Nonsteroidal anti-inflammatory drugs as a targeted therapy for bone marrow failure in Ghosal hematodiaphyseal dysplasia.

Advances in genomic diagnostics hold promise for improved care of rare hematologic diseases. Here, we describe a novel targeted therapeutic approach for Ghosal hematodiaphyseal dysplasia, an autosomal recessive disease characterized by severe normocytic anemia and bone abnormalities due to loss-of-function mutations in thromboxane A synthase 1 (TBXAS1). TBXAS1 metabolizes prostaglandin H2 (PGH2), a cyclooxygenase (COX) product of arachidonic acid, into thromboxane A2. Loss-of-function mutations in TBXAS result in an increase in PGH2 availability for other PG synthases. The current treatment for Ghosal hematodiaphyseal dysplasia syndrome consists of corticosteroids. We hypothesize that nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit COX-1 and COX-2, could ameliorate the effects of TBXAS1 loss and improve hematologic function by reducing prostaglandin formation. We treated 2 patients with Ghosal hematodiaphyseal dysplasia syndrome, an adult and a child, with standard doses of NSAIDs (aspirin or ibuprofen). Both patients had rapid improvements concerning hematologic parameters and inflammatory markers without adverse events. Mass spectrometry analysis demonstrated that urinary PG metabolites were increased along with proinflammatory lipoxygenase (LOX) products 5-hydroxyeicosatetraenoic acid and leukotriene E4. Our data show that NSAIDs at standard doses surprisingly reduced both COX and LOX products, leading to the resolution of cytopenia, and should be considered for first-line treatment for Ghosal hematodiaphyseal dysplasia syndrome.

Clinical Application for Diagnosis of Myelodysplatic/Myeloproliferative Neoplasm with Ring Sideroblasts and Thrombocytosis.

BACKGROUND: Myelodysplastic/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS/ MPN-RS-T) was newly introduced as a full entity in the 2016 revision of the WHO classification. In this study, we investigated the morphologic, laboratory, and clinical features of MDS/MPN-RS-T. METHODS: We reviewed the bone marrow and genetic studies of patients whose diagnoses were coded as "refractory anemia with ring sideroblasts (RARS)" or "MDS/MPN, unclassifiable" between January 2008 and April 2018. RESULTS: A total of 8 cases fulfilled the criteria for a diagnosis of MDS/MPN-RS-T. All of them had no specific symptoms. Half of the cases had less than 450 × 109/L platelet counts by an automated hematology analyzer; however, all platelet counts exceeded 450 × 109/L when performed manually. JAK2 mutation tests were performed in 7 cases, and a heterozygous mutation was detected in 1 case. SF3B1 mutations were present in 3 of the 4 cases tested. CONCLUSIONS: When RARS is suspected in patients without thrombocytopenia, manual platelet counts should be performed. For patients with suspected essential thrombocythemia, RS evaluation through careful observation of an iron-stained slide is crucial. Since the independent evaluation of RS was reflected in the revised classification, the ambiguous disease classification becomes clearer and more consistent.

Identification of circular RNAs as novel biomarkers and potentially functional competing endogenous RNA network for myelodysplastic syndrome patients.

Circular RNAs (circRNAs) have been identified to exert vital biological functions and can be used as new biomarkers in a number of tumors. However, little is known about the functions of circRNAs in myelodysplastic syndrome (MDS). Here, we aimed to investigate circRNA expression profiles and to investigate the functional and clinical value of circRNAs in MDS. Differential expression of circRNAs between MDS and control subjects was analyzed using circRNA arrays, in which we identified 145 upregulated circRNAs and 224 downregulated circRNAs. Validated by real-time quantitative PCR between 100 MDS patients and 20 controls, three upregulated (hsa_circRNA_100352, hsa_circRNA_104056, and hsa_circRNA_104634) and three downregulated (hsa_circRNA_103846, hsa_circRNA_102817, and hsa_circRNA_102526) circRNAs matched the arrays. The receiver operating characteristic curve analysis of these circRNAs showed that the area under the curve was 0.7266, 0.8676, 0.7349, 0.7091, 0.8806, and 0.7472, respectively. Kaplan-Meier survival analysis showed that only hsa_circRNA_100352, hsa_circRNA_104056, and hsa_circRNA_102817 were significantly associated with overall survival. Furthermore, we generated a competing endogenous RNA network focused on hsa_circRNA_100352, hsa_circRNA_104056, and hsa_circRNA_102817. Analyses using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes showed that the three circRNAs were linked with some important cancer-related functions and pathways.

Refractory anemia with ring sideroblasts (RARS) and RARS with thrombocytosis: "2019 Update on Diagnosis, Risk-stratification, and Management".

DISEASE OVERVIEW: Ring sideroblasts (RS) are erythroid precursors with abnormal perinuclear mitochondrial iron accumulation. Two myeloid neoplasms defined by the presence of RS, include refractory anemia with ring sideroblasts (RARS), now classified under myelodysplastic syndromes with RS (MDS-RS) and RARS with thrombocytosis (RARS-T); now called myelodysplastic/myeloproliferative neoplasm with RS and thrombocytosis (MDS/MPN-RS-T). DIAGNOSIS: MDS-RS is a lower-risk MDS, with single or multilineage dysplasia (SLD/MLD), <5% bone marrow (BM) blasts and ≥ 15% BM RS (≥5% in the presence of SF3B1 mutations). MDS/MPN-RS-T, now a formal entity in the MDS/MPN overlap syndromes, has diagnostic features of MDS-RS-SLD, along with a platelet count ≥450 × 10(9)/L and large atypical megakaryocytes. MUTATIONS AND KARYOTYPE: Mutations in SF3B1 are seen in ≥80% of patients with MDS-RS-SLD and MDS/MPN-RS-T, and strongly correlate with the presence of BM RS; MDS/MPN-RS-T patients also demonstrate JAK2V617F, ASXL1, DNMT3A, SETBP1, and TET2 mutations. Cytogenetic abnormalities are uncommon in both. RISK STRATIFICATION: Most patients with MDS-RS-SLD are stratified into lower-risk groups by the revised-IPSS. Disease outcome in MDS/MPN-RS-T is better than that of MDS-RS-SLD, but worse than that of essential thrombocythemia. Both diseases have a low risk of leukemic transformation. TREATMENT: Anemia and iron overload are complications seen in both and are managed similar to lower-risk MDS and MPN. The advent of luspatercept, a first-in-class erythroid maturation agent will tremendously boost the ability to manage anemia. Aspirin therapy is reasonable in MDS/MPN-RS-T, especially in the presence of JAK2V617F, but the value of platelet-lowering drugs remains uncertain.

Ghosal hematodiaphyseal dysplasia with autoimmune anemia in two adult siblings.

Ghosal hematodiaphyseal dysplasia (GHDD) is an autosomal recessive inherited disorder associated with biallelic mutations in the TBXAS1 gene located on the chromosome 7q33-34, which encodes thromboxane-A-synthase. GHDD is characterized by defective hematopoiesis due to bone marrow fibrosis and metadiaphyseal dysplasia of long bones. The accurate diagnosis of this rare syndrome is critical since it reduces the need of blood transfusions by corticosteroid therapy, leading to a significant improvement in anemia and bone changes. The aim of this study is to report two adult siblings diagnosed as GHDD, who admitted with pancytopenia and treated with steroids treatment in adult hematology clinic.

Chronic steroid-response pancytopenia and increased bone density due to thromboxane synthase deficiency.

Diagnosis of bone marrow failure (BMF) disorders is challenging but essential for optimal patient management. Here, we report a young adult from nonconsanguineous parents with progressive pancytopenia since childhood, bone pain, increased bone density, and haphazard ossification replacing hematopoiesis within the bone marrow. Sequencing revealed two novel biallelic variants of unknown significance within the thromboxane A synthase gene, TBXAS1 (c.266T > C; c.989T > C), bioinformatically predicted to disrupt the protein. TBXAS1 mutations result in Ghosal hematodiaphyseal dysplasia (OMIM 231095), the autosomal recessive syndrome associated with abnormal bone structure and BMF. Identification of the genetic defect prompted steroid therapy leading to resolution of symptoms.

Refractory anemia with ring sideroblasts (RARS) and RARS with thrombocytosis (RARS-T): 2017 update on diagnosis, risk-stratification, and management.

DISEASE OVERVIEW: Ring sideroblasts (RS) are erythroid precursors with abnormal perinuclear mitochondrial iron accumulation. Two myeloid neoplasms defined by the presence of RS, include refractory anemia with ring sideroblasts (RARS), now classified under myelodysplastic syndromes with RS (MDS-RS) and RARS with thrombocytosis (RARS-T); now called myelodysplastic/myeloproliferative neoplasm with RS and thrombocytosis (MDS/MPN-RS-T). DIAGNOSIS: MDS-RS is a lower risk MDS, with single or multilineage dysplasia (SLD/MLD), <5% bone marrow (BM) blasts and ≥15% BM RS (≥5% in the presence of SF3B1 mutations). MDS/MPN-RS-T, now a formal entity in the MDS/MPN overlap syndromes, has diagnostic features of MDS-RS-SLD, along with a platelet count ≥ 450 × 10(9)/L and large atypical megakaryocytes (similar to BCR-ABL1 negative MPN). MUTATIONS AND KARYOTYPE: Mutations in SF3B1 are seen in ≥80% of patients with MDS-RS-SLD and MDS/MPN-RS-T, and strongly correlate with the presence of BM RS; MDS/MPN-RS-T patients also demonstrate JAK2V617F, ASXL1, DNMT3A, SETBP1, and TET2 mutations; with ASXL1/SETBP1 mutations adversely impacting survival. Cytogenetic abnormalities are uncommon in both diseases. RISK STRATIFICATION: Most patients with MDS-RS-SLD are stratified into lower risk groups by the revised-International Prognostic Scoring System (R-IPSS). Disease outcome in MDS/MPN-RS-T is better than that of MDS-RS-SLD, but worse than that of essential thrombocythemia. Both diseases have a low risk of leukemic TREATMENT: Anemia and iron overload are complications seen in both and are managed similar to lower risk MDS and MPN. Aspirin therapy is reasonable in MDS/MPN-RS-T, especially in the presence of JAK2V617F, but the value of platelet-lowering drugs is uncertain.

Depletion of Sf3b1 impairs proliferative capacity of hematopoietic stem cells but is not sufficient to induce myelodysplasia.

Numerous studies have recently reported mutations involving multiple components of the messenger RNA (mRNA) splicing machinery in patients with myelodysplastic syndrome (MDS). SF3B1 is mutated in 70% to 85% of refractory anemia with ringed sideroblasts (RARS) patients and is highly associated with the presence of RARS, although the pathological role of SF3B1 mutations in MDS-RARS has not been elucidated yet. Here, we analyzed the function of pre-mRNA splicing factor Sf3b1 in hematopoiesis. Sf3b1(+/-) mice maintained almost normal hematopoiesis and did not develop hematological malignancies during a long observation period. However, Sf3b1(+/-) cells had a significantly impaired capacity to reconstitute hematopoiesis in a competitive setting and exhibited some enhancement of apoptosis, but they did not show any obvious defects in differentiation. Additional depletion of Sf3b1 with shRNA in Sf3b1(+/-) hematopoietic stem cells (HSCs) severely compromised their proliferative capacity both in vitro and in vivo. Finally, we unexpectedly found no changes in the frequencies of sideroblasts in either Sf3b1(+/-) erythroblasts or cultured Sf3b1(+/-) erythroblasts expressing shRNA against Sf3b1. Our findings indicate that the level of Sf3b1 expression is critical for the proliferative capacity of HSCs, but the haploinsufficiency for Sf3b1 is not sufficient to induce a RARS-like phenotype.

Aberrant splicing of genes involved in haemoglobin synthesis and impaired terminal erythroid maturation in SF3B1 mutated refractory anaemia with ring sideroblasts.

Refractory anaemia with ring sideroblasts (RARS) is distinguished by hyperplastic inefficient erythropoiesis, aberrant mitochondrial ferritin accumulation and anaemia. Heterozygous mutations in the spliceosome gene SF3B1 are found in a majority of RARS cases. To explore the link between SF3B1 mutations and anaemia, we studied mutated RARS CD34(+) marrow cells with regard to transcriptome sequencing, splice patterns and mutational allele burden during erythroid differentiation. Transcriptome profiling during early erythroid differentiation revealed a marked up-regulation of genes involved in haemoglobin synthesis and in the oxidative phosphorylation process, and down-regulation of mitochondrial ABC transporters compared to normal bone marrow. Moreover, mis-splicing of genes involved in transcription regulation, particularly haemoglobin synthesis, was confirmed, indicating a compromised haemoglobinization during RARS erythropoiesis. In order to define the phase during which erythroid maturation of SF3B1 mutated cells is most affected, we assessed allele burden during erythroid differentiation in vitro and in vivo and found that SF3B1 mutated erythroblasts showed stable expansion until late erythroblast stage but that terminal maturation to reticulocytes was significantly reduced. In conclusion, SF3B1 mutated RARS progenitors display impaired splicing with potential downstream consequences for genes of key importance for haemoglobin synthesis and terminal erythroid differentiation.

Breakpoint heterogeneity in (2;3)(p15-23;q26) translocations involving EVI1 in myeloid hemopathies.

Several chromosomal rearrangements involving band 3q26 are known to induce EVI1 overexpression. They include inv(3)(q21q26), t(3;3)(q21;q26), t(3;21)(q26;q22) and t(3;12)(q26;p13). Translocations involving the short arm of chromosome 2 and 3q26 have been reported in more than 50 patients with myeloid disorders. However, although the breakpoints on 2p are scattered over a long segment, their distribution had only been analyzed in 9 patients. We performed fluorescent in situ hybridization with a library of BAC (Bacterial Artificial Chromosome) clones in 4 patients with t(2;3)(p15-23;q26). Our results combined with those of the 9 previously reported patients showed scattering of the breakpoints in 2 regions. A 1.08Mb region in band 2p21 encompassing the MTA3, ZFP36L2 and THADA genes was documented in 5 patients. A second region of 1.83Mb in band 2p16.1 was identified in 8 patients. Four patients showed clustering around the BCL11A gene and the remaining 4 around a long intergenic non-coding RNA, FLJ30838. These regions are characterized by the presence of regulatory sequences (CpG islands and promoters) that could be instrumental in EVI1 overexpression.

Refractory anemia with ring sideroblasts and RARS with thrombocytosis.

DISEASE OVERVIEW: Ring sideroblasts (RS) are erythroid precursors with abnormal perinuclear mitochondrial iron accumulation. Two myeloid neoplasms defined by the presence of RS, include refractory anemia with ring sideroblasts (RARS) and RARS with thrombocytosis (RARS-T). DIAGNOSIS: RARS is a lower risk myelodysplastic syndrome (MDS) with dysplasia limited to the erythroid lineage, <5% bone marrow (BM) blasts and ≥15% BM RS. RARS-T is a provisional entity in the MDS/MPN (myeloproliferative neoplasm) overlap syndromes, with diagnostic features of RARS, along with a platelet count ≥450 × 10(9)/L and large atypical megakaryocytes similar to those observed in BCR-ABL1 negative MPN. Mutations and Karyotype: Mutations in the SF3B1 gene are seen in ≥80% of patients with RARS and RARS-T, and strongly correlate with the presence of BM RS; RARS-T patients have additional mutations such as, JAK2V617F (∼60%), MPL (<5%), and CALR (<5%). Cytogenetic abnormalities are uncommon in both RARS and RARS-T. RISK STRATIFICATION: Most patients with RARS are stratified into lower risk groups by the International Prognostic Scoring System (IPSS) for MDS and the revised IPSS. Disease outcome in RARS-T is better than that of RARS, but worse than that of essential thrombocytosis. Both RARS and RARS-T have a low risk of leukemic transformation. TREATMENT: Anemia and iron overload are complications in both diseases and are managed similar to lower risk MDS. Aspirin therapy is reasonable in RARS-T, especially in the presence of JAK2V617F, but the value of platelet-lowering drugs is uncertain. Case reports of RARS-T therapy with lenalidomide warrant additional studies.

Peroxiredoxin 2 expression is increased in neutrophils of patients with refractory cytopenia with multilineage dysplasia.

Myelodysplastic syndromes (MDS) are heterogeneous clonal disorders characterized by cytopenias that arise due to ineffective haematopoiesis and morphological dysplasia and carry an increased risk of incident acute myeloid leukaemia. The pathogenesis of marrow dysfunction in MDS is multifactorial and consistent with a multistep model and may lead to heterogeneity of MDS. We investigated the proteome profile of circulating neutrophils purified from patients with refractory cytopenia with multilineage dysplasia (RCMD) to identify proteins that have a role in the pathogenesis. Using 2-dimensional difference gel electrophoresis and protein identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we found that peroxiredoxin 2 (PRDX2), a member of the peroxiredoxin family that regulates reactive oxygen species, was markedly upregulated in neutrophils of RCMD patients compared to healthy donors. Increased PRDX2 expression in the neutrophils of RCMD patients was confirmed using quantitative reverse transcription polymerase chain reaction, immunoblotting and immunocytochemical analysis. In addition, white blood cell and neutrophil counts in RCMD patients correlated inversely with the PRDX2 expression of. Oxidative stress is a known factor involved in the pathogenesis of MDS, and PRDX2 is associated with tumourigenesis of several solid tumours. Accordingly, our results suggest that PRDX2 may perform an important function in the pathogeneis of RCMD.

SF3B1 mutations are prevalent in myelodysplastic syndromes with ring sideroblasts but do not hold independent prognostic value.

SF3B1 mutations were recently reported in myelodysplastic syndromes (MDSs), especially in the presence of ring sideroblasts (RSs). We sought to define the interaction between SF3B1 mutations, morphology, karyotype, and prognosis in MDS with more than or equal to 15% RS (MDS-RS). We studied 107 patients with MDS-RS, including 48 with refractory anemia with RS (RARS), 43 with refractory cytopenia with multilineage dysplasia (RCMD)-RS, 11 with refractory anemia with excess blasts-1 (RAEB1)-RS, and 5 with RAEB2-RS. SF3B1 mutations were detected in 53 (∼ 50%) patients: 35 RARS (73%), 16 RCMD-RS (37%), and 2 RAEB1-RS (18%). In univariate analysis, the presence of SF3B1 mutations was associated with better overall (P < .01) and leukemia-free (P < .01) survival; however, in both instances, significance was completely accounted for by World Health Organization morphologic risk categorization. In other words, when RARS and RCMD-RS were analyzed separately, there was no additional prognostic value from the presence or absence of SF3B1 mutations.

Molecular similarity between myelodysplastic form of chronic myelomonocytic leukemia and refractory anemia with ring sideroblasts.

Chronic myelomonocytic leukemia is similar to but a separate entity from both myeloproliferative neoplasms and myelodysplastic syndromes, and shows either myeloproliferative or myelodysplastic features. We ask whether this distinction may have a molecular basis. We established the gene expression profiles of 39 samples of chronic myelomonocytic leukemia (including 12 CD34-positive) and 32 CD34-positive samples of myelodysplastic syndromes by using Affymetrix microarrays, and studied the status of 18 genes by Sanger sequencing and array-comparative genomic hybridization in 53 samples. Analysis of 12 mRNAS from chronic myelomonocytic leukemia established a gene expression signature of 122 probe sets differentially expressed between proliferative and dysplastic cases of chronic myelomonocytic leukemia. As compared to proliferative cases, dysplastic cases over-expressed genes involved in red blood cell biology. When applied to 32 myelodysplastic syndromes, this gene expression signature was able to discriminate refractory anemias with ring sideroblasts from refractory anemias with excess of blasts. By comparing mRNAS from these two forms of myelodysplastic syndromes we derived a second gene expression signature. This signature separated the myelodysplastic and myeloproliferative forms of chronic myelomonocytic leukemias. These results were validated using two independent gene expression data sets. We found that myelodysplastic chronic myelomonocytic leukemias are characterized by mutations in transcription/epigenetic regulators (ASXL1, RUNX1, TET2) and splicing genes (SRSF2) and the absence of mutations in signaling genes. Myelodysplastic chronic myelomonocytic leukemias and refractory anemias with ring sideroblasts share a common expression program suggesting they are part of a continuum, which is not totally explained by their similar but not, however, identical mutation spectrum.

Efficacy of single-agent lenalidomide in patients with JAK2 (V617F) mutated refractory anemia with ring sideroblasts and thrombocytosis.

Patients with refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) are difficult to treat because the cytoreductive treatment might be beneficial for the thrombocytosis component but harmful for the RARS component. As lenalidomide has shown to be efficacious in both myelodysplastic syndromes and myeloproliferative neoplasms, we have treated 2 RARS-T patients, who were transfusion dependent, with lenalidomide. We report the results of lenalidomide treatment in these patients and show that lenalidomide has clinical activity in this rare disorder. Both patients became transfusion independent, and 1 of the patients attained indeed a complete molecular remission.

Apoptotic cells and clonally expanded cytotoxic T cells in bone marrow trephines of patients with myelodysplastic syndrome.

AIMS: There is increasing evidence that autoimmunity is involved in the pathogenesis of myelodysplastic syndromes (MDS). We examined the number of apoptotic cells, and analysed the T cells and the T cell receptor gene rearrangements in bone marrow trephines of patients with low-grade MDS [refractory anaemia (RA), refractory anaemia with ringed sideroblasts (RAS) and refractory cytopenia with multilineage dysplasia (RCMD)] to investigate the correlation between T cells and apoptosis. METHODS AND RESULTS: Bone marrow trephines from 30 patients with RA, seven patients with RCMD, four patients with RAS and 11 normal bone marrow donors were stained for CD3 and for apoptotic cells using immunohistochemistry and terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate (dUTP) nick end labelling (TUNEL) technique, respectively. The positive cells were quantified by computer-assisted image analysis. In addition, CD 8 and T cell-restricted intracellular antigen-1 (TIA-1)-positive cells were analysed by single staining and evaluated semiquantitatively by light microscopy. Junctional diversity of the T cell receptor (TCR) α-, ß- and γ-chains were analysed in 24 cases of RA and RCMD by reverse transcription-polymerase chain reaction (RT-PCR). In all cases of RA, RCMD and RAS an increase of apoptotic cells was accompanied by an increase of T cells, when compared to normal donors (P < 0.001). Expression of TIA-1 was found in 33 of 41 patients with low-grade MDS. In contrast, normal controls showed either no or only very weak expression. Furthermore, 14 of 24 cases with low-grade MDS showed clonal TCR gene rearrangement. CONCLUSION: These findings provide evidence that increased apoptosis in low-grade MDS correlates with increased numbers of cytotoxic T cells. A considerable proportion of the MDS cases showed clonal TCR rearrangement suggesting an antigen-driven selection of the T cells. We therefore speculate that cases of MDS can be accompanied by a presumably autoreactive T cell-mediated apoptosis induction in bone marrow cells.

Favorable outcome of patients who have 13q deletion: a suggestion for revision of the WHO 'MDS-U' designation.

To characterize bone marrow failure with del(13q), we reviewed clinical records of 22 bone marrow failure patients possessing del(13q) alone or del(13q) plus other abnormalities. All del(13q) patients were diagnosed with myelodysplastic syndrome-unclassified due to the absence of apparent dysplasia. Elevated glycosylphosphatidylinositol-anchored protein-deficient blood cell percentages were detected in all 16 with del(13q) alone and 3 of 6 (50%) patients with del(13q) plus other abnormalities. All 14 patients with del(13q) alone and 2 of 5 (40%) patients with del(13q) plus other abnormalities responded to immunosuppressive therapy with 10-year overall survival rates of 83% and 67%, respectively. Only 2 patients who had abnormalities in addition to the del(13q) abnormality developed acute myeloid leukemia. Given that myelodysplastic syndrome-unclassified with del(13q) is a benign bone marrow failure subset characterized by good response to immunosuppressive therapy and a high prevalence of increased glycosylphosphatidylinositol-anchored protein-deficient cells, del(13q) should not be considered an intermediate-risk chromosomal abnormality.

Marked upregulation of Survivin and Aurora-B kinase is associated with disease progression in the myelodysplastic syndromes.

BACKGROUND: Myelodysplastic syndromes are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis. Survivin is a member of the inhibitor of apoptosis family and suppresses apoptosis. Survivin also functions as a subunit of the chromosomal passenger complex for regulating mitosis with Aurora-B. Survivin and Aurora-B play an important role in maintaining genome stability. The aim of this study was to determine the role of Survivin and Aurora-B kinase in disease progression and prognosis of myelodysplastic syndromes. DESIGN AND METHODS: We evaluated the expression levels of these two genes in CD34(+) cells prepared from 64 patients with myelodysplastic syndrome or leukemic blasts from 50 patients with de novo acute myeloid leukemia using quantitative real-time PCR. RESULTS: Survivin and Aurora-B expression levels were highly correlated with the type of myelodysplastic syndrome, were much higher in refractory anemia with excess blasts-1, refractory anemia with excess blasts-2, and secondary acute myeloid leukemia following myelodysplastic syndrome than in normal control, and increased during disease progression. There was a significant correlation between these expression levels and the International Prognostic Scoring System. Interestingly, these levels were remarkably higher in patients with secondary acute myeloid leukemia following myelodysplastic syndromes than in those with de novo acute myeloid leukemia. CONCLUSIONS: This is the first report showing that high levels of Survivin and Aurora-B kinase expression in CD34(+) cells are distinctive molecular features of high-risk myelodysplastic syndromes and secondary acute myeloid leukemia following myelodysplastic syndrome. Marked upregulation of Survivin and Aurora-B kinase may contribute to genetic instability and disease progression of myelodysplastic syndromes. Our data may explain why patients with high-risk myelodysplastic syndromes frequently show complex chromosomal abnormality.

CD34+ gene expression profiling of individual children with very severe aplastic anemia indicates a pathogenic role of integrin receptors and the proapoptotic death ligand TRAIL.

UNLABELLED: BACKGROUND Very severe aplastic anemia is characterized by a hypoplastic bone marrow due to destruction of CD34(+) stem cells by autoreactive T cells. Investigation of the pathomechanism by patient-specific gene expression analysis of the attacked stem cells has previously been impractical because of the scarcity of these cells at diagnosis. DESIGN AND METHODS: Employing unbiased RNA amplification, patient-specific gene expression profiling was carried out for CD34(+) cells from patients newly diagnosed with very severe aplastic anemia (n=13), refractory anemia (n=8) and healthy controls (n=10). These data were compared to profiles of myelodysplastic disease (n=55), including refractory anemia (n=18). To identify possible targets of autoimmune attack, presence of autoreactive antibodies was tested in pre-therapeutic sera of patients with very severe aplastic anemia (n=19). RESULTS: CD34(+) gene expression profiling distinguished between healthy controls, children with aplastic or refractory anemia and clonal disease. Interferon stimulated genes such as the apoptosis inducing death ligand TRAIL were strongly up-regulated in CD34(+) cells of patients with aplastic anemia, in particular in patients responding to immunosuppressive treatment. In contrast, mRNA expression of integrin GPVI and the integrin complexes GPIa/IIa, GPIIb/IIIa, GPIB/GPIX/GPV was significantly down-regulated and corresponding antibodies were detected in 7 of 11 profiled patients and in 11 of 19 aplastic anemia patients. CONCLUSIONS As a potential diagnostic tool, patient-specific gene expression profiling of CD34(+) stem cells made it possible to make the difficult differential diagnosis of most patients with aplastic and refractory anemia. Profiling indicated a prognostic correlation of TRAIL expression and patient benefit from immunosuppressive therapy. Downregulation of integrin expression and concurrent presence of autoreactive anti-integrin-antibodies suggested a previously unrecognized pathological role of integrins in aplastic anemia.