Chromosomal Abnormalities In Myelodysplastic Syndrome Research Articles

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.

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<0.0001 and p<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 <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 <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 <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.

Techniques for detecting chromosomal aberrations in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are a group of heterogeneous hematologic diseases. Chromosomal aberrations are important for the initiation, development, and progression of MDS. Detection of chromosomal abnormalities in MDS is important for categorization, risk stratification, therapeutic selection, and prognosis evaluation of the disease. Recent progress of multiple techniques has brought powerful molecular cytogenetic information to reveal copy number variation, uniparental disomy, and complex chromosomal aberrations in MDS. In this review, we will introduce some common chromosomal aberrations in MDS and their clinical significance. Then we will explain the application, advantages, and limitations of different techniques for detecting chromosomal abnormalities in MDS. The information in this review may be helpful for clinicians to select appropriate methods in patient-related decision making.

Abnormal mRNA Expression Levels of Telomere-Binding Proteins Represent Biomarkers in Myelodysplastic Syndromes: A case control study

Objective:As evidence was shown that abnormal shortening of telomeres begins to accumulate in myelodysplastic syndrome (MDS) patients, this study was conducted to determine the relationship between the mRNA expression levels of telomere-binding proteins (TRF1/TRF2/TIN2/TPP1/POT1/RAP1) and the risk level in MDS.Materials and Methods:There were 40 patients with MDS and 40 normal controls in this study. Methods including telomere content assays and quantitative reverse transcription-polymerase chain reaction were used to examine the mRNA levels of TRF1/TRF2/TIN2/TPP1/POT1/RAP1 in patients with MDS.Results:Compared to the normal group used as a control, the mRNA expression levels of RAP1/POT1/TPP1 of the patients with MDS were decreased, whereas their levels of TRF1/TRF2 and TIN2 were increased. A positive correlation was found between the TRF1, TRF2, and TIN2 mRNA expression levels and the risk level of the International Prognostic Scoring System (IPSS) and the World Health Organization Prognostic Scoring System (WPSS) criteria; however, a negative correlation was found between RAP1/POT1/TPP1 mRNA expression levels and the risk levels of IPSS and WPSS criteria.Conclusion:Because the reduction of TRF1/TRF2/TIN2 mRNA expression and the increase of RAP1/POT1/TPP1 mRNA expression are closely related to the risk levels of the IPSS and WPSS criteria in MDS, it is thought that these telomere-binding proteins could lead to abnormal telomere length and function, which cause chromosomal abnormalities in MDS. With this evidence, we suggest that those proteins’ mRNA expressions could be used as biomarkers for the assessment of the risk degree of MDS patients.

Additional Information Offered By Snpa in Myelodysplastic Syndromes with Excess Blasts (MDS-EB) and Future Perspectives

Introduction: The detection of chromosomal abnormalities in myelodysplastic syndromes (MDS) supports the diagnosis, classification, prognostic stratification, therapy option, treatment monitoring and better understanding of the biology of disease. The main chromosomal abnormalities of MDS are losses and gains of genetic material, and these changes are among the most important prognostic parameters in IPSS-R. As a consequence, major advances have been achieved in the treatment and survival of patients. However, nearly half of the patients present a normal karyotype, which prevents their further characterization. Therefore, there is the desire to increase the abnormalities detection rate by other methods. Single nucleotide polymorphisms array (SNPa), also referred to as chromosomal microarray, is a sensitive technology used to perform high-resolution genome-wide DNA losses, gains and copy-neutral loss of heterozygosity (CN-LOH). The genomic DNA is hybridized to polymorphic probes which are SNP markers and non-polymorphic probes which are copy number markers, providing information about CN-LOH and copy number alteration (CNA), respectively. Previous studies have shown that the addition of the SNPa to karyotype (KT) increases by 28% the detection rate of cytogenetic abnormalities.Objective: To substantiate this idea we describe two cases of MDS to whom SNPa added valued information to karyotype.Methods: DNA was extracted from bone marrow cells for genomic clonal evaluation. The DNA was digested, amplified, fragmented, labeled and hybridized to the chip (Affymetrix - CytoScan HD®) containing the probes. The chip was scanned to detect the signals’ intensity emitted by the hybridized probes which were further analyzed by a software (ChAS) that allows visualization of CNA and CN-LOH. CNA´s analysis relies on the comparison of the obtained signals to a reference diploid DNA signal, and the difference encountered is characterized as loss or gain. CN-LOH analysis is based on two possible nucleotide signals (A or B), which are evaluated to discriminate three genotypes: AA, AB and BB. CN-LOH occurs when one allele is lost and duplicate another, resulting in genotypes AA or BB.Results: Case 1, 64y-male-patient, classified as MDS-EB (WHO 2016), Bone marrow histology showed grade II fibrosis. KT: 46,XY,del(5)(q15q33),del(17)(p11.2)[16] / 46,XY[4]. SNPa: 3p21.31p21.2 CN-LOH; 5q21.1q35.3 loss (CN:1.00); -7; +8; 12p13.33p12.3, 12p12.1p11.22, 12q22q23.3 loss (CN:1.00); -16; 17p13.3p11.2 mosaic loss (CN: 1.50) and -Y.Case 2, 74y-male-patient, classified as MDS-EB (WHO 2016), Bone marrow histology showed grade II/lll fibrosis. KT: 46,XY[15]. SNPa: 21q21.1q22.3 CN-LOH.Discussion and Conclusion: The SNPa has the advantage of detecting genomic alterations regardless of the cell cycle, even when the cell is quiescent or growth is defective. It also enables the identification of CN-LOH (also known as uniparental disomy, UPD), submicroscopic amplifications and deletions that are not detected by KT. On the other hand, SNPa does not allow the identification of balanced translocations and polyploidy. In case 1, after SNPa analysis, some chromosomal abnormalities (−7, +8, −16 and −Y) were found in sporadic metaphases during the KT reanalysis, but had not initially been described because they did not meet the criterion to be considered as a cytogenetic clone. The risk-stratification (IPSS-R) for this patient was intermediate, but the addition of the SNPa results the risk-stratification could be changed to very poor. Seven months after diagnosis the patient developed acute myeloid leukemia and died. In case 2, CN-LOH detected by SNPa could be responsible for homozygosity of mutations in critical genes located in the 21p region, such as RUNX1 that encodes a protein, which is a transcription factor critical in hematopoiesis. Indeed, sequencing of candidate genes in CN-LOH regions should be considered a priority in the search of driver mutations of MDS. Twenty-four months after diagnosis the patient died due to other non-hematologic causes. In Summary, SNPa analysis may add value to KT non-informative results and occasionally reveal cryptic abnormalities not recognized by karyotyping. However, SNPa analysis should be viewed as a complimentary tool.Acknowledgment: The SNPa test was supported by Grupo Fleury DisclosuresNo relevant conflicts of interest to declare.

Chromosomal Abnormalities in Myelodysplastic Syndromes Detected With Multiplex Ligation Dependent Probe Amplification

Detection of chromosomal abnormalities in myelodysplastic syndromes enables classification according to WHO Classification and risk stratification according to IPSS and R-IPSS, providing physicians with data regarding personal therapy approach. Detection of the chromosomal abnormalities in our cohort study was performed with the method Multiplex Ligation-dependent Probe Amplification is similar to multiplex PCR reactions, but in this case the target-sequences are amplificated, but MLPA probes hybridized for them. 70 patients, chromosomal abnormalities were detected in 32.9% patients, while 67.1% patients had normal findings. Patients were classified according to WHO and stratified according to IPSS and R-IPSS. Data obtained enabled us to make treatment strategy at diagnosis. There are different treatment options for different risk groups including allogeneic transplantation for high and very high risk patients as well as hypomethylating agents for those who are not candidates for transplantation. For low risk groups supportive treatment and growing factors were most often the choice.We can conclude that MLPA reliable method for detection of chromosomal abnormalities in myelodysplastic syndromes.

Karyotype analysis and fluorescence in situ hybridization detection of 122 patients with myelodysplastic syndrome

To assess the value of karyotype analysis and fluorescence in situ hybridization(FISH) assay for the diagnosis of myelodysplastic syndrome (MDS). The karyotypes of 122 initially treated MDS patients were analyzed with conventional R-banding and FISH using probes including GLP CSF1R/D5S23, D5S721, GLP EGR1/D5S23, D5S721, GLP D7S486/CSP7, GLP D7S522/CSP7, GLP D20S108, CSP8 and CSP X/Y. The detection rate of chromosomal abnormalities was 54.9% for the 122 patients. Among these, those involving 3 or more chromosomes are most common (16.4%), followed by +8(14.8%), -7/7q-(7.4%), -5/5q-(5.7%), 20q-(2.5%), and -Y in male patients (5.0%). Two MDS-RAEB II patients detected with t(8;21) should be diagnosed with acute myelocytic leukemia. FISH analysis showed that 54 patients were positive (44.3%). Among these, 30.3% had CSP8 amplification, followed by GLP D7S486/CSP7 and GLP D7S522/CSP7 deletion (12.3%), GLP CSF1R/D5S23, D5S721 and GLP EGR1/D5S23, D5S721 deletion (9.8%), GLP D20S108 deletion (7.4%), and CSPX/Y deletion (5%). With a detection rate of 54.9%, R-banding still constitutes the basic examination for MDS. As detection of interstitial chromosomal abnormalities in MDS can be greatly enhanced by FISH, combined karyotype analysis and FISH can improve the diagnosis of MDS and facilitate assessment of its prognosis.

Microarray-based genomic profiling and in situ hybridization on fibrotic bone marrow biopsies for the identification of numerical chromosomal abnormalities in myelodysplastic syndrome.

BackgroundMyelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematological malignancies. In MDS patients with a fibrotic bone marrow the aspiration of cells often fails (dry-tap), which hampers standard karyotyping. Obtaining genetic data from these fibrotic marrows is therefore challenging, and up till now in situ hybridization applied to bone marrow biopsies is the only option. The microarray-based genomic profiling technology has already proven its value for bone marrow aspirates and peripheral blood samples, but has never been applied to the technically challenging bone marrow biopsies. We describe an approach for microarray-based genomic profiling on bone marrow biopsies and demonstrate its ability to obtain clinically relevant cytogenetic aberrations. In addition the data were compared with those obtained by in situ hybridization and karyotyping.ResultsWe have evaluated the success rate of microarray-based genomic profiling by studying twenty-one bone marrow biopsies (7 fibrotic MDS, 12 non-fibrotic MDS and 2 reactive), by microarray-based genomic profiling and in situ hybridization (12 of 21 cases). The data obtained with these techniques were compared with conventional karyotyping data on corresponding bone marrow aspirates. Of the 15 copy number aberrations that were detected by in situ hybridization, 13 were concordant with microarray-based genomic profiling and karyotyping, whereas two hybridizations were misinterpreted. In 20 of 21 patients, the data obtained by microarray-based genomic profiling and karyotyping were identical or differences could be explained by the presence of marker chromosomes, complex karyotypes, clonal heterogeneity or disease progression.ConclusionsWe demonstrate that genome wide microarray-based genomic profiling performed on bone marrow biopsies has a similar success rate compared to in situ hybridization, and prevents misinterpretation of chromosomal losses as observed by FISH. In addition, equal to even higher resolutions were obtained with genomic profiling compared to conventional karyotyping. Our findings indicate that microarray-based profiling, even on bone marrow biopsies, is a valid approach for the identification of genetic abnormalities. This is a valuable substitution in cases of fibrotic MDS lacking cytogenetic results.

Genomic Copy Number Variations in the Myelodysplastic Syndrome and Acute Myeloid Leukemia Patients with del(5q) and/or -7/del(7q).

The most common chromosomal abnormalities in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are -5/del(5q) and -7/del(7q). When -5/del(5q) and -7/del(7q) coexist in patients, a poor prognosis is typically associated. Given that -5/del(5q) and/or -7/del(7q) often are accompanied with additional recurrent chromosomal alterations, genetic change(s) on the accompanying chromosome(s) other than chromosomes 5 and 7 may be important factor(s) affecting leukemogenesis and disease prognosis. Using an integrated analysis of karyotype, FISH and array CGH results in this study, we evaluated the smallest region of overlap (SRO) of chromosomes 5 and 7 as well as copy number alterations (CNAs) on the other chromosomes. Moreover, the relationship between the CNAs and del(5q) and -7/del(7q) was investigated by categorizing the cases into three groups based on the abnormalities of chromosomes 5 and 7 [group I: cases only with del(5q), group II: cases only with -7/del(7q) and group III: concurrent del(5q) and del(7q) cases]. The overlapping SRO of chromosome 5 from groups I and III was 5q31.1-33.1 and of chromosome 7 from groups II and III was 7q31.31-q36.1. A total of 318 CNAs were observed; ~ 78.3% of them were identified on chromosomes other than chromosomes 5 and 7, which were defined as 'other CNAs'. Group III was a distinctive group carrying the most high number (HN) CNAs, cryptic CNAs and 'other CNAs'. The loss of TP53 was highly associated with del(5q). The loss of ETV6 was specifically associated with group III. These CNAs or genes may play a secondary role in disease progression and should be further evaluated for their clinical significance and influence on therapeutic approaches in patients with MDS/AML carrying del(5q) and/or -7/del(7q) in large-scale, patient population study.

RPL27A Is a Target for Mir-595 and Contributes to Myelodysplastic Phenotype through Ribosomal Dysgenesis

Introduction: The importance of miRNAs in regulating gene expression has been addressed in several haematological cancers including myelodysplastic syndrome (MDS). In MDS, miR-145 and miR-146a have been associated with some of phenotypic features of 5q- syndrome. Monosomy 7/7q deletion is the second most common chromosomal abnormality in MDS and identifies a subgroup of patients with poor prognosis. Thus far, no studies have examined the role of miRNA dysregulation in MDS patients with monosomy 7. In this study we examined the functional consequences of deletion of miRNAs localized to 7q with particular focus on miR-595, which is localized to 7q36.3, which is commonly deleted region in 7q- and -7.Most of the available methodologies for miRNA target identification are based on computational algorithms. Therefore, there is a relatively high incidence of false positive target identification. To enable the identification of biologically relevant microRNA targets, a novel functional assay was developed in our lab, which is based on positive/negative selection (Gaken et al, 2012). Using this assay, we identified functional target of miR-595 and correlated this with the MDS phenotype.Method: The functional assay relies on miR-595, identified several targets including RPL27A, HSPA14, GRM5 and SEC63. We focused on ascertaining the biological function of RPL27A. RPL27A was validated as a target for miR-595, by examining the changes on RPL27A expression using quantitative RT-PCR and western blot analysis in cells transfected with plasmid expressing miR-595. We demonstrated a targeted reduction of RPL27A in several cell lines including HCT-116 and HEL cells (expressing p53) as well as HCT-116 p53-/- and K562 cells (not expressing p53) through retroviral expression of (shRNAs). RPS14 knocked down was used as a control. The knockdown of RPL27A and RPS14 was also performed in normal CD34+ (n=6) in two stages liquid culture using cytokines mixture to induce erythroid, myeloid and megakaryocyte differentiation. The effects on expressing erythroid cells were assessed and composed of CD71 and GlyA, myeloid and megakaryocyte cells using CD11b and CD41, respectively.Result: RPL27A was validated as target for miR-595 (Figure1). RPL27A knockdown resulted in a reduction in the large ribosomal subunit (60S) based on Polysomes fractionation, (Figure 2). On day 6 of the knockdown, the depletion of RPL27A led to p53 activation and induced apoptosis in all cell lines at different levels, 40-60% increases in apoptotic cells in HCT-116 and HEL cell lines and 20-30% increases in apoptotic cells in HCT-116 p53-/- and K562 cells (Figure 3). Interestingly, like RPS14 knockdown, the effect of RPL27A knockdown in 48 h was p53 dependent. The p53 independent effect, which was observed on day 6, was attributed to a defect in ribosome biogenesis based on the disruption of nucleolar staining in HCT-116 and HCT-116 p53-/- with depleted RPL27A using fibrillarin antibody and similar effects were seen with RPS14.To investigate whether the effect of RPL27A knockdown on erythroid differentiation is similar to the other ribosomal proteins, RPL27A knockdown (approximately 70% reduction at day 6) was performed in normal CD34+ and resulted in reduce viability and apoptosis within 10 days. Post-transcriptional p53 overexpression was also observed; this was associated with a marked increase in the mRNA expression of p53 targets, p21 and Bax. Following 10 days in liquid culture, RPL27A knockdown blocked the proliferation and differentiation of erythroid cells relative to myeloid and megakaryocyte lineages. Similar observations have been made in 5q- syndrome due to RPS14 haploinsufficiency and in DBA patients with mutations in large or small ribosomal subunit proteins.Conclusion: This study showed that haploinsufficency of miR-595 in patients with -7/7q del is important in pathogenesis of reduced erythropoiesis and a reduction in myeloid and erythroid progenitor populations. The effects are mediated via RPL27A, which is a ribosomal protein and its knockdown produces it effects via p53 dependent pathways, leads to ineffective erythropoiesis and arrest of the myeloid and megakaryocyte differentiation. These effects are particularly relevant in patients with complex chromosomal abnormalities that include haploinsufficiency of RPS14 due to -5/del 5q. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Detecting chromosomal aberrations in myelodysplastic syndrome with fluorescence in situ hybridization and conventional cytogenetic analysis

To detect chromosomal abnormalities in myelodysplastic syndrome (MDS) patients by fluorescence in situ hybridization (FISH) and conventional cytogenetic analysis (CCA). FISH and CCA were performed in 100 patients who were diagnosed with MDS by conventional detection of bone marrow smear and bone marrow biopsy, and were followed up. Forty-eight (48%) patients showed chromosomal abnormalities. The positive rate of -5/5q-, 20q-, +8, -7/7q-, and -Y was 16%, 15%, 12%, 11%, and 5%, respectively, and that of CCA was 11%. The positive rate of molecular genetics abnormalities detected by FISH was obviously higher than that of CCA (P<0.01) and the combination of FISH and CCA increased the detection rate to 49%. The follow-up showed that the prognosis of patients with normal FISH results was significantly better than the abnormal ones. A correlation between complex karyotypes and poor prognosis was observed. Abnormality of -7/7q- was found closely correlated with the higher risk of acute leukemia and death. Chromosomal abnormalities have been found in 49 MDS patients. Common chromosomal abnormalities in MDS patients include -5/5q-, 20q-, +8 and -7/7q-. FISH combined with CCA can improve the detection rate of chromosomal aberrations in MDS. FISH is more sensitive than CCA for detection and can be used as an important basis for prognostic assessment for MDS.

Clinical Significance of Cytogenetic Manifestations in Myelodysplastic Syndromes

Myelodysplastic syndrome (MDS) represents a heterogeneous hematopoietic stem-cell disorder that results in abnormal cellular maturation and peripheral blood cytopenias. MDS is characterized by progressive bone marrow failure, which can lead to bleeding, infections, and complications secondary to anemia. Approximately 35% to 40% of patients diagnosed with MDS progress to acute myeloid leukemia (AML), which confers a poorer prognosis. MDS may develop de novo without underlying risk factors or may be secondary, occurring after exposure to chemotherapeutic agents or ionizing radiation. The earliest attempt to classify MDS into various subgroups was by the French-American-British (FAB) group, which separated MDS by its morphologic and clinical characteristics, such as the percentage of blasts in bone marrow. The progression of disease in patients with MDS is extremely variable, however, reflecting the heterogeneity of this syndrome. Although the FAB classification system has been useful, it has proven to be inaccurate when determining a patient’s prognosis. As studies have evolved, the genetic heterogeneity of MDS has proven to be a clear reason that the FAB classification system is prognostically insufficient. More than 50% of patients with MDS have clonal cytogenetic abnormalities, and the molecular consequences of these abnormalities continue to be elucidated. The chromosomal abnormalities in MDS have helped to stratify the myelodysplastic syndromes into poor-, intermediate-, and good-prognosis groups.

The implications of myelodysplastic syndrome – associated chromosomal abnormalities in the development of graft‐versus‐host disease

There is a growing body of evidences that acquired chromosomal abnormalities in bone marrow (BM) cells are associated with clinical manifestations of myelodysplastic syndrome (MDS). However, to our knowledge, there are no reports that describe the association between chromosomal abnormalities in MDS and graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-SCT). Here, we describe two MDS cases with trisomy 8 and der(1;7)(q10;p10), who developed severe GVHD after allo-SCT. We analyzed cytokine production and cell survival of monocytes from these patients with MDS before allo-SCT, in comparison with healthy controls or an MDS patient with a different chromosomal abnormality, who has not developed GVHD. The monocytes from MDS patients with trisomy 8 and der(1;7)(q10;p10) produced a larger amount of pro-inflammatory cytokine, tumor necrosis factor-α, and a smaller amount of anti-inflammatory cytokine, interleukin-10, on stimulation with Toll-like receptor (TLR) ligands. In addition, the monocytes from MDS cases with GVHD showed a decrease in apoptotic cell death upon stimulation with TLR ligands. We also detected host-derived pro-inflammatory antigen-presenting cells (APCs) in skin GVHD lesions after allo-SCT. These data suggest that trisomy 8 and der(1;7)(q10;p10) may be associated with the development of severe GVHD, by prolonging survival of pro-inflammatory host-derived APCs in GVHD lesions.

Lenalidomide, p53 and del(5q) Myelodysplastic Syndrome: Ribosome Stress Relief

Whereas deletions involving the long arm of chromosome 5 are among the most common chromosomal abnormalities in myelodysplastic syndrome (MDS), isolated del(5q) MDS, which includes the 5q- syndrome, is rare and characterized by hypoplastic anemia and a moderate risk of transformation to acute myeloid leukemia (AML). The 5q- syndrome is now recognized as a ribosomopathy, and both the classic 5q- syndrome and del(5q) MDS are uniquely responsive to lenalidomide. However, the mechanism of action of lenalidomide is controversial and involves modulation of p53 activity, which may be beneficial in anemia remission but suggested to lead to malignant cell outgrowth. Here, we critically review the literature on this important controversy, which has obvious implications for therapy of del (5q) MDS.

A Comprehensive Genetic Analysis of MDS Patients From Peripheral Blood Combining FISH- and SNP-Array-Analysis

AbstractAbstract 4926 Introduction:Chromosomal banding analysis (CBA) of bone marrow metaphases is the gold standard to identify chromosomal abnormalities in myelodysplastic syndromes (MDS). We aim to comprehensively detect and follow chromosomal abnormalities during the course of the disease without the need of repeated bone marrow biopsies. In ongoing studies we attempt to achieve this goal by performing serial fluorescence in situ hybridization (FISH) analysis on CD34+ peripheral blood cells (PBC). The aim of this pilot study was to establish SNP-array-analysis (SNP-A) on CD34+ PBC to complement genetic analysis on peripheral blood by identifying chromosomal abnormalities not detectable by FISH and/or CBA. Methods:We immunomagnetically enriched CD34+ PBC of 20 patients (pts) with MDS (16 pts), suspected MDS (1 pts) and secondary acute myeloid leukemia (sAML, 3 pts). SNP-A was performed with arrays from Affymetrix (3x SNP 6. 0, 4x Cyto 2. 7, 13x CytoScanHD). Fresh or frozen CD34+ PBC of 10 pts and in methanol/acetic acid fixed CD34+ PBC of 9 patients were successfully processed. One whole genome amplified sample was included. CBA and FISH-A was done for all patients. Results:By CBA, 3 pts had no chromosomal abnormalities, 8 pts had one abnormality, 6 pts had 2–4 abnormalities and 3 pts had more than 6 abnormalities. By SNP-A on CD34+ PBC, additional abnormalities could be revealed in 13/20 pts. In two pts they were also confirmed by FISH-A. Most of them were micro-deletions not detectable by CBA. In addition, SNP-A revealed uniparental disomies (UPD) in 5/20 pts. Of the 3 pts with no detectable abnormalities in CBA, one had a micro-deletion in 4q24 (TET2). The other two had an insufficient number of metaphases. One of them showed a highly complex karyotype by FISH-A and SNP-A on CD34+ PBC. The other one had suspected MDS and did not show any abnormalities by SNP-A.The 17 pts with ≤ 6 abnormalities in CBA showed 55 abnormalities by CBA, FISH-A and SNP-A altogether. 34/55 (62%) abnormalities could be detected by SNP-A and/or FISH-A, but not by CBA. 24/55 (44%) abnormalities could only be detected by SNP-A. 4/55 (7%) of abnormalities were structural abnormalities or small clones and were only detected by CBA.Serial analysis indicated clonal evolution: A patient with 16 abnormalities detectable by CBA and additional three by FISH and SNP-A developed two further micro-deletions (del(2)(q31q32), del(4)(q24q26)) within four months. When a MDS patient with a known 20q-deletion (isolated by CBA and FISH) progressed to AML 25 months after first diagnosis we detected 3 micro-deletions by SNP-A of peripheral blood (0. 98 Mb on 4q, 1. 31 Mb on 12q, 2. 55 Mb on 12q) thus resulting in 4 cytogenetic alterations fulfilling the criteria of complex and prognostically unfavorable abnormalities. Conclusions:Recently it was shown that abnormalities detectable by SNP-A, but not by CBA, could worsen prognosis of MDS patients. We succeeded in detecting these additional abnormalities without the need of bone marrow biopsies out of peripheral blood. Nevertheless, by parallel FISH and SNP-A of CD34+ PBC, most abnormalities detectable by CBA of bone marrow metaphases could be detected. Comprehensive genetic analysis at close intervals thus is possible without the need of bone marrow biopsies to study clonal evolution. The information gained could be used for therapy decisions, to improve prognostication and to unravel genetic evolutionary steps towards acute leukemia. Disclosures:No relevant conflicts of interest to declare.

Chromosomal Abnormalities in Myelodysplastic Syndrome with Near-tetraploidy: A Case Report

eISSN 2093-6338 자들의 결함들이 축적되어서 급성골수성백혈병으로 전환되는 것 으로 여겨지고 있다[2]. 클론성 세포유전학적 이상은 새로(de novo) 발생한 골수형성이 상증후군에서 40%, 이차성 골수형성이상증후군에서는 80%가 발 견된다고 보고되었다[3]. International Prognostic Scoring System (IPSS)에서는 세포유전학적 이상을 급성골수성백혈병의 생존과 예후를 판정해주는 독립적인 인자로 인정하였으며, 그 중에서도 3 가지 이상의 세포유전학적 이상이 존재하는 복잡형 핵형(complex karyotype)은 골수형성이상증후군 중에서도 나쁜 예후를 시 사하는 소견이라고 정의하였다[4]. 여러 염색체 이상 중에서 50개 이상의 염색체수를 보이는 중과 이배수체증(massive hyperdiploidy)과 네배수체증(tetraploidy)은 골수성 종양에서 드물게 관찰되는 세포유전학적 이상으로 알려져 있으며[5], 이러한 세포유전학적 이상은 골수형성이상증후군에서 나쁜 예후와 관련이 있다고 하나 아직까지 보고된 예가 적어 임상 적인 의미가 확실하지 않다[5-9]. 저자들은 골수형성이상증후군을 진단받고 염색체 검사에서 근네배수체증(near-tetraploidy)을 보인 환자를 경험하여 문헌 고찰과 함께 보고하는 바이다. 서 론

Chromosomal Aberrations Identified in CD34+ Peripheral Blood Cells of MDS Patients by FISH- and SNP Array Analysis

Abstract 4872 Introduction:Chromosomal banding analysis (CBA) of bone marrow metaphases is the gold standard to identify chromosomal abnormalities in myelodysplastic syndromes (MDS). To detect and follow chromosomal abnormalities during the course of the disease without the need of repeated bone marrow biopsies, we are currently performing serial fluorescence in situ hybridization (FISH) analyses of CD34+ peripheral blood cells (PBC) in ongoing studies. To complement genetic analysis on peripheral blood we started a pilot study to establish SNP array analysis (SNP-A) on CD34+ PBC to identify chromosomal abnormalities not detectable by FISH. Methods:We analyzed eleven MDS and two AML-patients with known karyotypes and compared CBA with FISH and SNP-A of CD34+ peripheral blood and/or bone marrow cells. The FISH panel comprised up to 13 probes. The Affymetrix Genome-Wide Human SNP Array 6.0 and/or the Affymetrix Cytogenetics Whole-Genome 2.7M Array were used. We also included serial samples from a RAEB-II patient where bone marrow and/or peripheral blood were available from six time points collected over a period of 6 months. Results:In 13 patients analyzed using CBA, FISH, and SNP-A in parallel, 10/60 (17%) chromosomal abnormalities were exclusively detected by SNP-A. Using CBA and FISH we detected 28 chromosomal abnormalities in 12 patients. Additional SNP-A revealed 6 further aberrations (upd(7)(q11qter), upd(10)(q23.33q25.1), upd(17)(pterp11.2), del(9)(q22.33q31.1), del(13)(q12.3q22.2), del(15)(q15.1)). In one patient SNP-A increased the number of detectable chromosomal abnormalities from 22 to 26 (amplifications on 6p, del(15)(q11.2q21.1), del(18)(pterp11), upd(20)(q11.22q12)). Additional abnormalities were also detected in the serial sample: The major clone detectable by CBA at three different time points was 44,XX,del(5)(q13q33),-7,del(12)(p13p11.2),-17,-20,+der(20)t(17;20)(q10;p10). We could confirm all these abnormalities in CD34+ peripheral blood and bone marrow cells using a FISH panel that includes del(5q31)/EGR1, −7/CEP7, del(12p13)/TEL, del(17p13)/TP53, and del(20q12)/D20S108. FISH on CD34+ PBC confirmed a stable number of aberrant cells as 5q- was detectable in 94–98% of CD34+ PBC at all six available time points. We could also confirm all abnormalities of the major clone by SNP-A in CD34+ PBC in month 2 and in CD34+ bone marrow cells in month 6. Additional abnormalities occurring in sub-clones changed over time. A sub-clone exclusively detectable by CBA was identified in the first available sample in 2/25 (8%) metaphases: 44,idem,+der(3)t(3;6)(p10;q10),-6. Supplementary FISH and SNP-A revealed a 9.6 Mb del(13q14) in 44% of CD34+ PBC that was not detectable by CBA and had subsequently disappeared in the last available sample. SNP-A on CD34+ bone marrow cells of the last sample revealed two additional abnormalities in the absence of clinical signs of progression (del(2)(q31q32), del(4)(q24q26)). The del(4q)/TET2 could be confirmed by FISH. Conclusion:Detection and follow-up of chromosomal abnormalities during the course of the disease is possible without the need of bone marrow biopsies by parallel FISH and SNP-A of CD34+ peripheral blood cells. Detailed knowledge about the acquirement of chromosomal aberrations could be used to improve prognostication, to support therapy decisions and to unravel genetic evolutionary steps towards acute leukemia. Disclosures:No relevant conflicts of interest to declare.

Multiplex ligation-dependent probe amplification for detection of chromosomal abnormalities in myelodysplastic syndrome and acute myeloid leukemia

Current strategies for detecting chromosome abnormalities in MDS/AML include FISH or traditional cytogenetics. MLPA detects abnormalities in multiple loci simultaneously, with higher resolution and throughput. Peripheral blood from 50 healthy subjects was used to establish probe-specific reference ranges, increasing MLPA sensitivity and specificity. MLPA was then performed on 110 FISH-tested blood or bone marrow samples from suspected leukemia patients. Our novel MLPA analysis system combined maximum stringency with sensitive detection of low-frequency abnormalities. Accuracy/specificity of MLPA were excellent compared to FISH. Our MLPA analysis/interpretation method provides a clinically robust, high-throughput, high-resolution option for detection of abnormalities associated with MDS/AML.