Flow cytometric diagnosis of myelodysplasia and myeloproliferative disorders

The current diagnosis of both myelodysplastic syndromes and myeloproliferative disorders relies in large part on subtle and subjective morphologic findings and the presence of cytogenetic abnormalities. Consequently, diagnosis of these disorders is often difficult and tentative with diagnosis at early stages representing a particular challenge. There is a need for new diagnostic techniques to allow a more definitive and objective diagnosis for these diseases. The published literature relating to the potential diagnostic utility of flow cytometric immunophenotyping in the diagnosis of myelodysplastic syndromes and myeloproliferative disorders is reviewed, and the increasingly important contribution of this technique to the diagnosis of these disorders emphasized.     

骨髓增生异常综合征合并非霍奇金淋巴瘤一例病例报道并文献复习

目的:探讨骨髓增生异常综合征合并非霍奇金淋巴瘤的发病机制、诊断及治疗,并判断其预后情况。方法:回顾我院收治的一例骨髓增生异常综合征(MDS)患者同时诊断间变大细胞淋巴瘤(ALCL)患者的临床资料,总结其诊断及治疗经过,对其预后进行评价。结果:该患者诊断间变大细胞淋巴瘤后,经化疗对症治疗病情无显著改善,且患者ALK阳性,考虑预后较差,建议造血干细胞移植。结论:骨髓增生异常综合征合并非霍奇金淋巴瘤的病例在国内外十分罕见,治疗方法目前尚需进一步研究。     

Cytogenetic study of 20 cases of refractory anemia

The myelodysplastic syndromes are a group of hematological disorders not yet clearly defined. The authors describe the chromosomal aspects of 20 cases observed in the region of Clermont-Ferrand in comparison with the bibliographical data.     

SRSF2 Is Essential for Hematopoiesis,and Its Myelodysplastic Syndrome-Related Mutations Dysregulate Alternative Pre-mRNA Splicing

Myelodysplastic syndromes (MDS) are a group of neoplasms characterized by ineffective myeloid hematopoiesis and various risks for leukemia. SRSF2, a member of the serine/arginine-rich (SR) family of splicing factors, is one of the mutation targets associated with poor survival in patients suffering from myelodysplastic syndromes. Here we report the biological function of SRSF2 in hematopoiesis by using conditional knockout mouse models. Ablation of SRSF2 in the hematopoietic lineage caused embryonic lethality, and Srsf2-deficient fetal liver cells showed significantly enhanced apoptosis and decreased levels of hematopoietic stem/progenitor cells. Induced ablation of SRSF2 in adult Mx1-Cre Srsf2^flox/flox mice upon poly(I):poly(C) injection demonstrated a significant decrease in lineage⁻ Sca⁺ c-Kit⁺ cells in bone marrow. To reveal the functional impact of myelodysplastic syndromes-associated mutations in SRSF2, we analyzed splicing responses on the MSD-L cell line and found that the missense mutation of proline 95 to histidine (P95H) and a P95-to-R102 in-frame 8-amino-acid deletion caused significant changes in alternative splicing. The affected genes were enriched in cancer development and apoptosis. These findings suggest that intact SRSF2 is essential for the functional integrity of the hematopoietic system and that its mutations likely contribute to development of myelodysplastic syndromes.     

Analysis of human dysplastic haematopoiesis in long-term bone marrow culture

In this paper we have analysed the behaviour of myelodysplastic marrow in a long-term bone marrow liquid culture system (LTBMC) from eleven patients with myelodysplastic syndromes with regard to cellularity, day-7 and day-14 CFU-GM growth, cluster formation, adherent cells and CFU-F formation. An altered CFU-GM pattern was found in 64% of cases at diagnosis, while normal growth was seen in the remaining cases, all of which were affected by refractory anaemia. The levels of CFU-GM, as well as cellularity, were reduced in myelodysplastic marrows compared to normal controls over the whole duration of LTBMCs. Cases with a normal CFU-GM level at diagnosis also showed pathological behaviour when examined in LTBMC. The duration of dysplastic haematopoiesis was significantly shorter than that of controls. The proliferative ability of CFU-F was reduced in 50% of cases as shown by replating experiments. In conclusion, myelodysplastic marrow shows an abnormal behaviour in LTBMC, even in those cases which present normal CFU-GM growth at diagnosis.     

G-CSF and GM-CSF in clinical trials.

Hematopoietic growth factors have now been purified, cloned, and produced in bacteria and yeast. Those that are currently in clinical study include erythropoietin, GM-CSF, G-CSF, M-CSF (also called CSF-1), and multi-CSF (also called interleukin 3). Growth factor appear likely to enhance the recovery and function of circulating white cells after standard-dose cancer therapy and high-bone-dose cancer therapy with marrow transplant and to restore leukocyte numbers and competence in the acquired immune deficiency syndromes and myelodysplastic syndromes. Phase I, II trials in AIDS, in cancer patients receiving chemotherapy, in cases of myeloproliferative disease, and after bone marrow transplant have been published. The results of phase III studies are just becoming available.     

Myelodysplastic syndromes: their history, evolution and relation to acute myeloid leukaemia

The myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal disorders arising from a multipotent haemopoietic progenitor which share a leukaemic propensity, 30% of cases culminating in acute myeloid leukaemia (AML). Their pathogenesis probably entails multiple steps, phenotypic progression being determined by either expansion or evolution of the abnormal clone. The clonal origin of certain cases of de novo AML is analogous to that of MDS and evidence that they share a common pathogenesis and distinct biological characteristics is beginning to emerge.     

Mutations in ras genes in myelocytic leukemias and myelodysplastic syndromes

Acute myelocytic leukemias (AML) are characterized by a remarkably high incidence (approximately 30%) of point mutations affecting codons 12, 13, or 61 within ras genes. A predominant involvement of N-ras sequences has been established. Neither Philadelphia chromosome-positive chronic myelocytic leukemia nor other chronic myeloproliferative disorders show a similar frequency. However, a proportion of myelodysplastic syndromes, namely, the chronic myelomonocytic subtype (CMML) also show this molecular feature. The following is a brief discussion of the possible biologic and clinical implications of these observations.     

Human megakaryocytopoiesis--normal and abnormal.

Regulation of megakaryocyte and platelet production remains poorly understood. In culture system two separate activities are needed for maximum production of megakaryocyte progenitors: promotor of clonal expansion and promoter of maturation, other growth factors and cells also contribute to regulation of megakaryocytopoiesis. Increased proliferation of megakaryocytes is observed in myeloproliferative disorders and idiopathic thrombocytopenic purpura, and decreased proliferation is found in aplastic anaemia and hypomegakaryocytic thrombocytopenia. Dysmegakaryocytopoiesis is present in myelodysplastic syndromes and acute leukaemia, and a proliferation of immature megakaryocytes in acute megakaryoblastic leukaemia. Increased understanding of human megakaryocytopoiesis is beginning to help in rational clinical management.     

Mitochondrial ferritin

A novel ferritin type specifically targeted to mitochondria has been recently found in human and mouse. It is structurally and functionally similar to the cytosolic ferritins, well-characterized molecules found in most living systems which are designed to store and detoxify cellular iron. Cytosolic ferritins in mammals are ubiquitous while mitochondrial ferritin expression is restricted mainly to the testis, neuronal cells and islets of Langherans. In addition, it is abundant in the iron-loaded mitochondria of erythroblasts of patients with sideroblastic anaemia. The characterization of recombinant and transfected mitochondrial ferritin indicated that this protein has a role in protecting mitochondria from iron-induced damage. These data suggest that it is an interesting tool to study the iron metabolism in this organelle. In addition, it may be useful for the diagnosis of myelodysplastic syndromes and in protecting mitochondria from the toxic effects of excess iron.     

Inactivation of Chikungunya virus by 1,5 iodonapthyl azide

Myelodysplastic syndromes (MDS) are often accompanied by autoimmune phenomena. The underlying mechanisms for these associations remain uncertain, although T cell activation seems to be important. Human T-lymphotropic virus (HTLV-1) has been detected in patients with myelodysplastic syndromes, mostly in regions of the world which are endemic for the virus, and where association of HTLV-1 with rheumatological manifestation is not rare. We present here the case of a 58 year old man who presented with cytopenias, leukocytoclastic vasculitis of the skin and glomerulopathy, and was diagnosed as MDS (refractory anemia with excess blasts - RAEB 1). The patient also tested positive for HTLV-1 by PCR. After 8 monthly cycles of 5-azacytidine he achieved a complete hematologic remission. Following treatment, a second PCR for HTLV-1 was carried out and found to be negative. This is the first report in the literature of a HTLV-1-positive MDS with severe autoimmune manifestations, which was treated with the hypomethylating factor 5-azacitidine, achieving cytogenetic remission with concomitant resolution of the autoimmune manifestations, as well as HTLV-1-PCR negativity. HTLV-1-PCR negativity may be due to either immune mediated clearance of the virus, or a potential antiretroviral effect of 5-azacytidine. 5-azacytidine is known for its antiretroviral effects, although there is no proof of its activity against HTLV-1 infection in vivo.     

The promise of epigenetics in personalized medicine

Numerous preclinical and clinical trials, with older as well as some newer drugs, have demonstrated the targeting of aberrant epigenetic marks to be a viable means of preventing and treating certain human disorders, including myelodysplastic and leukemic syndromes and various hemoglobinopathies. These findings are encouraging, and although the risks associated with such therapy are largely unknown, precise maps of epigenetic marks are becoming increasingly available through advancements in sequencing protocols that combine chromatin immunoprecipitation and gene expression analyses. Indeed, progress in understanding gene regulation at promoter regions and chromatin organization in health and disease has been substantial. New insights into the proteins that are targeted by therapeutic agents that alter epigenetic programs may provide important inroads into personalized medicine.     

MicroRNAs in the midst of myeloid signal transduction

MicroRNA (miRNA) play important roles in the development and physiological function of hematopoietic stem/progenitor and mature cell lineages. In addition, deregulated miRNA expression and subsequent gene expression changes are associated with hematologic diseases including myelodysplastic syndromes and acute myeloid leukemia. This review focuses on myelopoiesis as a model to highlight the involvement of miRNA in the regulation of normal and malignant cellular signaling pathways. By incorporating miRNA regulation into well-established myeloid signal transduction pathways, we hope to shed light on targetable factors both upstream and downstream of miRNA signaling. These pathway-specific miRNA functions suggest scenarios wherein miRNA-based therapeutics may be beneficial either alone or in combination with current therapies.     

Clinical applications of human granulocyte-macrophage colony-stimulating factor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that stimulates myeloid cell proliferation and maturation and enhances the function of terminally differentiated effector cells. Phase I and II clinical trials have demonstrated mild to moderate toxicities at doses of less than 30 micrograms/kg/day. These studies suggest a potential role for this growth factor to stimulate myelopoiesis in patients with aplastic anemia, myelodysplastic syndromes, AIDS, chemotherapy-induced myelosuppression, chronic neutropenia, and following bone marrow transplantation. The potential clinical uses of GM-CSF will depend on results of studies designed to optimize its therapeutic efficacy.     

Clinical applications of recombinant human colony-stimulating factors.

The differentiation and maturation of hematopoietic progenitor cells are regulated by certain growth factors. Several of these glycoproteins have been characterized, and their amino acid sequences have been delineated. Modern DNA technology provides sufficient quantities of these hormones for testing in clinical trials. Erythropoietin (EPO) has been shown to increase the hemoglobin level and hematocrit in patients with end-stage renal disease. Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF) can increase the numbers of neutrophils and monocytes, in a dose-dependent fashion. The function of granulocytes and monocytes is also enhanced. Clinical studies of the toxicity and activity of G-CSF and GM-CSF have been conducted in patients with acquired immune deficiency syndrome, aplastic anemia, myelodysplastic syndromes, and neutropenia due to cancer and chemotherapy. In almost all patients the neutrophil count increased within 24 hours after the start of treatment. Side effects of G-CSF and GM-CSF are infrequent and usually mild. Combinations of CSFs may be even more effective.     

An expert system applied to the diagnosis of anemia with special reference to myelodysplastic syndromes

An expert system is described that includes interpretation of the results from a complete blood count as well as data from bone marrow aspiration. The system utilizes Bayes' rule. It has previously been tested on 180 cases of anemia including 20 benign and malignant hematologic disorders. On the data set, the system achieved 84% satisfactory diagnoses. In the present study, patients with myelodysplastic syndromes and with disorders of heme synthesis have been added to the test cases. For support, the expert system requires an IBM Personal Computer or equivalent. The program is available commercially (Coulter Electronics, Hialeah, FL).     

The assessment of proliferating cell nuclear antigen immunostaining in myelodysplastic syndromes and its prognostic significance

Several prognostic factors for patients with myelodysplastic syndromes (MDS) have been identified in previous years. In order to determine prognostic factors characterizing haematopoietic cell kinetics, bone marrow proliferative activity and serum TNF-a levels were measured in 51 cases of MDS. Cell proliferation was evaluated by employing a monoclonal antibody directed against the proliferating cell nuclear antigen (PCNA). The PCNA proliferating index (PCNA PI) and serum TNF-a levels showed significant differences between patients with MDS and normal controls (p<0.0001). PCNA PI and serum TNF-a were significantly higher in the high risk for leukemic transformation FAB subgroups (RAEB, RAEB-t and CMML) in comparison to the low risk group (RA and RARS) (p<0.001). PCNA PI and TNF-a also increased with increasing IPSS score (p<0.05). A positive correlation was noted between TNF-a concentrations and PCNA PI (r:0.36, p<0.008). Univariate analysis using the log-rank test showed that a higher PCNA PI was associated with a significantly shorter survival (p<0.001). We conclude that elevated PCNA PI and TNF-a serum levels are increased in high risk myelodysplastic disease and that a high PCNA PI is predictive of a shorter survival in this group of patients.     

Platelet dysfunction as the presenting feature of atypical myelodysplastic syndrome with monosomy 7, normal blood counts and no bleeding tendency

A 71-year-old male patient with atypical myelodysplastic syndrome showing monosomy 7 is described. He presented with severe foot pains, trophic skin and nail changes, loss of distal pulses, all compatible with peripheral arterial occlusive disease. He had completely normal blood counts and no bleeding tendency. Prolonged bleeding time was disclosed by chance, during routine haemostatic studies. An acquired platelet dysfunction was considered, with prolonged bleeding time and large platelets that failed to aggregate in response to arachidonic acid and that had impaired response to collagen and adrenaline. The bone marrow was hypercellular, with numerous dysplastic megakaryocytes and two other slightly dysplastic myeloid lines. Cytogenetic analyses of the bone marrow cells showed a mosaic karyotype: 46,XY/45,XY,-7. On angiography, bilateral thrombosis of the iliac, superficial femoral and popliteal arteries was disclosed. The patient was prepared with platelet transfusions. Arterial thrombectomy and amputation of the left calf were performed. Ten months later, his blood counts showed mild pancytopenia. He died at home. The authors discuss some clinical and pathogenetical aspects of such presentations of myelodysplastic syndromes.     

积分系统参与MDS诊断的可行性研究

目的:骨髓增生异常综合征(MDS)是一种起源于造血干细胞,以高风险向急性髓系白血病转化为特点的难治性血细胞质、量异常的异质性疾病。对不明原因血细胞减少或者有巨幼红细胞血症的老年人,如果得不到明确的诊断就不会获得有效的治疗。探讨对伴有不明原因血细胞减少或大红细胞血症患者采用一种积分系统来预测诊断为骨髓增生异常综合征(MDS)的可行性。方法:应用四个因子即年龄≥65,红细胞平均体积(MCV),红细胞分布宽度(RDW),和乳酸脱氢酶(LDH)的一个积分系统来计算诊断MDS的概率。结果:调查了303个病人,他们都因为不明原因的血细胞减少或者大红细胞血症在过去三年(2010~2012)进行了骨髓检查,最终三分之二患者被诊断为MDS,9%为疑似MDS,采用积分系统前后的MDS诊断率分别为12%和48%。结论:此四个因子的积分系统可以用于指导MDS患者的诊断。提高MDS的诊断率,减少不必要的骨髓穿刺,减轻病人的疼痛和医疗费用。