract Introduction: The acquisition of cytogenetic aberrations (CA) and/or of molecular mutations (clonal evolution, CE) is known to be associated with poor outcome in pts with myelodysplastic syndromes (MDS) (Jabbour et al, Am J Hematol, 2013; Cevik et al, abstract, Onkologie, 2013). The aim of our study was to identify additional cases with CE by taking cryptic CA, which cannot be detected by conventional chromosomal banding analysis (CCB), and their interaction with molecular mutations, into account. Methods: We retrospectively reviewed 103 pts with proven MDS (10x RCUD, 40x RCMD, 10x RAEB-1, 16x RAEB-2, 11x sAML after MDS, 3x MDS-U, 7x CMML, 6x MDS with unknown subtype). Karyotype from CCB and molecular karyotype from SNP-array analysis (SNP-A) were available for all pts. SNP-A and fluorescence in situ hybridization (FISH) analyses were used to detect cryptic CA. For 57 pts results from mutational analysis from a 17-genes Sanger and/or a 54-genes next generation sequencing panel were included. Genetic follow-up and survival data were available for 57 pts, 22 pts were genetically analyzed under treatment with disease modifying therapies. Genetic follow-up comprised analysis of bone marrow and/or frequent sequential genetic monitoring of CD34+ peripheral blood cells. Results: CA were detected at diagnosis in 65/103 pts (63%). By CCB (aberrant in 55/103 pts; 53%) the median number of CA was 1 (range: 0-4). One to 6 (median 1) cryptic CA were detected in 31/103 (30%) pts, 10/31 pts (32%) showed a normal karyotype in CCB. The cryptic CA included cryptic copy number variations (CNV) in 12 pts and CN neutral losses of heterozygosity (CN-LOH) in 19 pts as the sole cryptic CA (14x) or in addition to cryptic CNVs (5x). In 12 pts with CN-LOHs we could identify a homozygous molecular mutation or a cryptic deletion in the region of a CN-LOH. Genes mutated in the region of a CN-LOH were DNMT3A (2p23.3, 2 pts), EZH2 (7q36.1, 2 pts), CBL (11q23.3, 2 pts), RUNX1 (21q22.12, 2 pts), TET2 (4q24), IKZF1 (7p12.2), TP53 (17p13.1), and SRSF2 (17q25.1). The formation of this phenomenon was observed in consecutive samples of 3 pts. From the 57 pts with follow-up data available, 8 showed manifestations of antecedent CE at first diagnosis (2x sub-clones; 6x homozygous mutations in CN-LOHs), from which 7 showed further genetic progression during the course of the disease. CE after first diagnosis was also observed in 20 of the 49 other pts with available follow-up data. The new aberrations of the biggest clone at first CE after diagnosis were partial or complete LOH of chromosome (chr) 7 (6 pts), trisomy 8 (5 pts), chr 21 abnormalities (2 pts), chr 12 abnormalities (2 pts), CN-LOH in 4q, 11p-, 20q-, -X, an additional marker chromosome, tetraploid cells, and mutations in ASXL1 (2 pts), CDKN2A, ETV6, IDH1, and JAK2 . Further steps of CE in these pts are currently being evaluated. Median survival time from detection of the first CE after diagnosis (25 months, 12/20 deaths) did not differ significantly from median survival time from first diagnosis for pts with signs of antecedent CE at first diagnosis (25 months, 5/8 deaths, P=0.92, n.s.). Median survival time of pts without detection of CE was significantly longer (173 months, 8/29 deaths, P=0.005 and Conclusions: In our cohort of MDS pts the frequency of cryptic CA was 30%. A homozygous mutation in a region of CN-LOH, which indicates antecedent CE, was detected in 12% of pts. The presence of such evolutionary patterns at first diagnosis might be indicative of a genetically instable disease as these pts had a shorter survival time and a shorter time to further genetic progression. There is a clear tendency that cytogenetic LOHs preferentially affect regions with molecularly mutated genes, resulting in homozygous mutations. The development of these patterns seems to be an important step towards progression. Our data imply that the relevance of cryptic CA for CE and disease progression in MDS has been underestimated as yet. Cryptic CA should be taken into account for developing prediction models for clinical progress and for improving individualized patients care. Download : Download high-res image (124KB) Download : Download full-size image Disclosures Al-Ali: Gilead: Consultancy, Honoraria; Alexion: Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Metz: Celgene: Other: study projects, advanced training support; Novartis: Consultancy, Other: study projects, advanced training support. Germing: Janssen: Honoraria; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Platzbecker: Janssen: Consultancy, Honoraria, Research Funding; Acceleron: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Haase: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

The Clinical Significance of Chromosome 17 Abnormalities in Myelodysplastic Syndrome Treated with 5-Azacytidine. Results from the Hellenic 5-Azacytidine Registry

IntroductionAberrations of chromosome 17 have been correlated to poor prognosis and resistance to treatment in several hematologic malignancies. Their role in myelodysplastic syndrome (MDS) has not yet been fully elucidated. Inversion of chromosome 17 (inv17) has been characterized as an intermediate risk cytogenetic abnormality in the revised International prognostic scoring system (IPSS-R), but chromosome 17 monosomy and other chromosome 17 abnormalities have not yet been fully characterized. Moreover, mutations of TP53 located on chromosome 17p, have been correlated to poor prognosis and resistance to treatment.AimTo determine the incidence and clinical significance of acquired abnormalities of chromosome 17 in adult patients with MDS treated with 5-azacytidine, in the context of the Hellenic 5-azacytidine registry.Patients and methodsPatients diagnosed with myelodysplastic syndrome per the 2008 WHO classification were included in the study. Conventional cytogenetic testing was performed at diagnosis. All patients were treated with 5-azacytidine during the course of their disease according to common clinical practice. Demographic, clinical and laboratory data were recorded retrospectively (Hellenic 5-azacytidine registry). Statistical analysis was performed using IBM SPSS statistics, version 23.0 (IBM Corporation, North Castle, NY, USA).ResultsWe analyzed the results of the cytogenetic analysis of 465 patients with MDS at diagnosis. Patients with chronic myelomonocytic leukemia, MDS/myeloproliferative neoplasm (MDS/MPN), and low-blast acute myeloid leukemia were excluded from the analysis. The main characteristics of the patients are shown in Figure 1A. We found 23/465 (4.9%) patients with chromosome 17 aberrations (6 with chromosome 17 inversion, 13 with a complex karyotype including chromosome 17 monosomy, 3 with a complex karyotype with add(17) and 1 with t(17;19)). All cases of inv(17) were found as an isolated cytogenetic abnormality, while all cases of chromosome 17 monosomy were found in the context of a complex karyotype. Abnormalities of chromosome 17 were the 7th more common cytogenetic abnormalities after complex karyotype and abnormalities of chromosomes 7, 8, 3, 5 and 12.Patients with chromosome 17 abnormalities from our registry tended to be higher risk per both the IPSS and the IPSS-R. The median follow-up of the patients was 23.1 (0.7-203.8) months. Patients with chromosome 17 aberrations had a median overall survival (OS) of 20.7 months versus 35.3 months for patients without chromosome 17 aberrations (log Rank 2-sided p<0.0001, Figure 1B). Patients with any type of chromosome 17 abnormalities carried a worse prognosis than patients without abnormalities of chromosome 17 (OS was as follows: inv(17), 28.9 months; chromosome 17 monosomy, 20.7 months; add(17), 14.8 months; t(17;19), 5.7 months). Patients with a complex karyotype carried a poor prognosis irrespective of chromosome 17 aberrations. The presence of chromosome 17 abnormalities did not affect response to 5-azacytidine, and the rate of transformation to AML was comparable to that of the rest of the studied population. Moreover, t(17;19) has been correlated with poor prognosis in patients with acute lymphoblastic leukemia. Here we report for the first time the same chromosome abnormality in a patient with RAEB1 with poor prognosis.DiscussionThe present study evaluates the cytogenetic analysis of the largest, to our knowledge, cohort of patients with MDS treated with 5-azacytidine. Chromosome 17 abnormalities in patients with MDS comprise a group of cytogenetic disorders with poor prognosis since most of them are found in the context of a complex karyotype with the exception of inv(17) that occurs typically as a single cytogenetic abnormality. 5-azacytidine seems to be equally effective in patients with a complex karyotype with and without chromosome 17 abnormalities. Nevertheless, overall survival and survival after 5-azacytidine treatment remain short, although patients with inv(17) have a better prognosis from patients carrying other chromosome 17 abnormalities, with survival rates exceeding the historical control of 12 months for patients with high-risk MDS treated with best supportive care. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The diagnostic and prognostic significance of flow cytometric bone marrow assessment in myelodysplastic syndromes according to the European LeukemiaNet recommendations in single-centre real-life experience.

This analysis attempts to determine the diagnostic and prognostic value of bone marrow (BM) evaluation by multiparameter flow cytometry in patients with myelodysplastic syndrome (MDS). The study group consisted of patients who underwent diagnostic process in the years 2008-2017 due to cytopenia and finally were diagnosed with MDS (n=71). The comparative group included patients with cytopenia diagnosed in the same period, whose definitive diagnosis was other than MDS (n=39). Flow cytometric evaluation of BM was performed following the recommendations of the European LeukemiaNet (ELN) in all patients. The median number of immunophenotypic abnormalities found on granulocytes in the MDS group was significantly higher compared to the comparative group [2 (range 0-5) vs 0 (range 0-2); P<.0001]. Similarly, the median Ogata score was significantly higher in the MDS group [2 (range 0-4) vs 1 (range 0-3); P<.0001]. Since the disturbances of the CD11b/HLA-DR and CD11b/CD13 on granulocytes were significantly more common in MDS patients, the Ogata score was extended by these abnormalities, what resulted in its higher diagnostic sensitivity (82%) while preserving high specificity (87%). The positive correlation was found between risk score determined by the Revised International Prognostic Scoring System and the number of the BM immunophenotypic abnormalities (P=.017). Our results indicate that the diagnostic usefulness of the Ogata score may be increased by including the abnormal expression of CD11b/HLA-DR and CD11b/CD13 on granulocytes. Moreover, our findings suggest the prognostic significance of the number of BM cytometric abnormalities in MDS.

Tissue methylation and demethylation influence translesion synthesis DNA polymerases (TLS) contributing to the genesis of chromosomal abnormalities in myelodysplastic syndrome

AimsDNA methylation has its distribution influenced by DNA demethylation processes with the catalytic conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Myelodysplastic syndrome (MDS) has been associated with epigenetic dysregulation of...

Chromosomal Abnormalities in MDS Are Linked to Dysregulation of CDC20 and CEP55 Genes

Myelodysplastic syndrome (MDS) is a clonal hematopoietic disorder characterized by cytopenias and an increased risk of progression to acute myeloid leukemia (AML). Its pathogenesis is strictly linked to chromosomal instability, which in turn provides a valuable prognostic marker. Malignant cells develop alternative routes to escape mitosis checkpoints, overcoming the mitotic arrest imposed by Spindle Assembly Checkpoint (SAC), a process dependent on CDC20 inactivation. Abnormal levels of CDC20 can inhibit mitotic arrest, promoting premature exit from mitosis. Overexpression of CEP55 also facilitates the mitotic exit, resulting in polyploidy (an event called Mitotic Slippage). Since chromosomal abnormalities are one of the most important prognostic factors for patients with MDS, this study aimed to analyze the possible link between chromosomal abnormalities and CDC20 and CEP55 mRNA expression in MDS. We evaluated the bone marrow cells from 45 patients diagnosed as MDS according to 2016 WHO-classification (1 MDS-SLD, 15 MDS-RS-MLD, 5 MDS-MLD, 1 t-MDS, and 23 MDS-EB) and 5 bone marrow of healthy controls. Conventional Karyotyping was performed by G-banding of 20 metaphases whenever possible. TaqMan expression assays for CDC20 (Hs00426680_mH) and CEP55 (Hs01070181_m1) were performed in duplicate and the expression ratios were calculated using the 2−ΔCq method. Normality was evaluated by Shapiro-Wilk test. Outliers were removed. The Student's t-test or one-way ANOVA with Tukey/Games Howell post-hoc test was used to analyze the influence of relative expression regarding variables. Patients with MDS showed increased expression of CDC20 and CEP55 compared to healthy individuals (p&amp;lt;0.0001 and p&amp;lt;0.0001). Regarding karyotype, there was the overexpression of CDC20 and CEP55 in patients with altered karyotype and aneuploid karyotype when compared to patients with normal karyotype (p &amp;lt;0.0001 and p =0.001; p = 0.013 and p = 0.022, respectively) (Figure 1A-D). CDC20 and CEP55 have fundamental functions in controlling the progression of metaphase to anaphase and both, when upregulated, induce chromosomal instability. Additionally, patients with del(7q) and complex karyotype showed hyperexpression of CEP55 when compared with patients with normal karyotype (p = 0.005 and p = 0.019) (Figure 1E-F), while patients with deletion (5q) had an increased expression of CDC20 when compared with patients with normal karyotype (p &amp;lt;0.0001). Our group previously demonstrate that high CDC20 protein expression is associated with complex karyotype in MDS patients. Thus, we hypothesized that the deregulation of CDC20 and CEP55 expression induces chromosomal changes, each one in its way. Both can cause disturbances in crucial phases of mitosis (anaphase and cytokinesis, respectively). Finally, we detected a strong correlation between CDC20 and CEP55 (r = 0.646; p &amp;lt;0.0001), suggesting both genes may play a synergistic role during chromosomal abnormalities in MDS, creating possible new targets to be evaluated in MDS. Our data suggest CDC20 and CEP55 as possible new therapeutic targets in MDS. There is a need for further studies, validations and urgent in-depth investigations in cell lines/primary samples or murine models. Disclosures No relevant conflicts of interest to declare.

Favorable prognostic phenotype in myelodysplastic syndrome with der(1;7)(q10;p10).

Unbalanced translocation der(1;7)(q10;p10) is a characteristic chromosomal abnormality in myelodysplastic syndrome (MDS). The current study revealed that among 13 MDS patients with der(1;7)(q10;p10), seven cases with no apparent dysplasia also had low numbers of myeloblasts in the bone marrow and a 3‐year survival rate of 86%; in contrast, the remaining six cases had a 3‐year survival rate of 0% (P = .003). It was therefore suggested that MDS patients with der(1;7)(q10;p10) are classified into a distinct group with a favorable prognosis and another distinct group with a very poor prognosis.

Expression, Regulation and Function of microRNA as Important Players in the Transition of MDS to Secondary AML and Their Cross Talk to RNA-Binding Proteins.

Myelodysplastic syndromes (MDS), heterogeneous diseases of hematopoietic stem cells, exhibit a significant risk of progression to secondary acute myeloid leukemia (sAML) that are typically accompanied by MDS-related changes and therefore significantly differ to de novo acute myeloid leukemia (AML). Within these disorders, the spectrum of cytogenetic alterations and oncogenic mutations, the extent of a predisposing defective osteohematopoietic niche, and the irregularity of the tumor microenvironment is highly diverse. However, the exact underlying pathophysiological mechanisms resulting in hematopoietic failure in patients with MDS and sAML remain elusive. There is recent evidence that the post-transcriptional control of gene expression mediated by microRNAs (miRNAs), long noncoding RNAs, and/or RNA-binding proteins (RBPs) are key components in the pathogenic events of both diseases. In addition, an interplay between RBPs and miRNAs has been postulated in MDS and sAML. Although a plethora of miRNAs is aberrantly expressed in MDS and sAML, their expression pattern significantly depends on the cell type and on the molecular make-up of the sample, including chromosomal alterations and single nucleotide polymorphisms, which also reflects their role in disease progression and prediction. Decreased expression levels of miRNAs or RBPs preventing the maturation or inhibiting translation of genes involved in pathogenesis of both diseases were found. Therefore, this review will summarize the current knowledge regarding the heterogeneity of expression, function, and clinical relevance of miRNAs, its link to molecular abnormalities in MDS and sAML with specific focus on the interplay with RBPs, and the current treatment options. This information might improve the use of miRNAs and/or RBPs as prognostic markers and therapeutic targets for both malignancies.