Hypoplastic form of myelodysplastic neoplasm.

BACKGROUND: Hypoplastic myelodysplastic neoplasm (MDS-h) is a rare hematopoietic disorder characterized by peripheral cytopenia, hypoplasia (cellularity ≤ 25%) and dysplastic changes in the bone marrow. Compared to normo- /hypercellular MDS, in addition to hypocellularity, MDS-h patients have more profound neutropenia and thrombocytopenia, a lower percentage of blasts, and less frequent abnormal karyotype. It is difficult to distinguish MDS-h from aplastic anemia in differential diagnosis. Abnormal karyotype is found in 15-50% of MDS-h patients and the most common chromosomal aberrations include -5/del (5q), -7/del (7q), +8, 17pLOH, del (20q), UPD at 4q, 11q, 13q, and 14q. Approximately 35% of MDS-h patients harbour somatic mutations that are most often detected in PIGA, TET2, DNMT3A, RUNX1, NPM1, ASXL1, STAG2, and APC genes. An autoimmune destruction of hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) mediated by abnormally activated T cells plays a key role in the pathophysiology of MDS-h. Expanded T cells overproduce proinflammatory cytokines (IFN- g and TNF-a), which inhibit proliferation and induce apoptosis of HSC/HPCs. The antigens that trigger the immune response are not known, but potential candidates have been suggested such as WT1 protein and HLA class I molecules. MDS-h does not represent a phenotypically homogeneous subtype of MDS, but rather it is a mixed entity comprising both patients showing features similar to myelodysplastic neoplasm and patients with features of non-malignant bone marrow failure. Determining the prevailing phenotype in MDS-h is important for choosing the optimal treatment and prognosis prediction. PURPOSE: The aim of this article is to point out an interesting hypoplastic MDS, the diagnosis of which is difficult, and to provide an overview of its main clinical-pathological features, genetic background, and mechanisms of aberrant immune response.

Microarray profiling defines circulating microRNAs associated with myelodysplastic syndromes.

Circulating microRNAs (miRNAs) are non-coding RNAs secreted into body fluids, and aberrant levels of these miRNAs correlate with diseases of various origins, making them highly potential clinical biomarkers. We investigated the spectrum of circulating miRNAs in the plasma of myelodysplastic syndrome (MDS) patients to identify miRNAs showing discriminatory levels in the patients with different prognosis. Plasma samples were analyzed with microarrays to define miRNA profiles, and the deregulated miRNAs were further studied using droplet digital PCR. With regard to the prognosis, the levels of miR-27a-3p, miR-150-5p, miR-199a-5p, miR-223-3p and miR-451a were reduced in higher-risk MDS. Multivariate analysis indicated miR-451a level as an independent predictor of progression-free survival (HR = 0.072, P = 0.006) and revealed a significant association of miR-223-3p level with overall survival (HR = 0.039, P = .032). Our data demonstrate that plasma levels of specific miRNAs are associated with MDS patient outcome and may add information beyond the currently used scoring systems.

Up-regulation of ribosomal genes is associated with a poor response to azacitidine in myelodysplasia and related neoplasms.

Azacitidine (AZA) is a hypomethylating drug used to treat disorders associated with myelodysplasia and related neoplasms. Approximately 50 % of patients do not respond to AZA and have very poor outcomes. There is thus great interest in identifying predictive biomarkers for AZA responsiveness. We searched for specific genes whose expression level was associated with response status. Using microarrays, we analyzed gene expression patterns in bone marrow CD34+ cells in serial samples from 32 patients with myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia with myelodysplasia-related changes before and during the AZA therapy. At baseline, a comparison of the responders and non-responders showed 52 differentially expressed genes (P < 0.01). Functional annotation of the deregulated genes revealed categories primarily related to ribosomes and pathways associated with proliferation. The expression level of RPL28 correlated with overall survival. We identified altered expression in 167 genes in responders, 26 genes in non-responders with stable disease, and 13 genes in non-responders with disease progression using paired t test of expression levels in patients before and during treatment. Our data indicate that AZA treatment failure is associated with the up-regulation of ribosomal genes/pathways that are likely related to intensive proteosynthesis in proliferative/neoplastic cells of non-responders.

5-azacitidine in aggressive myelodysplastic syndromes regulates chromatin structure at PU.1 gene and cell differentiation capacity.

Epigenetic 5-azacitidine (AZA) therapy of high-risk myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) represents a promising, albeit not fully understood, approach. Hematopoietic transcription factor PU.1 is dynamically regulated by upstream regulatory element (URE), whose deletion causes downregulation of PU.1 leading to AML in mouse. In this study a significant group of the high-risk MDS patients, as well as MDS cell lines, displayed downregulation of PU.1 expression within CD34+ cells, which was associated with DNA methylation of the URE. AZA treatment in vitro significantly demethylated URE, leading to upregulation of PU.1 followed by derepression of its transcriptional targets and onset of myeloid differentiation. Addition of colony-stimulating factors (CSFs; granulocyte-CSF, granulocyte-macrophage-CSF and macrophage-CSF) modulated AZA-mediated effects on reprogramming of histone modifications at the URE and cell differentiation outcome. Our data collectively support the importance of modifying the URE chromatin structure as a regulatory mechanism of AZA-mediated activation of PU.1 and induction of the myeloid program in MDS.

Monitoring of methylation changes in 9p21 region in patients with myelodysplastic syndromes and acute myeloid leukemia.

Epigenetic de novo methylation of CpG islands is an important event in malignant transformation. Two genes are frequently methylated: cyclin-dependent kinase inhibitor 2B (CDKN2B) and cyclin-dependent kinase inhibitor 2A (CDKN2A). In our study methylation of these genes was studied in 63 patients with myelodysplastic syndromes (MDS), 2 with myelodysplastic/myeloproliferative neoplasms (MDS/MPN) and 13 with acute myeloid leukemia (AML). Five patients were monitored during 5-azacytidine treatment. Twenty-six healthy donors were tested in a control group. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) method with all associated techniques was used for detection. Aberrant methylation was present in the CDKN2A gene in 38% and in the CDKN2B gene in 77% of the patients in MDS group. The level of methylation was higher in the group of AML patients - 77% in CDKN2A gene and 100% in CDKN2B gene. In MDS patients, an aberrant methylation was associated with a tendency to disease progression towards more advanced forms according to the World Health Organization (WHO) classification and the International Prognostic Scoring System (IPSS). Significant differences in methylation level were observed between early and advanced forms of MDS in CDKN2B gene (P value < 0.05) but not for CDKN2A gene. The trend of methylation in patients treated with azacitidine was analyzed in CDKN2B gene and correlated with the course of the disease. Increased methylation was connected with disease progression. We concluded that the methylation level of CDKN2B gene might be used as a marker of leukemic transformation in MDS. Our study indicates the role of hypermethylation as an important event in the progression of MDS to AML.

Differential gene expression of bone marrow CD34+ cells in early and advanced myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a hematopoietic stem cell disorder characterized by ineffective hematopoiesis and dysplasia in one or more blood cell lines. Because it often progress to poor outcome stages or acute leukemia we searched for candidate genes associated with disease progression. Using microarrays we performed gene expression profiling in CD34+ cells of 4 early and 4 advanced MDS patients and identified 286 significantly differentially expressed genes between these two categories. Out of these, 136 genes were up-regulated and 150 down-regulated in early MDS compared to advanced MDS. Using clustering analysis those two patient categories were clearly differentiated. Further, we selected three genes (ADAM8, BIRC5, MPL) for gene expression validation by qRT-PCR in an additional set of 29 MDS and sAML patients. We confirmed decreasing trend for BIRC5 expression from early to advanced stages of MDS, with the lowest levels in sAML patients. On the contrary, higher ADAM8 and MPL expression was observed in most advanced MDS patients compared to the early MDS patients. Association between gene expression levels and bone marrow blast proportion was tested, but only BIRC5 expression showed negative correlation (r=-0.83 at p<0.001). This study demonstrates stage-specific expression of some genes that may have potential prognostic significance.

[Cell clonality in myelodysplastic syndrome]. / Bunecná klonalita u myelodysplastického syndromu.

INTRODUCTION: Myelodysplastic syndromes (MDS) are classified as oncohematologic, clonal diseases. However, some MDS subtypes lack malignant clinical features. Literary data on the clonality of blood cells in MDS are controversial. Therefore we examined the clonality in the group of our own MDS patients. We were especially interested in the comparison of the clonality of myeloid and lymphoid cell line-ages. METHODS AND MATERIAL: Using X-chromosome inactivation methods based on polymorphism in human androgen receptor gene (HUMARA), iduronate sulphatase gene (IDS) and protein p55 gene, we assessed the clonality of granulocytes, monocytes and T-lymphocytes of 49 female patients with MDS and 12 control women of various ages. RESULTS: Though the results were heterogeneous, certain trends were observed: most frequently monoclonality of monocytes was found (51%), granulocytes were monoclonal in 33% of cases, monoclonal T lymphocytes only in 8% of cases. There was no case with monoclonal T lymphocytes and simultaneously polyclonal myeloid cell fractions. Results differ in relation to the subtypes of MDS. Among controls we observed monoclonality of myeloid cells in one case of an old lady. WE CONCLUDE: The results confirm the presence of malignant clone of myeloid cells in MDS, which can be of variable size. The examination of the clonality in MDS can be significant for the therapy decision. Monoclonal myeloid cells are found least frequently in refractory anemia, most frequently in RAEB-T. Monoclonality of cells in sideroblastic anemia is interesting. The prevailing polyclonality of T lymphocytes in MDS can be explained by the presence of a long-lived cell population originating from the period before the development of MDS. The role of the polyclonal memory T cells in the antitumor immunity in MDS is discussed.

Genes involved in the destruction of leukaemic cells by induced photosensitivity.

Gene expression changes were observed in the HEL and HL-60 cell lines after the stimulation of protoporphyrin IX synthesis by ALA administration and photodynamic process induction. Isolated ribonucleic acids were radiolabelled by reverse transcription, and the cDNA obtained was hybridized to membrane macroarrays (Clontech 7742-1) containing 588 gene probes. Besides changes in the activity of genes supposed to be involved in the programmed cell death and DNA reparation processes, increased or diminished transcription activity was also observed in several other genes; the reason for this phenomenon was not clear. The activation of programmed-cell-death genes appeared after the ALA load application, indicating the toxic effect of ALA. The gene expression changes observed in the two cell lines differed substantially, only a few of them were common for both cell lines.

Clonal stability of blood cell lineages indicated by X-chromosomal transcriptional polymorphism.

The idea that stem cells oscillate between a state of activity and dormancy, thereby giving rise to differentiating progeny either randomly or in orderly clonal succession, has important implications for understanding normal hematopoiesis and blood cell dyscrasias. The degree of clonal stability in individuals also has practical implications for the evaluation of clonal lymphomyeloproliferative diseases. To evaluate the clonality pattern of the different types of blood cells as a function of time we have validated the applicability, sensitivity, and reproducibility of a thermostable ligase reaction to detect transcripts of the G6PD allele on the active X-chromosome in normal heterozygous females. While the ratio of the two X-chromosome-derived allelic transcripts varied widely among hemopoietic tissues in a given individual, this allelic ratio was virtually identical in all types of mature myeloid and lymphoid cells. Longitudinal studies indicated constancy of the G6PD allelic ratio in blood cells over a 912-d period of observation in healthy females. The individual variability observed in this allelic ratio suggests that the progeny of a relatively small number of original embryonic hemopoietic stem cells, approximately eight, contribute to the sustained production of all types of blood cells in healthy individuals.

A novel clonality assay based on transcriptional polymorphism of X chromosome gene p55.

The clonal nature of cell populations in malignant and myeloproliferative disorders can be determined in female subjects by random-inactivation assays of the X chromosome. Assays utilizing either expression of the G6PD isozymes or DNA-methylation differences between the active and inactive X chromosomes have significant short-comings. We developed a test based on nucleotide #1311 exonic polymorphism of G6PD that allows detection of clonality by determining the transcriptional polymorphism of the active X chromosome using a reverse transcription-polymerase chain reaction-ligase detection reaction (rtPCR-LDR). Since only 18% of females in the United States are informative (heterozygous) for this chromosome with this assay, we searched for other exonic X chromosome polymorphisms. Concentrating on a discrepancy (G or T at cDNA #358) of published sequences of ubiquitously expressed gene, palmitoylated membrane protein for p55, we confirmed this conservative polymorphism at the cDNA level. To detect the genotype of this polymorphism, we established the intron/exon boundary of the first 5' exons and determined the whole sequence of the second intron. We found that the polymorphic site is at the third exon, nine nucleotides downstream from the 294-bp second intron. This close proximity to the intron necessitated the development of a separate PCR-LDR reaction with oligonucleotides for cDNA and genomic DNA. Furthermore, we determined that the p55 gene is subject to X chromosome inactivation. Based on these observations, we developed a novel p55 clonality assay that is reproducible, quantitative, and very sensitive. Screening of 37 randomly selected healthy females of Caucasian, African-American, and Asian origin revealed that 38% of females are informative when this assay is used. We demonstrate a multiple crossover between the G6PD and the p55 polymorphisms (separated by approximately 200 kb), suggesting that these two polymorphisms are in linkage disequilibrium; thus, approximately 50% of female subjects are informative for clonality studies using the two assays.

Clonality in juvenile chronic myelogenous leukemia.

Juvenile chronic myelogenous leukemia (JCML) is a myeloproliferative disease in which morbidity and mortality are primarily caused by nonhematopoietic organ failure from myelomonocytic infiltration or by failure of the normal bone marrow. Morphologic evidence of maturation arrest, karyotypic abnormalities, and progression to blast crisis are infrequent events. Viral infections and other reactive processes can initially mimic the clinical course of JCML, creating diagnostic problems. Because of the rarity of JCML and technical limitations, formal clonality studies have not been reported previously. Nine female JCML patients were identified by clinical criteria, characteristic 'spontaneous' in vitro cell growth, and negative cultures and titers for various viral agents. Peripheral blood and bone marrow samples were obtained at the time of diagnosis for cell separation and RNA and DNA isolation. To assess clonality, X-chromosome inactivation patterns were evaluated using three different, recently developed polymerase chain reaction-based clonality assays. All nine female JCML patients showed evidence for monoclonal origin of mononuclear cells at the time of diagnosis. Cell separation studies further traced the monoclonal origin back to at least the most primitive myeloid progenitor cell. Reversion to a polyclonal state was demonstrated after bone marrow transplant and also in one patient following treatment with 13-cis retinoic acid. This demonstration of clonality in JCML delineates it from the reactive processes and provides a basis for molecular genetic strategies to identify causally associated mutations.

Clonal hematopoiesis and acquired thalassemia in common variable immunodeficiency.

BACKGROUND: Common variable immunodeficiency (CVID) is defined by hypogammaglobulinemia and increased susceptibility to infections. The gene defect responsible for CVID remains unknown. METHODS: During the course of their CVID disease, a female and three male patients developed microcytic anemia. The investigation of this anemia forms the basis for this report. RESULTS: Reticulocyte globin chain synthesis studies revealed the abnormal alpha/beta ratios that are pathognomonic of thalassemia. Through transcriptional analysis of the glucose-6-phosphate-dehydrogenase (G6PD) locus of the active X-chromosome in blood cells, we determined that the female patient has clonal reticulocytes, platelets, granulocytes, and B and T lymphocytes. CONCLUSIONS: The simultaneous presence of globin synthesis abnormalities and panhypogammaglobulinemia suggests that a common insult at the stem cell level could contribute to the development of CVID and acquired thalassemia.