The cell body space occupied by the nucleus during the cell differentiation in human lymphocytic, granulocytic and erythroid cell lineages.

The present nuclear and cell body diameter measurements demonstrated size differences of the approximate cell space estimate occupied by the cell nucleus during the cell differentiation in lymphocytic, granulocytic and erythroid cell lineages. These lineages were used as convenient models because all differentiation steps were easily identified and accessible in diagnostic peripheral blood or bone marrow smears of blood donors (BDs), patients suffering from chronic lymphocytic leukemia (CLL), patients with chronic myeloid leukemia (CML) and refractory anemia (RA) of the myelodysplastic syndrome (MDS). The cell space occupied by the nucleus was constant and did not change during the cell differentiation in the lymphocytic cell lineages of BDs and CLL patients despite the decreased cell size. In contrary, the cell space occupied by the nucleus markedly decreased in differentiating cells of granulocytic and erythroid lineages of patients suffering from CML. In the erythroid cell lineage in patients with RA of MDS the small reduction of the cell space occupied by the nucleus during the differentiation was not significant. The measurements also indicated that in progenitor cells of all studied cell lineages nuclei occupied more than 70 % of the cell space. Thus, the nucleus-cytoplasmic morphological and functional equilibrium appeared to be characteristic for each differentiation step and each specific cell lineage.

Novel compound heterozygous variants of TBXAS1 presenting with Ghosal hematodiaphyseal dysplasia treated with steroids.

BACKGROUND: Homozygous or compound heterozygous pathogenic variants in the thromboxane A synthase 1 (TBXAS1) gene are associated with Ghosal hematodiaphyseal dysplasia (GHDD) which is characterized by defective hematopoiesis and increased bone density of long bones. METHODS: Patients 1 and 2 are identical twins, who presented with red blood cell transfusion-dependent normocytic anemia and thrombocytopenia with bone marrow fibrosis and cortical bone thickening of long bones on plain radiograph. To clarify the etiology of their anemia and thrombocytopenia, whole blood was used for the DNA extraction and analyzed using next-generation sequencing (NGS) on an in-house bone marrow failure syndrome panel. RESULTS: The NGS results indicated that these two patients carried two heterozygous variants in TBXAS1, exon7, c.583_584del, p.Ala195Leufs*12, and exon12, c.1420G>T, p.Gly474Trp, which were inherited from their mother and father, respectively. Patients 1 and 2 have been on chronic oral steroids with normalization of hemoglobin and platelet count after steroid initiation. Patient 3 is their sister who has normal blood counts but also has the same variants in TBXAS1 as her brothers. Radiographs showed cortical bone thickening and she has not required any treatment or transfusion. CONCLUSION: We report three Caucasian siblings from non-consanguineous parents with novel compound heterozygous variants of TBXAS1 presenting with the phenotypes of GHDD. These three cases illustrate the variable clinical expressivity of the GHDD from two-compound heterozygous pathogenic variants of TBXAS1.

Bevacizumab for treating Hereditary Hemorrhagic Telangiectasia patients with severe hepatic involvement or refractory anemia.

OBJECTIVE: To report our clinical experience with bevacizumab in a cohort of Hereditary Hemorrhagic Telangiectasia (HHT) patients with severe hepatic involvement and/or refractory anemia. METHODS: Observational, ambispective study of the Institutional Registry of HHT at Hospital Italiano de Buenos Aires. Patients were treated with bevacizumab due to iron deficiency refractory anemia secondary to nasal/gastrointestinal bleeding and/or high output cardiac failure. We describe basal clinical data, bevacizumab schedules, efficacy outcomes and adverse events. Wilcoxon signed ranks test and longitudinal analysis were conducted. RESULTS: Twenty adult patients were included from July 2013 to June 2019. Clinical indications were: 13 for anemia, 4 for heart failure and 3 for both. In the anemia group, median pretreatment hemoglobin was 8.1 g/dl [IQR: 7.2-8.4] and median transfusion requirement was 4 units [2-6]. In heart failure group, pretreatment median cardiac index was 4.5 L/min/m2 [4.1-5.6] and cardiac output was 8.3 L/min [7.5-9.2]. Bevacizumab 5 mg/kg/dose every 2 weeks for 6 applications was scheduled. By the end of induction, median hemoglobin at 3 months was 10.9 g/dl [9.5-12.8] (p = 0.01) and median transfusion requirement 0 units [0-1] (p<0.01), and this effect was more or less sustained during a year. Regarding heart failure group, two patients had complete hemodynamic response and achieved liver transplantation and two had partial response. No serious adverse events were registered. CONCLUSION: Bevacizumab is a promising line of treatment for HHT patients with refractory anemia. For patients with high output cardiac failure, bevacizumab may be useful as bridge therapy awaiting for liver transplantation.

Successful treatment with daratumumab for post-HSCT refractory hemolytic anemia.

Autoimmune cytopenias (AIC) following allogeneic hematopoietic stem cell transplantation (HSCT) may cause significant morbidity and mortality and are often challenging to treat. We present a case of a pediatric patient with primary myelofibrosis of infancy caused by VPS45 protein deficiency, who developed severe refractory hemolytic anemia and immune-mediated thrombocytopenia 3.5 months following HSCT. After the failure of several treatments, he received daratumumab, an anti-CD38 specific antibody, and demonstrated fast and sustained response. The only side effect was delayed recovery of humoral immunity. Daratumumab, by targeting antibody-producing plasma cells, may be a valid treatment option for refractory post-HSCT AIC.

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.

Ghosal Type Hematodiaphyseal Dysplasia.

BACKGROUND: Ghosal Type Hematodiaphyseal Dysplasia is an autosomal recessive disorder characterized by refractory anemia and diaphyseal bone dysplasia. CASE CHARACTERISTICS: A 3 y 9 mo-old male child presented with progressive anemia and bowing of thighs. Child was found to have a previously reported homozygous point mutation c.1238G>A, (p.Arg413Glu) in Exon 16 of TBXAS1 gene. OUTCOME: Low dose steroid therapy resulted in normalization of hemoglobin and prevented further progression of bony changes. MESSAGE: Refractory anemia in association with bony deformities should prompt pediatricians to investigate for inherited bony dysplasia.

Spectrum of the WHO Classification De Novo Myelodysplastic Syndrome: Experience from Southern Pakistan.

BACKGROUND: Myelodysplastic syndrome (MDS) is a clonal disorder of hemopoeitic stem cells, characterized by infective hematopoiesis, peripheral cytopenias along with hypercellularity of marrow and marked dysplastic features. Our aim was to study the spectrum of the WHO classification in adult Pakistani patients with MDS at disease presentation. MATERIALS AND METHODS: This retrospective descriptive study was conducted at Liaquat National Hospital and Medical College, extending from January 2010 to December 2014. Patient data were retrieved from the maintained archives. RESULTS: Overall, 45 patients were diagnosed at our institution with de novo MDS during the study period. There were 28 males and 17 females. Age ranged between 18 and 95 years with a mean of 57.6±17.4 years. The male to female ratio was 1.7:1. According to the WHO classification, 53.3% had refractory cytopenia with multilineage dysplasia, 22.2% had refractory cytopenia with unilineage dysplasia, 4.4% each had refractory anemia with excess of blasts-1 and II and 15.5% had MDS unclassified. The main presenting complaints were generalized fatigue (60%), fever (33.3%), dyspnea (15.5%), bleeding (13.3%) and weight loss (11.1%). Physical examination revealed pallor in 37.7%, followed by petechial and purpuric rashes in 20% of patients. Hemoglobin was <10 g/dl in 41 (91.1%). Pancytopenia and bicytopenia were noted in 18 (40%) and 14 (31.1%) respectively. CONCLUSIONS: MDS in our patients presents at a relatively young age. Refractory c ytopenia with multilineage dysplasia was the dominant disease variant in our setting.

Role of microRNA-29b in myelodysplastic syndromes during transformation to overt leukaemia.

Chromosome 7q32 is a frequently deleted region in myelodysplastic syndromes (MDSs) and encodes the microRNAs (miRNAs) miR-29a/miR-29b. Both miR-29s down-regulate the anti-apoptotic protein myeloid cell leukaemia 1 (MCL-1) in acute myeloid leukaemia. Thus, to investigate the role of miR-29s in the transformation of MDS to overt leukaemia (OL), we analysed the relationship between miR-29 expression and MCL-1 expression. MiR-29b expression was down-regulated in refractory anaemia and OL bone marrow as compared to that in control bone marrow. MCL-1 expression level in OL was significantly higher than that in refractory anaemia with excess blasts and a negative correlation was observed between miR-29b and MCL-1 messenger RNA expression levels in OL samples. Immunohistochemical analysis showed that the MCL-1 positive rate among MDS bone marrow CD34 positive cells significantly increased during transformation to OL. Additionally, MCL-1 positive cells were negative for cleaved caspase 3, which indicated that these cells avoided apoptosis. Reduced miR-29b expression in MDS bone marrow cells might trigger transformation to OL via overexpression of MCL-1 in blastic cells.

Abnormalities in the T cell receptor Vδ repertoire and Foxp3 expression in refractory anemia with ringed sideroblasts.

Immune dysregulation has been implicated in myelodysplastic syndrome (MDS) pathogenesis. Refractory anemia with ringed sideroblasts (RARS) is a low-risk subtype of MDS. Our previous study has reported an abnormal γδ T cell receptor (TCR) repertoire in patients with intermediate or high-risk MDS. To characterize the status of T cell immunity in RARS, we investigated the distribution and clonality of the TCR Vδ repertoire and the expression of Foxp3 in patients with RARS. The number of expressed Vδ subfamily members in the RARS group (4.8±2.25) was significantly lower than that in the control group (7.6±0.52, p=0.0012). A significantly lower expression frequency for the Vδ4 (p=0.007), Vδ5 (p=0.0049) and Vδ7 subfamilies (p=0.0225) could be detected in the RARS group. The most frequent clonally expanded T cell subfamily member in the RARS group was Vδ7 (100%, 3/3). Foxp3 mRNA expression was significantly lower and higher than that in the controls in 60% and 40% RARS patients, respectively. In conclusion, marked restriction of the TCR Vδ subfamily expression pattern and great heterogeneity in Foxp3 expression were characteristics found in RARS. These results provide data regarding the immunodeficiency and immune reactive characteristics of patients with RARS, which may provide a basis for immunotherapy options.

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.

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.

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.

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.