Flow cytometry evaluation of erythroid dysplasia in patients with myelodysplastic syndrome.

Erythroid dysplasia is the pathologic hallmark of myelodysplastic syndromes (MDS). To develop a quantitative flow-cytometry approach to its evaluation, we analyzed the expression of CD71, CD105, cytosolic H-ferritin (HF), cytosolic L-ferritin (LF) and mitochondrial ferritin (MtF) in erythroblasts from 104 MDS patients, 69 pathologic control patients and 19 healthy subjects. Six-parameter, 4-color flow cytometry was employed, and data were expressed as mean fluorescence intensity. Compared with pathologic and healthy controls, MDS patients had higher expression of HF (P < 0.001) and CD105 (P < 0.001), and lower expression of CD71 (P < 0.001). MtF was specifically detected in MDS with ringed sideroblasts, and there was a close relationship between its expression and Prussian blue staining (r = 0.89, P < 0.001). In vitro cultures of myelodysplastic hematopoietic progenitors showed that both HF and MtF were expressed at a very early stage of erythroid differentiation, and that MtF expression is specifically related to mitochondrial iron loading. A classification function based on expression levels of HF, CD71 and CD105 allowed us to correctly classify > 95% of MDS patients. This flow-cytometry approach provides an accurate quantitative evaluation of erythroid dysplasia and allows a reliable diagnosis of sideroblastic anemia, and may therefore be a useful tool in the work-up of patients with MDS.     

NOD/SCID mice transplanted with marrow from patients with myelodysplastic syndrome (MDS) show long-term propagation of normal but not clonal human precursors

Sublethally irradiated NOD/SCID mice were transplanted with hematopoietic progenitor cells obtained from the marrow of patients with myelodysplastic syndromes (MDS). Engraftment of MDS cells, as determined by flow cytometry, was delayed compared to marrow from normal donors. Human CD38(+)CD34(-) cells were prominent in marrows and spleens of MDS chimeras. CD34(+)CD38(-), CD34(+)CD38(+) and T cells were also easily detected. Human myeloid cells (CD33(+); CD15(+)) were present in low proportions. No clonal precursors were identified by fluorescent in situ hybridization (FISH) or by molecular analysis of polymorphic X-linked markers in mice with documented engraftment of human cells more than 2 months after transplantation. These data indicate that human cells present in murine MDS chimeras, at the levels of sensitivity of our assays, were derived from residual normal cells in human MDS marrow, and suggest that the NOD/SCID environment was not conducive to the expansion of clonal MDS precursors. This model may allow identification of factors relevant for sustaining or expanding clonal precursors.     

骨髓增生异常综合征免疫表型的研究

目的：评价免疫表型测定在骨髓增生异常综合征(MDS)诊断及分型中的价值。方法：应用单克隆抗体方法对55例MDS患者进行免疫表型检测。结果：MDS患者髓系抗原表达明显增高,FAB亚型的抗原表达呈现规律性改变,随着RA向RAEB／RAEB-t转化,较早期的髓系抗原(CD33)逐渐增加,较成熟的CD15抗原和T-淋巴细胞抗原逐渐减少;同时在RAEB/RAEB—t阶段CD34^ 细胞数明显增高;较早期的骨髓细胞表面抗原(CD38、HLA-DR)在MDS表达明显增加。结论：免疫表型的检测对MDS更精确的诊断和分型有重要意义。     

Effects of recombinant human granulocyte colony stimulating factor and granulocyte-monocyte colony stimulating factor on in vitro hemopoiesis in the myelodysplastic syndromes.

We evaluated the effects of recombinant human granulocyte colony stimulating factor (rhG-CSF) and granulocyte-monocyte colony stimulating factor (rhGM-CSF) on the in vitro proliferative, differentiative, and regenerative responsiveness of marrow cells from myelodysplastic syndrome patients (MDS) in comparison to those from normal individuals. Our studies showed decreased primary clonogenicity of myeloid (CFU-GM) and erythroid (BFU-E) hemopoietic progenitor cells from the MDS patients. rhGM-CSF had more potent stimulatory effects than rhG-CSF for MDS marrow CFU-GM growth; no enhanced cellular proliferation in the MDS patients was observed in liquid culture with either rhGM-CSF or rhG-CSF. Decreased myeloid clonal cell self-generation and/or recruitment occurred in the MDS patients upon exposure to either rhG-CSF or rhGM-CSF. rhG-CSF demonstrated more potent granulocytic differentiation effects than rhGM-CSF both for normals and MDS patients using marrow enriched for immature myeloid cells with lesser differentiation noted for MDS. Cytogenetic abnormalities, present with or without additional normal karyotypes in native marrow of four MDS patients, persisted after culture with rhG-CSF, indicating induced differentiation of both normal and abnormal clones. Although proliferative and differentiative effects were seen with both factors these data show MDS marrow cells in vitro to have predominantly differentiative responsiveness to rhG-CSF and proliferative responsiveness to rhGM-CSF.     

Myeloproliferative defects following targeting of the Drf1 gene encoding the mammalian diaphanous related formin mDia1

Rho GTPase-effector mammalian diaphanous (mDia)-related formins assemble nonbranched actin filaments as part of cellular processes, including cell division, filopodia assembly, and intracellular trafficking. Whereas recent efforts have led to thorough characterization of formins in cytoskeletal remodeling and actin assembly in vitro, little is known about the role of mDia proteins in vivo. To fill this knowledge gap, the Drf1 gene, which encodes the canonical formin mDia1, was targeted by homologous recombination. Upon birth, Drf1+/- and Drf1-/- mice were developmentally and morphologically indistinguishable from their wild-type littermates. However, both Drf1+/- and Drf1-/- developed age-dependent myeloproliferative defects. The phenotype included splenomegaly, fibrotic and hypercellular bone marrow, extramedullary hematopoiesis in both spleen and liver, and the presence of immature myeloid progenitor cells with high nucleus-to-cytoplasm ratios. Analysis of cell surface markers showed an age-dependent increase in the percentage of CD11b+-activated and CD14+-activated monocytes/macrophages in both spleen and bone marrow in Drf1+/- and Drf1-/- animals. Analysis of the erythroid compartment showed a significant increase in the proportion of splenic cells in S phase and an expansion of erythroid precursors (TER-119+ and CD71+) in Drf1-targeted mice. Overall, knocking out mDia1 expression in mice leads to a phenotype similar to human myeloproliferative syndrome (MPS) and myelodysplastic syndromes (MDS). These observations suggest that defective DRF1 expression or mDia1 function may contribute to myeloid malignancies and point to mDia1 as an attractive therapeutic target in MDS and MPS.     

Evaluation of CD7 and terminal deoxynucleotidyl transferase (TdT) expression in CD34+ myeloblasts from patients with myelodysplastic syndrome

There is an emerging use of flow cytometry to evaluate patients with myelodysplastic syndrome (MDS). We have studied CD7 and TdT expression in the CD34+ myeloid blast cell population in 55 bone marrow samples of patients with MDS. CD7 and/or TdT were detected in 38 out of 55 patients (69%). CD7 expression was not related to other bad prognosis data but conversely, we found an association between TdT+ CD34 myeloblasts and high-risk MDS patients according to the International Prognostic Scoring System. Therefore, CD7 and TdT may help to establish the diagnosis of MDS and, TdT expression also seems to be a useful marker in distinguishing risk groups.     

Acute panmyelosis with myelofibrosis: clinical, immunophenotypic and cytogenetic study of twelve cases

The clinical, cytogenetic, and immunophenotypic features in 12 adult patients with acute panmyelosis with myelofibrosis (APMF; ICD-0-3: 9931/3; C42.1) are reported (median age: 57 years; f/m = 1.4). The white cell count (WBC) was normal in 3 patients; 9 had leucopenia. The median hemoglobin value was 64.5 g/l, and median platelet count 12 x 10(9)/l. Bone marrow biopsy showed a hypercellular marrow in 10/12 patients with a significant infiltration of pathological blasts (range: 30 - 60%). All the cases had marked reticulin fibrosis. Immunophenotyping of bone marrow blast cells showed the expression of early (CD34) and lineage-unspecified antigens (HLA-DR) in 6/7, and 7/7 patients, respectively. "Early" myeloid antigens (CD13, CD33) were seen in 6/7 and 4/6 patients respectively. Monocyte antigen (CD14) was expressed in 3/7 patients. Megakaryocyte antigen (CD61) and erythroid cell antigen (GpA) were each expressed in only 1 patient. Two patients had expression of CD34, HLA-DR and "early" myeloid antigens by their bone marrow blast cells and 1 of these also had a co-expression of the antigens from a differentiated monocytic cell proliferation (lysozyme+, CD68+). Nonspecific chromosomal aberrations were recorded in 8/10 patients. The median survival was 2 months. These findings suggest an immature myeloid phenotype of blast cells in APMF. In 6/9 patients a leukemic cell differentiation into monocytic, megakaryocytic or erythroid lineage was also demonstrated.     

In vitro suspension culture reactions to 1,25 dihydroxyvitamin D3 in relation to bone marrow morphology and prognosis in patients with myelodysplastic syndromes.

Thirty-four patients with MDS or AML following MDS were studied with regard to survival, peripheral blood values and bone marrow morphology. The effects of 1,25 dihydroxyvitamin D3 (D3) on differentiation (NBT positivity) and proliferation (3H-thymidine incorporation) were studied in suspension cultures of bone marrow cells. Twelve bone marrow donors served as controls. Normal cells showed spontaneous differentiation in vitro, but only 2/12 were induced to differentiation by D3. Myelodysplastic cells did not differentiate spontaneously, but cells from 18/34 patients differentiated after incubation with D3. Normal cells showed increased proliferation, myelodysplastic cells showed a heterogeneous response and leukemic cells reacted with decreased proliferation after D3 incubation. Poor survival was associated with low platelet counts, high percentage of bone marrow blasts (BM blast %), low spontaneous in vitro proliferation and absence of hypogranulation of myeloid cells. Platelet counts and hypogranulation retained their predictive value in a multi-variate analysis. Progression to AML was predicted by a high BM blast % and low scores for erythroid and total dysplasia. In conclusion, the pattern of in vitro proliferation showed prognostic value while the pattern of vitamin D3-induced differentiation failed to correlate to other parameters. An estimation of bone marrow dysplasia can be used to predict the development of AML. Our results add to the information about the biology of MDS and may be important for the evaluation of therapeutic trials.     

Toll-like receptor-4 is up-regulated in hematopoietic progenitor cells and contributes to increased apoptosis in myelodysplastic syndromes.

PURPOSE: To investigate the function and expression of Toll-like receptors (TLR) in bone marrow cells of myelodysplastic syndrome (MDS) patients and to examine their involvement in the apoptotic phenomenon characterizing MDS hematopoiesis. EXPERIMENTAL DESIGN: TLR mRNA and protein expression was investigated in bone marrow cell populations of MDS patients and controls. TLR-4 ability to recognize lipopolysaccharide and up-regulate self mRNA and protein expression was examined. Tumor necrosis factor involvement in the constitutive and lipopolysaccharide (LPS)-induced TLR expression was also evaluated. Possible correlation between TLR-4 overexpression and apoptosis was investigated by simultaneous staining with Annexin V and TLR-4. RESULTS: TLR-2 and TLR-4 are expressed in almost all bone marrow cell lineages including megakaryocytes, erythroid cells, myeloid precursors, monocytes, and B lymphocytes and are up-regulated in MDS patients compared with controls. In hematopoietic CD34(+) cells, TLR-4 is also expressed and significantly up-regulated at both the mRNA and protein levels. Treatment with an anti-tumor necrosis factor antibody reduces both constitutive and LPS-induced TLR-4 levels. Increased TLR-4 expression correlates with increased apoptosis as TLR-4 is almost exclusively found in apoptotic bone marrow mononuclear and CD34(+) cells. The addition of the TLR-4 ligand LPS further enhances the apoptosis of these cells. CONCLUSIONS: TLR-4 and other TLRs are significantly up-regulated in MDS patients whereas TLR-4 is involved in promoting apoptosis, possibly contributing to MDS cytopenia.     

Multiple distinct clones may co-exist in different lineages in myelodysplastic syndromes

Using single nucleotide polymorphism (SNP) microarray with unfractionized bone marrow specimens, recent studies have demonstrated that multiple cytogenetically cryptic genomic aberrations, uniparental disomy (UPD) and/or copy number (CN) aberration, are present in patients with myelodysplastic syndromes (MDS). We hypothesize that various hematopoietic lineages in MDS may carry different cytogenetically cryptic genomic aberrations leading to lineage-specific manifestations of MDS. Flow cytometry sorting was performed to sort 12 MDS marrow samples into blastic, erythroid, immature myeloid and lymphoid fractions. The fractions with enough DNA underwent 250K SNP microarray analysis. Of importance, different chromosomal regions of UPD, deletions and/or gains were present in different fractions of same patients in all samples. Only small percentages (6.7%) of genomic aberrations were present in all fractions from same patients. These results suggest that multiple distinct clones may co-exist in different lineages in MDS and may contribute to cytopenias in specific lineages and the significant clinical heterogeneity observed in these patients. Further studies are warranted to confirm our findings and to investigate the lineage specific genomic lesions in MDS.     

细胞发育异常在骨髓增生异常综合征患者预后评估中的价值

目的:探讨骨髓细胞病态造血在骨髓增生异常综合征(MDS)向急性白血病转化及预后评估中的价值。方法:回顾性分析82例MDS患者骨髓细胞形态学、细胞遗传学及流式细胞术等相关资料,分析患者骨髓细胞病态造血特点及患者预后转归。结果:82例MDS患者中,25例转化为急性髓系白血病(AML),其中45例存在原始细胞增多中20例转化为急性白血病,37例无原始细胞增多中5例转化为急性白血病,转化率差异有统计学意义(P=0.002);20例存在Auer小体者中13例转化为急性白血病,62例无Auer小体者中12例转化为急性白血病,两组转化率差异有统计学意义(P=0.000 1);31例小巨核病态改变者中15例转化为急性白血病,51例无小巨核病态改变者中10例转化为急性白血病,转化率差异有统计学意义(P=0.006)。其余病态造血未发现与转化为急性白血病有相关性(均P>0.05)。82例MDS患者中,27例死亡。其中存在原始细胞增多、Auer小体以及小巨核者,其生存时间较短。结论:原始细胞增多、Auer小体、小巨核病态改变与转化为急性白血病及其预后有相关性,对指导预后有一定意义。     

Megakaryocytopoiesis and apoptosis in patients with myelodysplastic syndromes

In order to investigate simultaneously the megakaryocytopoiesis and apoptotic characteristics in bone marrow in patients with myelodysplastic syndromes (MDS), we used CD41 immunoenzyme (alkaline phosphatase anti-alkaline phosphatase) and DNA in situ end-labeling techniques on plastic embedded bone marrow biopsy sections of 29 MDS patients. Fourteen patients with iron deficiency anemia served as controls. The results showed that CD41-positive cells in MDS marrow numbered 26.2 +/- 18.2/mm2 (mean +/- standard deviation) compared with 15.6 +/- 7.1/mm2 in controls (P < 0.05). Numbers of cells with the morphology of micro-megakaryocytes in MDS marrow were significantly higher than in controls (P < 0.01). Furthermore, megakaryocytes in MDS marrow were frequently distributed along trabeculae (in 27 cases) and formed clusters (in 25 cases). Apoptotic megakaryocytes in MDS marrow accounted for just 4.4 and 9.3% of all CD41-positive cells and all apoptotic cells, respectively (P > 0.05 compared with controls), but apoptosis occurred only in micro-megakaryocytes. Based on these observations, we conclude that megakaryocytosis and dysmegakaryocytosis are the features of dyshematopoiesis in MDS marrow. Decreased thrombocyte production and thrombocyte release coming from increased dys(micro)megakaryocytes and abnormally located megakaryocytes perhaps play a more important role in peripheral thrombocytopenia than megakaryocytic apoptosis itself. Apoptosis of micro-megakaryocytes may be a protective biological mechanism to remove useless megakaryocytes.     

骨髓增生异常综合征中CD34+细胞CXCR4的表达及其与细胞迁移的相关性

  目的  探讨不同危险度骨髓增生异常综合征（myelodysplasticsymdromes,MDS）中骨髓CD34+细胞CXCR4的表达情况及其与细胞迁移率的相关性。   方法  收集40例骨髓增生异常综合征患者的骨髓标本,根据IPSS积分系统进行危险度分组。低危组20例:IPSS积分0~1.5分;高危组20例:IPSS积分≥1.5分;同时采集10例健康者的骨髓标本作为对照。分离纯化骨髓CD34+细胞,通过流式细胞术检测CXCR4膜蛋白的表达;研究SDF-1α趋化作用下CD34+细胞的迁移率及CD34+细胞对骨髓基质细胞的迁移率。   结果  高危组MDS患者CD34+细胞CXCR4的表达率明显高于低危组和正常对照组（P < 0.000 1）;低危组和正常对照组之间CXCR4的表达率无显著性差异（P>0.05）。高危组CD34+细胞对SDF-1α及骨髓基质细胞的迁移率显著高于低危组及正常组（均P < 0.000 1）,且其对骨髓基质细胞的迁移率与CXCR4的表达呈正相关（P=0.000 1）。   结论  高危组MDS患者CD34+细胞CXCR4的表达量及其对骨髓基质细胞的迁移率均明显高于低危组患者,且其迁移率随CXCR4表达量的增加而升高,不同风险组的MDS患者存在SDF-1及其受体CXCR4表达和功能上的差异,SDF-1及其受体CXCR4在MDS发病中具有重要作用。      

Copper deficiency with increased hematogones mimicking refractory anemia with excess blasts

We describe a 19-year-old male patient with a previous diagnosis of familial Mediterranean fever (FMF), nephrotic syndrome and secondary amyloidosis, who presented with anemia and leukopenia. The bone marrow assessments showed dysplastic precursors including vacuolated myeloid and erythroid precursors and increased proportion of immature cells up to 19%. The patient received erythropoietin and G-CSF for myelodysplastic syndrome (MDS). No response was observed. During his evaluations copper deficiency was detected. One month after oral copper replacement, the peripheral blood counts and bone marrow findings became completely normalized. An evaluation to identify the cause of copper deficiency, revealed intestinal amyloidosis. Based on our experience we recommend serum copper determination in the diagnostic workup of MDS in patients with comorbidities.     

Angiogenesis in relation to clinical stage, apoptosis and prognostic score in myelodysplastic syndromes

The International Prognostic Scoring System (IPSS), based on the number of cytopenias, percentage of bone marrow blasts and cytogenetics, is an important prognostic tool for patients with myelodysplastic syndrome (MDS). In addition, factors such as high bone marrow cellularity and lactate dehydrogenase levels have been associated with an adverse outcome, spontaneously and after chemotherapy. Recently, increased bone marrow angiogenesis, measured as, e.g. microvascular density (MVD), was reported to be more intense in high-risk than in low-risk MDS. To assess the prognostic role of MVD in MDS, a cohort of 56 patients, thoroughly investigated for various clinical and morphological parameters, were followed-up for survival > or =60 months after the diagnostic analysis. As a group MDS patients had higher MVD compared to healthy controls (p<0.02). The highest median MVD value was observed in the RAEB group, but there was no overall significant difference between the FAB groups. No significant correlations were observed between MVD and peripheral blood counts, bone marrow cellularity, percentage of bone marrow blasts and CD34 positive cells, apoptotic index (TUNEL), proliferation index (MIB-1), erythroid index, FAB group and IPSS score. MVD was not correlated to overall survival. In contrast, bone marrow blast count <5%, low or normal cellularity, as well as a high erythroid index, indicated a favorable survival. Thus, our data do not support an important prognostic role of angiogenesis, reflected by microvessel density, in the myelodysplastic syndromes.     

Reduction in multi-lineage and erythroid progenitors distinguishes myelodysplastic syndromes from non-malignant cytopenias

We studied the diagnostic role of CFC assays in myelodysplastic syndromes (MDS) using CFC data from bone marrow (BM) and peripheral blood (PB) of 221 MDS patients, 51 patients with non-malignant causes of cytopenia and/or dysplasia and 50 normal controls. A consistent decrease in BM but not PB multi-lineage and erythroid progenitor frequencies was seen in patients with MDS compared to controls (P < 0.05). Automated distinction showed a sensitivity of 87 ± 6% and a specificity of 71 ± 11% in classifying MDS patients. In conclusion, a defect in early hematopoietic progenitor activity, in particular erythroid activity, distinguishes MDS from non-MDS.     

世界卫生组织标准在骨髓增生异常综合征诊断分型中的应用

 【摘要】　目的　依据世界卫生组织（WHO）标准对249例骨髓增生异常综合征（MDS）进行诊断分型。方法　根据WHO（2008）标准对249例MDS行细胞形态学、细胞遗传学、免疫表型分析及骨髓病理活组织检查诊断。结果　所有MDS病例外周血成熟红细胞均见有巨大形与椭圆形改变;幼红、幼粒细胞、Pelger样核异常细胞与无颗粒中性粒细胞检出率随分型进展而增高;骨髓发育异常细胞特点及比例随MDS亚型进展而更明显、更高; 经动态随访观察, MDS随分型进展其转急性白血病（AL）率增高（P＜0.05）;148例患者行免疫表型分析,随分型进展髓系特异性抗原（CD33）表达逐渐增加;138例患者行染色体检查,其中异常核型表达53例（38.7 %）,主要见于20q-和+8,复杂异常核型16例（28 %）,其中5q- 2例;180例患者同时行骨髓活组织检查,19例具有形态异常,符合MDS诊断,42例伴有骨髓纤维化。结论　依据WHO标准多指标联合检测MDS有助于更准确的分型及诊断,进行长期追踪随访有助于判断预后。     

骨髓增生异常综合征345例病态造血特点分析

 目的　分析骨髓增生异常综合征（MDS）病态造血特点。方法　收集2003年7月4日至2007年3月14日原因不明血常规异常的成年患者标本716例,以WHO MDS分类标准为诊断金标准,分别进行细胞形态学、细胞化学染色、骨髓病理检查、细胞遗传学、流式细胞术等检测。分析骨髓细胞学检查中病态造血特征在判断克隆性和非克隆性疾病中的诊断价值,计算灵敏度和特异度。结果　MDS病态造血形态学诊断的主要依据：粒系Auer小体、核出芽、微核有其中之一者;红系核出芽;外周血片中出现巨核细胞;外周血片中出现原粒细胞或早幼红细胞;环状铁粒幼细胞>1 %。次要依据：粒系假Pelger-Hǔet 异常、不能分叶中性粒细胞、同一细胞内核发育不同步、环形核、核染色质聚集;红系的多核、奇数核、子母核、核碎裂、空泡、成熟红细胞大小悬殊;微巨核。结论　细胞形态学是诊断MDS的基础,但也存在一定的局限性,尤其是对于早期MDS细胞形态学改变不典型时,需要结合其他检测手段分析。     

骨髓增生异常综合征和再生障碍性贫血患者骨髓CD34+细胞测定

 目的 检测骨髓增生异常综合征（MDS）和再生障碍性贫血（AA）患者骨髓CD+34细胞占单个核细胞（MNC）的比率,以探讨二者可能的发病机制。方法 用流式细胞术（FCM）检测22例MDS患者、13例AA患者及12例非血液病患者骨髓CD+34细胞占MNC的比率。结果 AA组与对照组、AA组与MDS-RA组、AA组与MDS-RAEB组、MDS-RA组与MDS-RAEB组的骨髓MNC中CD+34细胞的比率的比较差异有统计学意义（P＜0.05）。大多数重型AA（SAA）患者（3／4）及很少慢性AA（CAA）患者（1／9）的骨髓MNC中的CD+34细胞的比率＜0.1 %。结论 骨髓CD+34细胞的检测有助于判断AA患者病情及MDS患者的预后,亦可用于鉴别AA和MDS。