Chromosomal Abnormalities In Myelodysplastic Syndrome Research Articles

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.

Molecular Characterization of Chromosomal Abnormalities In Myelodysplastic Syndrome and Acute Myeloid Leukemia: Validation of An MLPA Protocol and Analysis Method for Use In a Diagnostic Setting

AbstractAbstract 4849 Introduction:Current diagnostic screening strategies for copy number variations (CNVs) in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) include fluorescence in situ hybridization (FISH) or karyotyping, both of which are time-consuming, costly, laborious, and lacking in resolution. Multiplex ligation-dependent probe amplification (MLPA) can be used to detect copy number changes in multiple loci simultaneously in a single PCR reaction, and boasts a resolution down to single exons. To adapt MLPA for use in routine clinical diagnostics, we have developed and validated a protocol for automatic data analysis and interpretation of common chromosomal abnormalities in MDS/AML. Patients and Methods:The study used a training set of 45 healthy subjects to establish a normal reference range for each individual probe. Using these ranges we built an automated Excel spreadsheet-based analysis system, which included multiple quality checks, and flagged samples failing these quality controls. Each probe was given a call of “no mutation detected,” “deletion,” or “gain,” based on whether the normalized ratio fell within or outside of the empirically-determined normal range for that probe. We then analyzed over 100 leukemia cases tested by FISH, including both suspected myeloid leukemia samples and suspected chronic lymphocytic leukemia (CLL) samples. Documented chromosomal abnormalities in CLL include 11q-, 17p- (loss of TP53), and trisomy 12, all of which had the potential to be detected by the probes in the MDS MLPA probemix. The greater prevalence of CLL and its associated CNVs provided additional positive controls for the validation of the MDS MLPA probemix and our analysis method. Results:The empirically-determined normal ranges demonstrated that some probes varied widely (3 standard deviation [3SD] normal range of 0.46–1.54), while others were extremely reliable (3SD normal range of 0.84–1.16). The MLPA assay demonstrated excellent overall accuracy (>90%) and specificity (>93%) for both suspected myeloid and CLL samples when compared to FISH. The sensitivity of the MLPA assay is somewhat lower than that of FISH, requiring a probe-dependent 20–40% positivity for a given CNV to be detected. However in several cases, the MDS MLPA assay was able to detect additional lesions too small to be seen by FISH. Conclusions:For MLPA, the total process-to-report time, including data analysis, is 2–3 days, versus the 7–10 days required for FISH analysis. In addition, the MLPA assay is substantially cheaper and considerably less labor-intensive than FISH. Our improved MLPA assay protocol and analysis method provides a clinically robust, multiplexed, high-throughput, high-resolution, and low-cost solution for detection of copy number changes in MDS/AML, and can therefore be used as a first-line screening test in a clinical laboratory. Disclosures:Donahue: Quest Diagnostics Inc.: Employment. Abdool: Quest Diagnostics Inc.: Employment. Wohlgemuth: Quest Diagnostics Inc.: Employment. Yeh: Quest Diagnostics Inc.: Employment.

Do we need to do fluorescence in situ hybridization analysis in myelodysplastic syndromes as often as we do?

Although conventional cytogenetics is considered the gold standard to detect chromosomal abnormalities in myelodysplastic syndromes (MDS), fluorescence in situ hybridization (FISH) is being increasingly used additionally. However, the real contribution of FISH analysis in the cytogenetic diagnosis of MDS has not been well defined. The aim of this study was to evaluate whether FISH studies are able to reveal chromosomal abnormalities in MDS patients undetected by conventional cytogenetics. One hundred seventy-four FISH studies were performed on bone marrow samples of 60 patients with MDS. The number of FISH studies in each patient was variable (1–5). FISH studies confirmed the G-banding cytogenetic findings in 99.4% (153/154) of samples and detected cytogenetic abnormalities in 25% (5/20) of cases in which the conventional cytognetic study failed. These results indicate that FISH studies provide relevant information in MSD in which the conventional cytogenetic analysis was unsuccessful but add little value to a normal katyotype in conventional cytogenetic analysis.

Evaluation of Conventional Cytogenetics Versus Fluorescence in Situ Hybridization Analysis in the Prognostic Scoring of Patients with Myelodysplastic Syndromes.

Abstract 1757Poster Board I-783 BackgroundThe myelodysplastic syndromes (MDS) encompass a heterogeneous group of progressive bone marrow insufficiency disorders characterized by chronic cytopenias and a risk of progression to acute leukemias. Non-random clonal chromosomal abnormalities, seen in almost 50% of patients with MDS, are associated with clinically and biologically distinct forms of the disease with prognostic and therapeutic implications. Currently, conventional cytogenetics (CC) and fluorescence in situ hybridization (FISH) techniques are commonly used to detect the chromosomal abnormalities in MDS. In this retrospective study, an effort is made to evaluate the merit of concomitant CC and FISH analyses for prognostic scoring in MDS. MethodsA cohort of 86 MDS patients with both CC and FISH results were analyzed. The FISH technique focused on chromosomes 5, 7, 8 and 20. Data was collected on patient age, sex, WHO (World Health Organization) classification, CC and FISH findings, histologic transformation and transfusion dependence. For each patient, IPSS scores were calculated based on either CC (IPSS-CC) or FISH (IPSS-FISH). A weighted kappa statistic was calculated to measure agreement between the CC and FISH methods. Fisher's Exact tests were performed to measure the association between concordance/discordance in scores while survival time for concordant and discordant scores was calculated using Kaplan-Meier estimation and compared using a log-rank test. A significance test of 0.05 was assumed for all comparisons. ResultsA cohort of 86 MDS patients with 64% male and median age of 68.5 years at diagnosis was studied. According to the WHO classification, 34% of the patients had refractory anemia (RA), 11% had refractory anemia with ringed sideroblasts (RARS), 13% had refractory anemia with excess blasts –1 (RAEB-1), 20% had refractory anemia with excess blasts –2 (RAEB-2) and 20% had refractory cytopenia with multilineage dysplasia (RCMD).Of the 86 patients, 74 had concordant IPSS-CC and IPSS-FISH scores. The p-value for weighted kappa statistic was 0.87 with 95% confidence interval (0.80, 0.94) indicating that there is strong agreement beyond chance between the IPSS-CC and IPSS-FISH. Of the remaining 12 patients with discordant scores, 9 had IPSS-CC > IPSS-FISH while 3 patients (eliminated from comparative analysis for small size) had IPSS-FISH > IPSS-CC. Of the several variables compared between the concordant and discordant groups, only WHO subgroup turned out to be statistically significant. Concordant scores were significantly more likely to be in the RA (37%) and RCMD (23%) cohort, while all discordant patients were in the RAEB-1 (43%), RAEB-2 (29%), and RARS (14%) subgroups (p=0.004).The 9 discordant patients were compared to 27 patients with concordant scores belonging to WHO subgroups RAEB-1, RAEB-2 and RARS. The prognostic variables for comparison were patient survival, leukemic transformation and transfusion dependence. 33% of the discordant patients had leukemic transformation as compared to 11% of concordant patients (p=0.15). Although the difference was large, the sample size was too small to be statistically significant. ConclusionCurrently CC and FISH analyses are both being employed to detect the chromosomal abnormalities in MDS patients. This retrospective study indicates that there is statistically significant concordance between cytogenetic and FISH analyses. Conceivably, doing one or other test would most likely be sufficient for prognostic scoring of the vast majority of MDS patients especially those in RA and RCMD subclasses. The discordant scores are significantly more likely to occur in the RAEB-1, RAEB-2 and RARS classes hence justifying this subgroup of patients as potential candidates for both CC and FISH analyses. In our 9 patients with discordant scores (IPSS-CC > IPSS-FISH), a trend was observed towards higher leukemic transformation when compared to WHO subgroup matched concordant patients. While further validation of the current findings by larger prospective studies is awaited, this study unravels the MDS patient sub-population where CC and FISH could be complementary and not superfluous. DisclosuresNo relevant conflicts of interest to declare.

Conventional and Molecular Cytogenetic Responses in Patients with Myelodysplastic Syndrome (MDS) with Deletion 5q Treated with Lenalidomide.

Abstract 1746Poster Board I-772 Background and objectivesInterstitial deletions of the long arm of chromosome 5 - del (5q) - are the most common chromosomal abnormalities in MDS. Del (5q) is detected in 14 % of the patients. Lenalidomide induces complete cytogenetic remissions and abolishes transfusion dependence in a significant proportion of these patients, apparently correlated with the malignant clone suppression. The objectives of this study were to assess clinical and cytogenetic responses to Lenalidomide and to establish the relationship between the number of 5q deleted cells detected by conventional cytogenetics (CG) and by fluorescence in situ hybridization (FISH) previously and after treatment. Due to its larger sensitivity, the FISH technique might be able to predict an eventual loss of response to treatment. Patients and methodsBone marrow samples were collected at diagnosis and every 6 months from the beginning of treatment with lenalidomide. CG and FISH (with LSI EGR1 (5q31)SO/D5S23, D5S721 (5p15.2) probe, from Vysis) studies were performed in all samples, even in the cases that a complete cytogenetic remission was confirmed by karyotype during the treatment follow-up. ResultsWe have treated 9 transfusion dependent MDS patients with lenalidomide, with a median age of 76 years (45-92). Four of the patients had one additional cytogenetic anomaly besides 5q deletion. Due to high clinical and cytological suspicion, one of the patients had a trisomy 9 by karyotype (2/20 metaphase) and was diagnosed of del (5q) by FISH, with a low clonal load (9,4% of deleted nuclei). Six patients were low IPSS risk, while 2 were Int-1 and one was Int-2 risk IPSS. All patients achieved complete erythroid response and transfusion independence (in a median of 8 weeks of treatment) and are still in response. Treatment was stopped in two patients after achieving complete hematologic response, maintaining normal hemoglobine levels for > 24 months. Five patients achieved cytogenetic remission, but 2 of them relapsed in a latter phase of the treatment (25 and 30 months). FISH analysis could not predict cytogenetic relapse in these two cases. ConclusionsOur results support the established high efficacy of lenalidomide in MDS with del (5q). In our experience a single additional cytogenetic anomaly did not compromise hematologic response. Moreover, in our study FISH analysis did not bring additional information to CG analysis in prediction of response and follow-up in the majority of the cases. DisclosuresNo relevant conflicts of interest to declare.

Use of single nucleotide polymorphism (SNP) array karyotyping to detect clonal chromosomal abnormalities in myelodysplastic syndrome (MDS) and refractory anemia with ringed sideroblasts (RARS)

7090 Background: The current standard for identification of chromosomal abnormalities in MDS is metaphase cytogenetics (MC). However, this technique has low resolution and requires bone marrow samp...

Acute Myelomonocytic Leukemia with Dysplastic Bone Marrow Eosinophils Showing t(5; 17)(q13;q11) and a Secondary Chromosomal Aberration, inv(16)(p13q22)

inv(16)(p13q22) is associated with de novo acute myelomonocytic leukemia with dysplastic bone marrow eosinophils (AMML Eo), which has a relatively favorable clinical course with a longer remission duration and better survival prospects. On the other hand, t(5; 17)(q13;q11), although relatively rare, has been reported to be a component of complex chromosomal abnormalities in myelodysplastic syndromes and secondary acute myeloid leukemia (AML). We treated a 29-year-old woman with the first reported case of de novo AMML Eo with inv(16)(p13q22) in addition to t(5; 17)(q13;q11). Although she attained complete remission (CR) immediately after induction therapy, the disease recurred 1 year after the completion of consolidation therapies. She underwent HLA-matched unrelated allogeneic bone marrow transplantation (UBMT), together with a myeloablative conditioning regimen, after achieving a second CR and has survived without a recurrence for more than 24 months since UBMT. In general, certain secondary chromosomal abnormalities are associated with the phenotype of the disease, which retains its essential biologic characteristics established by the primary abnormality. Accordingly, the primary nature of the leukemic cells in this case differs from the findings for core-binding factor AML with inv(16)(p13q22). We believe this report is the first of de novo AMML Eo with t(5; 17)(q13;q11) showing as a secondary chromosomal aberration with inv(16)(p13q22).

Point Mutations in Two EVI1 Zn Fingers Abolish EVI1-GATA1 Interaction and Allow Erythroid Differentiation of Murine Bone Marrow Cells

EVI1 is an aggressive nuclear oncoprotein deregulated by recurring chromosomal abnormalities in myelodysplastic syndrome (MDS). The expression of the corresponding gene is a very poor prognostic marker for MDS patients and is associated with severe defects of the erythroid lineage. We have recently shown that the constitutive expression of EVI1 in murine bone marrow results in a fatal disease with features characteristic of MDS, including anemia, dyserythropoiesis, and dysmegakaryopoiesis. These lineages are regulated by the DNA-binding transcription factor GATA1. EVI1 has two zinc finger domains containing seven motifs at the N terminus and three motifs at the C terminus. Supported by results of assays utilizing synthetic DNA promoters, it was earlier proposed that erythroid-lineage repression by EVI1 is based on the ability of this protein to compete with GATA1 for DNA-binding sites, resulting in repression of gene activation by GATA1. Here, however, we show that EVI1 is unable to bind to classic GATA1 sites. To understand the mechanism utilized by EVI1 to repress erythropoiesis, we used a combination of biochemical assays, mutation analyses, and in vitro bone marrow differentiation. The results indicate that EVI1 interacts directly with the GATA1 protein rather than the DNA sequence. We further show that this protein-protein interaction blocks efficient recognition or binding to DNA by GATA1. Point mutations that disrupt the geometry of two zinc fingers of EVI1 abolish the protein-protein interaction, leading to normal erythroid differentiation of normal murine bone marrow in vitro.

Alterações cromossômicas em síndrome mielodisplásica

O estudo das alterações cromossômicas em síndrome mielodisplásica (SMD) é de extrema importância porque auxilia no diagnóstico, prognóstico, classificação, acompanhamento evolutivo, escolha terapêutica e melhor entendimento da biologia da doença. As alterações cromossômicas são observadas em 30% a 50% dos casos de SMD primária, ao diagnóstico, e entre 80% e 90% das secundárias. Os cromossomos 5, 7, 8, 11, 13, 17, 20, 21 e X são os mais freqüentemente envolvidos. As alterações cromossômicas são variáveis independentes de valor prognóstico e correlacionam-se com o curso clínico da doença e com a transformação. Destacam-se como indicativos de bom prognóstico: cariótipo normal, nulissomia Y, del(5q), del(12p), del(11q) e del(20q) isoladas; como de prognóstico intermediário: trissomia 8, rearranjos envolvendo 3q21q26, translocações 11q, del(17p), trissomia 18 e trissomia 19; e como de prognóstico desfavorável: cariótipo complexo, monossomia 7, del(7q) e i(17q). Em relação à classificação, a Organização Mundial da Saúde tornou obrigatória a análise do cariótipo para a completa avaliação da SMD recém diagnosticada e definiu a síndrome 5q- como entidade específica. No tocante ao acompanhamento, alterações podem ser detectadas evolutivamente, definindo agressividade ou transformação da doença. Atualmente alguns tratamentos já são delineados com base nas alterações cromossômicas. Não se sabe se as alterações cromossômicas detectadas em SMD são eventos iniciais que levam ao desenvolvimento da doença (causa) ou se são apenas fenômenos secundários (conseqüência). De qualquer forma, elas apontam que, em nível molecular, há uma série de aspectos ainda por serem entendidos.

Myelodysplastic syndromes with isolated deletion of the long arm of the chromosome X as a sole cytogenetic change

Deletions of Xq are extremely rare events in myelodysplastic syndromes (MDS) patients and were previously described in five patients, in two of them as a sole chromosome abnormality. We found isolated del(Xq) in 3 of 127 MDS patients with clonal chromosome changes. Detailed analysis of clinical and morphological data of presented and previously published cases indicates the following: (1) del(X)(q24) and del(X)(q13) are nonrandom chromosomal abnormalities in MDS; (2) MDS with deletions of Xq affect exclusively females ages 46–65; and (3) deletions of Xq are associated with refractory anemia with excess blasts (RAEB) and indicate an unfavorable prognosis.

Comprehensive Promoter Methylation Analysis of Genes on 5q31.1 in MDS and AML.

Interstitial deletion or loss of chromosome 5 is a frequent chromosomal abnormality in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Previous studies uncovered a 1.5–2Mb region on 5q31.1 to be a common deleted region (CDR) in those diseases. It is believed that there exists one or more of critical tumor suppressor gene(s) in this CDR. Mutation analysis, however, failed to identify target gene(s). With a hypothesis that epigenetic silencing may play a significant role as a “second hit” in MDS/AML, we performed a comprehensive methylation analysis of genes in this CDR on 5q31.1. Among 25 genes that are located in this region (SPOCK-AF116688, covering CDR determined by Horrigan et al. Blood 2000 and Fang et al. Genomics 2000), 14 genes had promoter regions in CpG islands that were therefore susceptible to methylation dependent silencing. Methylation status of multiple CpG sites in promoter regions of these those 14 genes (SPOCK, HNRPA1, PKD2L2, C5orf5, KIF20A, GFRA3, CDC25C, C5orf6, JMJD1B, C5orf19, EGR1, ETF1, HSPA9B, CTNNA1, SIL1) was analyzed using bisulfite-PCR followed by pyrosequencing. Analyzed samples were 13 leukemic cell lines as well as peripheral blood mononuclear cells from 5 patients with AML with 5q deletion (5qAML), 9 AML without 5q deletion (non-5qAML), 17 MDS with 5q deletion (5qMDS), 9 MDS without 5q deletion (non-5qMDS) and 10 individuals without leukemia. Significant hypermethylation (>15% by pyrosequencing) was observed in 5 genes in patient samples (SPOCK, PKD2L2, GFRA3, HSPA9B, and ETF1, see Table 1) and 3 other genes in cell lines only (CDC25C, C5orf19, CTNNA1). In 5qMDS, the most frequently methylated genes were ETF1 (5/17), GFRA3 (4/17), and HSPA9B (11/17), but HSPA9B also showed methylation in normal blood. We then evaluated the expression levels of 5 genes that showed significant methylation in patients' samples using quantitative RT-PCR. SPOCK, PKD2L2, GFRA3 were not expressed in normal PBMC. HSPA9B expression levels did not correlate with the degree of methylation. On the other hand, ETF1 expression was inversely correlated with the degree of methylation. ETF1 is a eukaryotic translation termination factor, which plays a role in recognizing stop codon in mRNA and terminates translation. Based on specific hypermethylation and silencing in 5qMDS, we propose ETF1 as a candidate tumor suppressor gene in the 5q31.1 region. Functional analysis of ETF1 in leukemic cells is underway.Frequency of promoter CpG sites methylationSamplesNSPOCKPKD2L2GFRA3HSPA9BETF1Cell lines1362%46%77%38%0%5qAML50%0%0%20%0%Non-5qAML933%0%0%0%0%5qMDS176%6%24%65%29%Non-5qMDS911%11%33%33%11%Normal100%0%0%90%0%

Clinical features and chromosomal abnormality in Myelodysplastic Syndrome of the Refractory Anaemia Subtype

Carmen Silvia Passos Lima, MD, PhDDepartamento de Clinica Medica – Faculdade de Ciencias Medicas da Universidade Estadual de CampinasRua Alexandre Fleming no 181 – Cidade Universitaria “Zeferino Vaz”Distrito de Barao Geraldo13083-970 – Campinas-SP – BrasilTel.: (19) 3788 7098; Fax: (19) 3289 4107 – e-mail: carmenl@fcm.unicamp.br

Molecular mechanisms of myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a family of clonal disorders of hematopoietic stem cells that are characterized by ineffective hematopoiesis and susceptibility to acute myelogenous leukemias and are shown to be strikingly refractory to current therapeutic modalities. A substantial proportion of these complex diseases arises in the setting of exposures to environmental or occupational toxins, including cytotoxic therapy for a prior malignancy or other disorder. The conversion of a normal stem cell into a preleukemic and ultimately leukemic state is a multistep process requiring the accumulation of a number of genetic lesions. At the genomic level, MDS is typified by losses and translocations involving certain key gene segments, with disruption of the normal structure and function of genes that control the balance of proliferation and differentiation of hematopoietic precursors. More than half of the chromosomal abnormalities in MDS comprise deletions of chromosomes 5, 7, 11, 12, 13 and 20. This evidence suggests that as yet unidentified tumor-suppressor genes should have important roles in the molecular mechanisms of MDS. Further molecular approaches to such genetic lesions will identify the relevant tumor-suppressor genes. Over the past years, major signal transduction molecules have been identified and their genetic alterations have been extensively analyzed in both MDS and leukemias. These include receptors for growth factors, RAS signaling molecules, cell cycle regulators and transcription factors. Notable among them are transcription factors that regulate both proliferation and differentiation of hematopoietic stem cells. The disruption of the normal flow of the signal transduction pathways involving these molecules translates into ineffective multilineage hematopoiesis and bone marrow failure. Therefore, MDS provides a fertile testing ground on which we could study the molecular dissection implicated in the multistep leukemogenesis.

Molecular pathogenesis of MDS.

Myelodysplastic syndromes (MDS) are considered to be a family of clonal disorders of hematopoietic stem cells that are characterized by ineffective hematopoiesis and susceptibility to acute myelogenous leukemias, and are shown to be strikingly refractory to current therapeutic modalities. A substantial proportion of these complex diseases arise in the setting of exposures to environmental or occupational toxins, including cytotoxic therapy for a prior malignancy or other disorder. The conversion of a normal stem cell into a preleukemic and ultimately leukemic state is a multistep process requiring the accumulation of a number of genetic lesions. On the genomic level, MDS is typified by losses and translocations involving certain key gene segments, with disruption of the normal structure and function of genes that control the balance of proliferation and differentiation of hematopoietic precursors. More than a half of the chromosomal abnormalities in MDS comprise deletions of chromosomes 5, 7, 11, 12, 13 and 20. This evidence suggests that as yet unidentified tumor suppressor genes should have important roles in the molecular mechanisms of MDS. Further molecular approaches to such genetic lesions will identify the relevant tumor suppressor genes. Over the past years, major signal transduction molecules were identified and their genetic alterations were extensively analyzed in MDS as well as leukemias. These include receptors for growth factors, RAS signaling molecules, cell cycle regulators, and transcription factors. Among them, notable is transcription factors that regulate both proliferation and differentiation of hematopoitic stem cells. The disruption of the normal flow of the signal transduction pathways involving these molecules translates into ineffective multilineage hematopoiesis and bone marrow failure. Therefore, MDS provides a fertile testing ground on which we could study the molecular dissection implicated in the multistep leukemogenesis.

Der(1)t(1;19)(p12;p11): A new nonrandom chromosomal abnormality in myelodysplastic syndrome

The der(1)t(1;19)(p12;p11) has not been previously reported in myelodysplastic syndrome (MDS). Fluorescence in situ hybridization (FISH) using chromosome 1- and chromosome 19-specific probes, performed on the bone marrow (BM) cells of this patient confirmed the initial karyotype, i.e., 47,XY,+der(1) t(1;19)(p12;p11).

5 Primary chromosomal abnormalities in myelodysplastic syndromes (MDS)

5 Primary chromosomal abnormalities in myelodysplastic syndromes (MDS)