Histological and cytogenetic characterization of bone marrow in relation to prognosis and diagnosis of myelodysplastic syndromes

Bone marrow (BM) histology of 102 myelodysplastic syndromes (MDS) patients was analyzed retrospectively. All the cases were reclassified according to the World Health Organization (WHO) classification. Karyotype study was conducted for all except one. Fifteen of the MDS cases were hypoplastic. The cellularity in bone marrow histology is sometimes ineffective in the differential diagnosis of MDS and aplastic anemia (AA). Nonetheless, a marked decrease in the number of megakaryocytes (average, 0.3/mm(2); range, 0-2/mm(2)) even in the hyperplastic foci of the marrow of AA was the most important histological feature differentiating AA from MDS, whereas the number of megakaryocytes increased in most MDS cases (44/mm(2); range, 1-240/mm(2)) and also in hypoplastic MDS (14/mm(2); range, 8-26/mm(2)). Hyperplastic marrow had a significantly high frequency of progress to acute myeloid leukemia (AML) and hypoplastic MDS had a lower rate of progress to AML. Severe myelofibrosis had a significantly poor prognosis. An increase in CD34-positive cells in MDS indicated a high rate of progress to AML. As for the patients with refractory cytopenia with multilineage dysplasia (RCMD; the new category under the WHO classification), the increased number of megakaryocytes was correlated with poor prognosis.     

p53 expression in myeloid cells of myelodysplastic syndromes. Association with evolution of overt leukemia.

To assess p53 expression in the hematopoietic cells of the bone marrow in premalignant as well as malignant conditions, we examined immunohistochemically bone marrow biopsies from patients with myelodysplastic syndromes (MDS, n = 51), acute myeloid leukemia (n = 42) and as a nonneoplastic condition, aplastic anemia (n = 20) and samples from individuals who had no hematological disorder (control, n = 12). Nuclear accumulation of p53 protein was found in seven of 51 patients with MDS (14%) and two of 42 acute myeloid leukemia patients (5%), whereas patients with aplastic anemia and control subjects were uniformly negative for p53 protein. In the bone marrow of patient with MDS, p53-positive cells constituted about 5 to 30% of the total bone marrow cells. Two-color immunohistochemical analysis revealed that the p53-positive cells were also positive for the myeloid cell marker. Half of the MDS cases that evolved to overt leukemia (seven of 14) exhibited positive p53 reaction in the bone marrow at the time of initial diagnosis. This frequency (50%) was significantly higher than that in de novo acute myeloid leukemia cases. All of the seven MDS cases that exhibited p53 expression at the time of initial diagnosis developed overt leukemia later, and p53 expression was maintained throughout the progression of MDS. The results suggest that p53 mutations that occur in the myeloid cells in MDS may confer a growth advantage to these cells resulting in the progression to overt leukemia. Thus, immunohistochemical examination for p53 is very useful for predicting the evolution to overt leukemia from MDS.     

Immunohistochemical assessment of human bone marrow macrophages in hematologic disorders

Changes in bone marrow macrophages may be associated with abnormal hematopoiesis in various hematologic disorders. We immunohistochemically evaluated the density of macrophages in bone marrow trephine biopsies. In reactive erythroid hyperplasia (hemolytic anemia and megaloblastic anemia), the macrophages slightly increased in density, extending their cytoplasmic processes between hematopoietic cells. In erythroid hypoplasia (pure red cell aplasia), they became rounded and frequently had hemosiderin granules. There was no significant difference in the macrophage density in the hematopoietic area between erythroid hyperplasia and hypoplasia. The macrophages increased in density in myeloproliferative disorders (polycythemia vera, chronic myelogenous leukemia and primary thrombocythemia). In myelofibrosis, some macrophages became extremely elongated along the line of the fibroblastic cells. In contrast, in conditions in which myelopoietic activity is considerably impaired (aplastic anemia, acute leukemia and multiple myeloma), they significantly decreased in density. These results suggest that the morphologic change in bone marrow macrophages is associated with erythropoietic activity and that there is a correlation between macrophage density and myelopoietic activity.     

Haematological changes in HIV infection

HIV infection is associated with a wide range of hematological abnormalities. The peripheral blood findings and the morphological abnormalities in the bone marrow can simulate myelodysplastic syndrome, myeloproliferative disorders, and T cell lymphoma. We studied the peripheral blood smear and bone marrow findings of 42 patients with HIV infection over a 3-year period with the aim of recognising the morphological findings sufficiently characteristic of HV infection. The salient peripheral blood smear findilngs were anemia, bicytopenia and pancytopenia. The bone marrow revealed trilineage dysplasia, plasma cells and eosinophils, increased megakaryocytes, increased iron and reticulin fibrosis. In two cases the bone marrow revealed granulomata.     

Idiopathic aplastic anemia: Diagnosis and classification

Aplastic anemia (AA) is a disease characterized by pancytopenia and hypoplastic bone marrow caused by the decrease of hematopoietic stem cells. The pathogenesis of AA is complex and involves an abnormal hematopoietic microenvironment, hematopoietic stem cell/progenitor cell deficiencies and immunity disorders. Survival in severe aplastic anemia (SAA) has markedly improved in the past 4 decades because of advances in hematopoietic stem cell transplantation, immunosuppressive and biologic drugs, and supportive care. Herein, we will update the main issues concern AA according to our literature review.     

p53 protein overexpression in bone marrow biopsies of patients with Shwachman-Diamond syndrome has a prevalence similar to that of patients with refractory anemia

CONTEXT: Shwachman-Diamond syndrome (SDS) is a rare inherited disorder characterized by pancreatic insufficiency, neutropenia, and in some patients, metaphyseal dysostosis. Patients with SDS are at a high risk for development of bone marrow failure, myelodysplastic syndrome, and acute leukemia. The p53 gene plays a major role in cell-cycle regulation, particularly in the presence of a genetic alteration in DNA, a critical step for the initiation of leukemogenesis. p53 gene up-regulation and p53 protein overexpression may occur as a cellular reaction to significant DNA damage. Shwachman-Diamond syndrome and refractory anemia patients have close similarities in the prevalence of acute leukemia and in cell-cycle changes in bone marrow cells. This similarity was further investigated for p53 protein overexpression using archived tissue from patients with hematologic diseases having various leukemic propensities, including SDS and refractory anemia. METHODS: Immunohistochemical staining for p53 protein overexpression was performed on bone marrow biopsies from 9 patients with SDS. These specimens were compared with biopsies from 71 patients with acquired hematologic disorders with variable risk levels for leukemia, including acquired aplastic anemia (n = 14), refractory anemia (n = 46), and various acquired cytopenias (n = 11), as well as 37 control subjects. RESULTS: p53 protein overexpression was identified only in patients with SDS and in patients with refractory anemia; these groups exhibited comparable prevalences of 78% and 72%, respectively. None of the patients with acquired aplastic anemia, acquired cytopenias, or in the control group showed overexpression of p53 protein. CONCLUSION: The prevalence of p53 protein overexpression in SDS is significantly different from that in acquired aplastic anemia and acquired cytopenias, but it is similar to the prevalence in refractory anemia. We speculate that p53 protein overexpression in this bone marrow failure syndrome may represent an early indicator of significant DNA genetic alteration, which is a crucial step in the process of leukemogenesis.     

Cytogenetics and bone marrow transplantation

The authors report hematologic and cytogenetic data on 19 patients treated with allogeneic bone marrow transplantation (BMT) for severe hematologic disorders: 8 patients with chronic myelogenous leukemia, 6 with acute leukemia, 3 with severe aplastic anemia, 1 with refractory anemia, and 1 with beta-thalassemia major. Cytogenetic assays were performed on marrow cells before conditioning, 30 days after BMT, and at subsequent times. The authors discuss the role of cytogenetic studies in the evaluation of bone marrow engraftment, leukemic transformation of the graft, and disease relapse.     

Bone marrow biopsies of patients with hematopoietic and lymphoid disorders - epidemiology, chromosomal aberrations and molecular pathology

Bone marrow biopsy of the iliac crest is the first and most important step in the diagnostics of hematopoietic disorders. The biopsies of the years 2006 and 2007 from the Institute of Pathology of the Jena University Hospital were retrospectively analyzed for clinicopathological parameters. In addition, the Mitelman database was retrieved for chromosomal aberrations. The analysis of 2820 reports from 1185 patients revealed that lymphomas, plasma cell myeloma and acute leukemia were most frequent. Males predominated in myeloproliferative neoplasms and lymphoma subtypes, particularly CLL, except for plasma cell myeloma and acute leukemia. A peak incidence was seen between 61 and 70 years of age with a varying pattern for single entities. The database search revealed that ALL, AML, CLL and CML were mainly diploid while Hodgkin lymphoma, mature B-cell lymphoma and multiple myeloma mostly carried hyperdiploid chromosome numbers. Numerical aberrations like chromosome 8 gains in hyperdiploid CML were prominent in specific subgroups. Molecular testing is exemplified in CML, plasma cell myeloma and hairy cell leukemia. The study highlights typical clinicopathological characteristics and new genetic findings in hematopoietic and lymphoid neoplasms with relevance for the new WHO classification and beyond. We hope that it may help in the differential diagnosis of bone marrow biopsies.     

荧光定量聚合酶链反应检测GATA-1、GATA-2、过氧化物酶体增殖物激活受体γ和干细胞因子基因在再生障碍性贫血患者及正常人骨髓间充质干细胞中的表达

背景：GATA-1、GATA-2、过氧化物酶体增殖物激活受体γ和干细胞因子基因在骨髓造血及成脂调控机制中起重要作用。 目的：观察GATA-1、GATA-2、过氧化物酶体增殖物激活受体γ和干细胞因子基因在再生障碍性贫血患者及正常人间充质干细胞中的表达。 方法：收集6例再生障碍性贫血患者及6位正常人骨髓液各10 mL,Ficoll贴壁法分离培养骨髓单个核细胞,达70%~80%融合后扩增传代。取传至第3代的间充质干细胞,应用流式细胞仪进行鉴定。提取总RNA,比较各组基因与内参基因之间的差别。荧光定量聚合酶链反应检测GATA-1、GATA-2、过氧化物酶体增殖物激活受体γ和干细胞因子基因在再生障碍性贫血患者及正常人间充质干细胞中的表达量。 结果与结论：与正常人间充质干细胞相比,再生障碍性贫血患者间充质干细胞的GATA-2和干细胞因子基因表达量降低(P=0.012,0.039),过氧化物酶体增殖物激活受体γ基因表达量升高(P=0.035);两组GATA-1基因表达量差异无显著性意义。提示GATA-2、干细胞因子基因的异常表达可能影响AA患者骨髓微环境的造血调控作用;过氧化物酶体增殖物激活受体γ基因的异常表达可能解释再生障碍性贫血患者骨髓易成脂的原因。     

Bone marrow transplantation in Ramathibodi Hospital: progress report.

Bone marrow transplantation has become the accepted treatment for several hematologic disorders. We have done 3 autologous and 6 allogeneic bone marrow transplantations at Ramathibodi Hospital since July 1989 in patients with acute lymphoblastic leukemia, acute non-lymphocytic leukemia, chronic myeloid leukemia, non-Hodgkin's lymphoma and severe aplastic anemia. Only one patient with aplastic anemia had late graft rejection, but the rest of them engrafted and did well during the median follow up period of 317 days (range: 39 to 962 days) post transplantation. None of the allogeneic BMT had graft-versus-host disease. We use cyclosporin and short course methotrexate for post transplantation immunosuppression.     

561例血液病患者染色体核型分析

目的探讨血液病患者有关核型改变及其意义。方法抽取骨髓标本2～5ml,采用骨髓细胞直接法、24h短期培养法制备染色体标本,用G显带技术进行核型分析。结果 561例血液病患者中,常规细胞遗传学发现异常核型282例（50.3%）,其中结构异常116例（20.7%）,数目异常84例（15.0%）,同时有结构异常和数目异常82例（14.6%）。不同类型血液病患者染色体检测结果不同,在急性髓系白血病（AML）、急性淋巴细胞白血病（ALL）、慢性粒细胞白血病（CML）、骨髓增生异常综合症（MDS）、再生障碍性贫血（AA）、骨髓增殖性疾病（MPD）、淋巴瘤、血小板减少症中异常核型分别为56.5%（109/193）、44.9%（57/127）、74.7%（62/83）、56.7%（34/60）、3.7%（1/27）、0%（0/15）、72.7%（8/11）、0%（0/5）,在急性混合细胞白血病、特发性血小板减少性紫癜（ITP）、多发性骨髓瘤（MM）、慢性粒单细胞白血病（CMML）、淋巴瘤白血病、慢性淋巴细胞白血病（CLL）、纯红再障、恶性组织细胞病等较少患者中,异常核型分别为25.0%（2/8）、0%（0/8）、20%（1/5）、0%（0/5）、100%（4/4）、40%（2/5）、0%（0/3）、100%（2/2）。M2伴t（8;21）,M3伴t（15;17）,M4伴inv（16）、t（16;16）,ALL伴t（9;22）CR率分别为88.2%、94.7%、80%、45.5%。结论染色体核型分析在恶性血液病诊断、分型、预后判断和指导治疗中具有重要意义。     

Bone marrow as an organ of erythrocyte destruction in hematological disorders

Bone marrow of patients with hematological diseases contains a great number of erythroclasic clusters characterized by exocytic lysis of the constituent erythrocytes by cluster-forming myelocaryocytes including erythrocaryocytes. The content of erythroclasic clusters with exocytic lysis of erythrocytes varied from 21% of total erythroclasic clusters in bone marrow of patients with aplastic anemia to 81% in bone marrow of patients with an active phase of acute lymphoblastic leukemia showing high intensity of hemolysis in the bone marrow. Most intensive lysis of erythrocytes in erythroclasic clusters took place in the bone marrow of patients in an active stage of acute lymphoblastic leukemia and patients with chronic myeloid leukemia. At the time of the investigation tens of thousands of erythrocytes were undergoing destruction in erythroclasic clusters in one mcl of bone marrow of such patients. These findings confirm the idea of the bone marrow as an organ of erythrocyte destruction.     

Clinical features of a new category, myelodysplastic/myeloproliferative diseases, defined by WHO classification

The WHO classification published in 2001 defined a new category of hematological disease, myelodysplastic/myeloproliferative diseases (MDS/MPD), that have both myelodysplasia and myeloproliferation at the time of initial presentation. This category consists of four subclasses, chronic myelomonocytic leukemia (CMML), atypical CML(aCML), juvenile chronic myelogenous leukemia and MDS/MPD-unclassifiable (MDS/MPD-u). In order to clarify the clinical features of these diseases, we analyzed clinical data of tentatively diagnosed MDS/MPD cases in the past ten years accumulated from affiliated hospitals. By reviewing the data of each case according to the criteria, we diagnosed 31 cases of MDS/MPD, including 22 cases of CMML, 5 cases of aCML and 4 cases of MDS/MPD-u. Male predominance and high age were common among these three subclasses. The prognosis of CMML was poor compared to other subclasses because of the high incidence of blast crisis. It is noteworthy that blast crisis in CMML exclusively occurred within one year after diagnosis. Young age, a high percentage of blasts in the peripheral blood, splenomegaly, lymphadenopathy and clonal cytogenetic abnormality were associated with blast crisis. It is suggested that there are two subgroups in CMML which differ in disease progression. Thus, these indicators may be useful in deciding the therapeutic strategy including hematopoietic cell transplantation for the high risk subgroup. There were four MDS/MPD cases with a history of preceding hematological diseases, such as aplastic anemia, MDS or malignant lymphoma. Among these, three cases with a long-term history of treatment with metenolone acetate developed CMML. It is suggested that the long-term effect of androgen plays a role in the pathophysiology of CMML.     

Clinical impact of dysplastic changes in acquired aplastic anemia: A systematic study of bone marrow biopsies in children and adults

Aplastic anemia (AA) is a rare disorder characterized by suppression of bone marrow function, which can progress to myelodysplastic syndrome (MDS) or to acute myeloid leukemia (AML). To determine if there are characteristics in bone marrow biopsies in children and adults previously diagnosed with acquired AA, which could predict progression to MDS, we evaluated 118 hypocellular bone marrow biopsies from adults (76 patients) and children (42) diagnosed initially with acquired AA previously to any treatment. Histology was reviewed according to a detailed protocol including Bennett and Orazi criteria for hypocellular myelodysplastic syndrome (h-MDS) and Bauman et al. criteria for refractory cytopenia of childhood (RCC). Twelve patients (10.2%; 6 children and 6 adults) progressed to MDS after a median time of 56 months. Criteria described by Bennett and Orazi suggestive of h-MDS in bone marrow biopsies were detected in 16 cases (13.5%; 8 adults and 8 children), and none in patients that progressed to MDS/AML. Twenty adults' biopsies (26.3%) had the histological criteria used for the diagnosis of pediatric RCC, and none showed MDS/AML evolution. Ten children (23.8%) were reclassified morphologically as RCC, and only one progressed to MDS. In this population with acquired aplastic anemia (AAA), no histological/immunohistochemical (H/IHC) bone marrow findings could discriminate patients with higher risk for myeloid clonal progression, which questions the diagnosis of h-MDS/RCC based only on the finding of dysplasia in the cases without increased blasts and/or the characteristic genetic abnormalities.     

肝移植患者围手术期骨髓细胞学改变的意义

背景：晚期肝病及肝移植后造血系统受累相关报道较少见。 目的：通过对肝移植患者围手术期骨髓细胞学的观察,探讨移植前造血功能的改变及移植后多种并发症对骨髓造血功能的影响。 方法：选择20例实施原位肝移植患者,因与血液系统疾病相关,需行骨髓细胞学检查进行诊断及鉴别诊断,其中移植前2例,移植后18例。 结果与结论：原位肝移植患者移植前容易出现骨髓细胞学改变为脾功能亢进、大红细胞性贫血,移植后出现：移植物抗宿主病、浆细胞白血病、反应性粒细胞增高、原发性血小板减少性紫癜、嗜血细胞综合征、粒细胞缺乏症、急性造血细胞停滞、转移癌、反应性血小板增高。说明通过骨髓细胞学检查,监测肝移植围手术期各种并发症对骨髓造血功的影响,特别对移植物抗宿主病的患者,实施及时抢救有重要意义。     

真性红细胞增多症患者骨髓单个核细胞植入再生障碍性贫血小鼠

背景：真性红细胞增多症患者骨髓的高增殖低凋亡特性使其造血干细胞植入NOD/SCID小鼠后成功分化出粒红系细胞,但其能否植入并改善再生障碍性贫血小鼠造血功能,目前国内外尚未有报道。 目的：探讨JAK2阳性真性红细胞增多症患者骨髓单个核细胞植入后对再生障碍性贫血小鼠造血重建的影响。 方法：应用注射用重组人γ-干扰素联合白消安的方法建立再生障碍性贫血小鼠模型,随机分为实验组(n=10)和对照组(n=10),给药结束后第5天实验组经尾静脉输注真性红细胞增多症患者骨髓单个核细胞悬液,对照组同法输注等容积生理盐水。输注后第14天检测小鼠外周血常规、骨髓细胞形态、骨髓组织病理变化以及小鼠外周血和骨髓内CD45+细胞的百分含量。 结果与结论：输注后第14天,实验组小鼠血细胞计数三系减少,骨髓涂片可见散在淋巴细胞和早期造血细胞,骨髓组织活检可见骨髓增生减低,少量粒系细胞及幼红细胞,未见巨核细胞,与对照组比较,造血功能无明显改善。对照组小鼠外周血及骨髓均未检测到CD45+细胞,实验组小鼠外周血及骨髓均可检测到人源化的CD45+细胞,且骨髓中CD45+细胞比外周血高,提示JAK2V617F阳性真性红细胞增多症患者骨髓单个核细胞能成功植入再生障碍性贫血小鼠体内,但血常规、骨髓涂片及活检结果显示未能明显改善骨髓造血功能。  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Cytogenetic analysis in human bone marrow transplantation

Chromosome and sex chromatin studies have contributed significantly to the evaluation of human bone marrow transplants. In addition to their use in documenting bone marrow engraftment following transplantation, they have been used to (1) evaluate whether recurrent leukemia occurs in donor or recipient cells and whether recovery in aplastic anemia results from growth of host or donor cells, (2) demonstrate the bone marrow origin of pulmonary macrophages and hepatic Kupffer cells, (3) show that bone marrow fibroblasts have an origin different from that of the hematopoietic bone marrow cells, (4) evaluate twin zygosity in preparation for transplantation, and (5) show that the defect in Fanconi's anemia is intracellular.     

Donor-cell myelodysplastic syndrome developing 13 years after marrow grafting for aplastic anemia

Donor-cell-derived hematopoietic malignancy is a rare event after bone marrow transplantation. Most cases in the literature occurred within the first year. We present a rare case of a female patient who had a bone marrow transplant for severe aplastic anemia (SAA) at the age of two and a half years from her human leukocyte antigen-identical brother. She developed a myelodysplastic syndrome (refractory cytopenia with multilineage dysplasia) 12 years later. Initially, the malignant clone was of recipient origin, but within several months, progression to a clinically more aggressive refractory anemia with excess blasts (RAEB) was accompanied by the outgrowth of a new clone of donor origin. In this report we provide evidence proving that the patient's final malignant clone arose in donor cells: cytogenetic analysis of the marrow showed a male karyotype and a t(3;21)(q26;q21) in all 62 metaphases analyzed. Interphase fluorescence in situ hybridization showed that all identifiable cells contained the Y chromosome. We conclude that donor-cell-derived hematopoietic malignancy after bone marrow transplantation can occur even after many years. We believe that the 13 years that elapsed between the transplant and the development of RAEB in our case represent the longest latency period in the literature.     

Malignant lymphoma and leukemia concepts in new WHO classification

The new WHO classification is based on the principles of REAL classification of lymphoma and expands to myeloid, mast cell and histiocytic/dendritic neoplasms. The distinct diseases are defined according to a combination of morphology, immunophenotype, genetic features, and clinical syndromes, and the cell origin is postulated. Lymphatic leukemia is included in lymphoma. The lymphoid malignancies are grouped into B cell lymphoma, T/NK cell lymphoma and Hodgkin lymphoma, and the myeloid neoplasm are grouped into 4 categories; chronic myeloproliferative diseases(chronic myelogeneous leukemia, polycythemia vera, chronic idiopathic myelofibrosis, essential thrombocythemia etc.), myelodysplastic/myeloproliferative diseases (chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia etc.), myelodysplastic diseases(perfactory anemia, refractory anemia with ringed sideroblasts etc.) and acute myeloid leukemia.     

Evaluation of angiogenesis and vascular endothelial growth factor expression in the bone marrow of patients with aplastic anemia

It is generally appreciated that bone marrow function and growth of myelopoietic cells depends on an intact microvasculature. A pivotal regulator of angiogenesis is vascular endothelial growth factor (VEGF). Here, we describe analysis of VEGF expression and microvessel density in the bone marrow of patients with aplastic anemia by immunohistochemistry. Bone marrow was examined at diagnosis and at the time of hematological remission after immunosuppressive therapy using anti-thymocyte globulin, cyclosporin A, and glucocorticoids or allogeneic stem cell transplantation. At diagnosis, both VEGF expression and microvessel density were found to be significantly lower in aplastic anemia compared to normal bone marrow (aplastic anemia, 1.1 +/- 0.7 events per field, versus controls, 5.9 +/- 3.0 events per field; P < 0.05). In response to successful therapy, VEGF and microvessel density in the bone marrow increased substantially. Serum VEGF levels were also found to be significantly lower at diagnosis in aplastic anemia compared to healthy controls (aplastic anemia, 51 +/- 35 pg/ml versus controls, 444 +/- 220 pg/ml; P < 0.05). VEGF in the serum increased substantially after successful immunosuppressive therapy or stem cell transplantation (P < 0.05). Taken together, these data show that aplastic anemia is associated with reduced angiogenesis and reduced VEGF expression.