骨髓增生异常综合征患者骨髓细胞 凋亡的初步探讨

采用TUNEL法对26例骨髓增生异常综合征（MDS）、11例巨幼细胞贫血（MegA)、8例阵发性血红蛋白尿（PNH）、6例Evans综合征患者和10例正常健康志愿者的单个核细胞（BMMNC）凋亡进行探讨,结果发现,50％MDS呈现凋亡过度,MegA、PNH和Evans综合征患者与对照组无显著差异。MDS患者凋亡累及粒、红、巨三系各阶段造血细胞,随病情演变,凋亡程度渐下降,提示凋亡过度为MDS无效造血机制之一,病情演变化可能与异常克隆逃逸凋亡有关,抗凋亡疗法可试用于早期MDS。     

Effects of various recombinant human hemopoietic growth factors (rhEpo,rhG-CSF,rhGM-CSF,rhIl-3) on the growth of peripheral blood progenitor cells (BFU-E,CFU-GM)

Summary The effects of rhEpo 1, rhG-CSF, rhGM-CSF and rhIl-3 on the growth of both CFU-GM and BFU-E from normal human adult peripheral blood have been studied in plasma clot cultures. Using optimal concentrations of all growth factors, alone and in combination with all other factors, rhIl-3 showed the highest activity in regard to growth of both CFU-GM and BFU-E, whereas rhGM-CSF treatment resulted only in half-maximal colony growth compared to rhIl-3. No synergism or additive effect was seen with the combination of rhIl-3 and rhGM-CSF. Treatment with rh-G-CSF had no additional effect with optimal concentrations of rhIl-3 and/or rhGM-CSF. When suboptimal concentrations of rhGM-CSF and rhIl-3 were applied, however, they showed a marked synergism on both BFU-E and CFU-GM. RhGCSF, added to a suboptimal concentration of rhGM-CSF, resulted in a marked growth increase of CFU-GM but had no effect on BFU-E.Abbreviations BFU-E Burst forming unit-erythroid - CFU-GEM Colony forming unit-granulocytic/erythroid/monocytic/megacaryocytic - CFU-GM Colony forming unit-granulocytic/monocytic - rhEpo Recombinant human erythropoietin - rhG-CSF Recombinant human granulocyte-colony stimulating factor - rhGM-CSF Recombinant human granulocyte/monocyte-colony stimulating factor - RhIl-3 Recombinant human Il-3     

Cytogenetic findings in 179 patients with myelodysplastic syndromes

Cytogenetic analyses were performed on 266 bone marrow and peripheral blood samples from 179 patients with myelodysplastic syndromes (MDS). According to the FAB classification, 42 patients presented with RA, 18 with RARS, 37 with RAEB, 22 with CMML, and 29 with RAEB-T. Nine patients showed a secondary MDS (S MDS). FAB classification was not available for 22 patients. Clonal karyotype anomalies were found in 92 patients (51.4%). Complex chromosome abnormalities occurred in 17 (18.5%) of them. An evolution of the karyotype was detected in 16 cases (17.4%). Cytogenetically independent cells or cell clones were found in eight patients. Nonclonal chromosome abnormalities were uncovered in 29 (16.2%) of the 179 MDS patients. Consecutive studies were performed in 48 patients and revealed a good correlation of initial karyotype and clinical course. The most frequent single anomalies were 5q- in 29 (31.5%), –7 in 22 (23.9%), trisomy 1q in 14 (15.2%), and +8 in 13 (14.1%) of 92 patients respectively. Our cytogenetic findings are presented in detail and discussed in relation to patients' age, morphological classification, clinical course, and prognostic impact. The contribution of cytogenetic findings to the delineation of multistep pathogenesis of MDS with special emphasis to karyotype instability is demonstrated.     

Management of high-risk myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are a group of clonal disorders characterized by one or more cytopenias secondary to bone marrow dysfunction. The percentage of bone marrow blasts, the number of cytopenic cell lines and cytogenetics define more precisely clinical risk groups. In the high-risk MDS, the time for evolution to acute myeloid leukemia (AML) is between 0.2 and 1.1 years and the median survival has been evaluated between 0.4 and 1.2 years. Progress in the understanding the biology of MDS and the development of accurate prognostic classification systems have allowed a risk-adapted treatment strategy in individual patients. Some high-risk MDS patient categories may benefit from intensive cytotoxic treatment. Allogeneic stem cell transplantation (alloSCT) from donors remains the treatment of choice for younger patients. Autologous stem cell transplantation (ASCT) may provide a suitable alternative for those patients without a sibling donor or for older patients' categories. New regimens using non-myeloablative stem cell transplantation followed by donor lymphocyte infusions (DLI) are underway and have achieved promising results in HLA-identical transplantation, resulting in reduced morbidity and mortality and confirming that this approach is feasible in patients ineligible for conventional allografting due to age and/or organ toxicity. Other therapeutic strategies include new low-dose treatments, antiapoptotic agents such as amifostine and anticytokine therapy, which are currently under investigation and deserve further evaluation. More insights into the biology of the disease, the discovery of new therapeutic approaches and the search for better ways to use existing strategies may lead to more effective treatments.     

Feasibility of peripheral blood progenitor cell harvest and transplantation in patients with poor-risk myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are a group of clonal haematological disorders with a highly unfavourable prognosis. Allogeneic bone marrow transplantation offers the sole possibility for cure and prolonged survival, but is only available for a minority of patients. Therefore, we investigated the feasibility of PBPC collection and transplantation in 11 patients with high-risk myelodysplasia who were not eligible for allogeneic bone marrow transplantation. In six patients, PBPC were harvested after mobilization with G-CSF alone. Five patients were harvested during the recovery phase of intensive chemotherapy combined with G-CSF. This resulted in seven patients in an adequate CD34 progenitor yield >1 × 10⁶/kg. Six patients obtained a CFU-GM content of the PBPC harvest >10 ×10⁴/kg. Five patients were subsequently transplanted following a standard BuCy4 regimen. The median to ANC (absolute neutrophil count) ≥0.5 and 1.0 × 10⁹/l was respectively 14 d (range 10–18) and 16 d (range 11–25). Platelets were self-supporting ≥20 × 10⁹/l after a median of 41 d (range 8–144). One patient had a persistent lack of platelet engraftment unresponsive to infusion of back-up bone marrow.
These data demonstrate that in selected patients with high-risk MDS, adequate PBPC collection appears feasible, enabling the harvest of sufficient cell numbers required for rapid and stable engraftment after reinfusion. Improvement in mobilization efficiency may enable the collection of higher CD34⁺ progenitor cell numbers required for more rapid platelet engraftment. PBPC transplantation may be an alternative treatment option for patients who lack an allogeneic marrow donor. Follow-up is, however, still too limited to draw any conclusion regarding the long-term cure rate.     

Correlations between cytogenetics and morphology in myelodysplastic syndromes

Summary In order to detect possible relationships between cytogenetic abnormalities and morphologic features in myelodysplastic syndromes (MDS), 48 patients with MDS were investigated. Clonal cytogenetic abnormalities were present in bone marrow cells from 27 patients (56%). The most frequent single anomaly was del (5 q) (10 cases), followed by monosomy 7 (3 cases), trisomy 8 (3 cases) and del (20 q) (2 cases). Complex anomalies were present in 6 patients. Morphologically, according to the French-American-British (FAB) classification: 17 cases were considered as refractory anemia (RA), 17 as RA with excess of blasts (RAEB), 2 as RAEB in transformation, 2 as acquired idiopathic sideroblastic anemia and 10 as chronic myelomonocytic leukemia. With regard to the FAB classification, del (5 q) was often associated with RA and complex cytogenetic anomalies with RAEB. When myelodysplasia was studied in individual myeloid lineages, del (5 q) was associated with hypolobulated megakaryocytes, monosomy 7 with micromegakaryocytes and complex chromosomal anomalies with the association of two or more features of dysmegakaryocytopoiesis. Del (11 q) was associated with increased iron storage and del (20 q) with marked dyserythropoiesis. No correlation was observed between cytogenetic anomalies and features of dysgranulocytopoiesis.     

Chromosome and molecular abnormalities in myelodysplastic syndromes

Cytogenetic abnormalities are seen in approximately 50% of cases of myelodysplastic syndrome (MDS) and 80% of cases of secondary MDS (following chemotherapy or radiotherapy). These abnormalities generally consist of partial or complete chromosome deletion or addition (del5q, -7, +8, -Y, del20q), whereas balanced or unbalanced translocations are rarely found in MDS. Fluorescence hybridization techniques (fluorescence in situ hybridization [FISH], multiplex FISH, and spectral karyotyping) are useful in detecting chromosomal anomalies in cases in which few mitoses are obtained or rearrangements are complex. Ras mutations are the molecular abnormalities most frequently found in MDS, followed by p15 gene hypermethylation, FLT3 duplications, and p53 mutations, but none of these abnormalities are specific for MDS. The rare cases of balanced translocations in MDS have allowed the identification of genes whose rearrangements appear to play a role in the pathogenesis of some cases of MDS. These genes include MDS1-EVI1 in t(3;3) or t(3;21) translocations, TEL in t(5;12), HIP1 in t(5;7), MLF1 in t(3;5), and MEL1 in t(1;3). Genes more frequently implicated in the pathogenesis of MDS cases, such as those involving del5q, remain unknown, although some candidate genes are currently being studied. Cytogenetic and known molecular abnormalities generally carry a poor prognosis in MDS and can be incorporated into prognostic scoring systems such as the International Prognostic Scoring System.     

骨髓增生异常综合征多指标综合诊断的前瞻性研究

目的建立多指标综合诊断骨髓增生异常综合征(MDS)诊断标准。方法以前瞻性方法验证从回顾性研究中得出的综合诊断MDS的8项指标。观察中国医学科学院血液病医院2000年1月至2004年6月住院MDS患者,其中多指标诊断组38例,同期对照组79例,既往对照组123例,对比分析3组患者转化为白血病的情况。结果随访期间多指标诊断组18例转化为白血病,转白率为47.37%,中位转白时间6(1～33)个月。同期对照组18例转化为白血病,转白率为22.78%(与多指标诊断组相比,P<0.01),中位转白时间6(1～33)个月。既往对照组16例转化为白血病,转白率为13.01%(与多指标诊断组相比P<0.01),中位转白时间5(1～23)个月。多指标转白的相对危险度:骨髓原始粒细胞和单核细胞≥0.020的相对危险度(RR)为9.11,髓系细胞分化指数≥1.8的RR为6.50,有淋巴样微巨核RR=4.55,外周血中出现幼稚粒、单核细胞RR=4.40,骨髓有核红细胞糖原染色阳性RR=4.26,染色体核型异常RR=2.87,骨髓细胞体外培养粒、单核细胞系集簇与集落比值≥4.0的RR为2.14。结论多指标综合诊断较全面反映MDS恶性造血克隆的生物学本质,能更准确地诊断MDS患者。     

细胞凋亡与骨髓增生异常综合征疾病演变的关系

目的探讨骨髓增生异常综合征（ＭＤＳ）骨髓细胞凋亡特征及其病理学意义。方法用原位末端脱氧核糖核苷酸转移酶介导的ｄＵＴＰ缺口末端标记（ＴＵＮＥＬ），ＤＮＡ梯子和体外培养等方法对３８例ＭＤＳ患者骨髓单个核细胞（ＢＭＭＣ）凋亡进行研究。结果ＭＤＳ患者ＢＭＭＣ的ＴＵＮＥＬ凋亡细胞阳性指数（ＰＩ）为２０．１９％±１１．０７％，显著高于正常人、ＭＤＳ转化的白血病、急性髓细胞白血病（ＡＭＬ）及缺铁性贫血（ＩＤＡ）组，（Ｐ＜０．００１和Ｐ＜０．０１）。ＴＵＮＥＬ和ＣＤ４１碱性磷酸酶抗碱性磷酸酶免疫酶标法（ＡＰＡＡＰ）双标记证明ＭＤＳ骨髓病态小巨核细胞发生了凋亡。动态观察１０例ＭＤＳ患者，转为白血病后凋亡细胞ＰＩ值显著下降，尚未转为白血病患者随病情恶化凋亡呈下降趋势。７例ＭＤＳ患者ＢＭＭＣ体外培养后，晚期ＰＩ（ＬＰＩ）显著增高，３例出现ＤＮＡ梯子，ＡＭＬ和ＩＤＡ组ＬＰＩ未见明显升高，未出现ＤＮＡ梯子。结论ＭＤＳ骨髓细胞凋亡过度与演变和转为白血病有关。原位检测细胞凋亡可作为监测ＭＤＳ预后指标     

Emerging immunological concepts in the pathogenesis of myelodysplastic syndromes

The involvement of T-lymphocytes in the pathogenesis of myelodysplastic syndromes (MDS) is now well documented by relevant clinical and experimental findings. This brief review will focus on the T-cell repertoire pattern typical of MDS patients as well as on the potential role exerted by specific T-cell subsets in this context. Future investigations should further explore the specific role played by different T-cell subsets in the bone marrow milieu typical of MDS, further clarifying which of the described changes represent either an epiphenomenon or rather a real causative factor in the pathogenesis of these disorders.     

Alterations of CD43 expression in transfusion-dependent myelodysplastic syndromes

CD43 is a sialylated glycoprotein expressed on the surface of most haemopoietic cells and has been implicated in cell adhesion and signaling. It has previously been shown that CD43 expression is altered in patients with myelodysplastic syndrome (MDS). This raised the question of whether the alteration is associated with transfusions in these patients. We studied the expression of this antigen on peripheral blood leucocytes in two groups of patients with refractory anaemia, 22 transfused and 20 non-transfused. We found decreased expression of CD43 on the monocytes and neutrophils of patients receiving transfusions. Other activation molecules were studied (CD11b, CD18) and were found up-regulated suggesting the existence of activated leucocytes in these patients. The increased levels of soluble vascular cellular endothelial molecule after transfusions in these patients suggested vascular endothelial activation in the absence of infection. Given together, these results show that decreased CD43 in the transfused group of MDS patients is associated with an activated endothelial phenotype.     

风湿性疾病合并骨髓增生异常综合征九例临床分析

目的 了解风湿性疾病合并骨髓增生异常综合征（MDS）的临床特点及可能的发病机制。方法 对住院确诊的9例风湿性疾病合并MDS的临床特征进行统计分析。结果 9例MDS患者中有4例诊断MDS前已有风湿病史，9例确诊MDS时均有活动期风湿病表现，伴有红细胞沉降率增快、不同程度血清免疫球蛋白升高（主要为IgG和IgM）及多种不同的自身抗体阳性，其中2例合并类风湿关节炎（RA）和RA＋抗磷脂综合征（APS），4例系统性红斑狼疮（SLE）和SLE＋多发性肌炎（PM），1例成人斯蒂尔病，1例伴有急性关节炎、皮肤血管炎等风湿病表现，1例伴有多浆膜腔积液、蛋白尿、血清补体下降、Commb’s试验阳性等狼疮样表现。结论 风湿性疾病合并MDS时多有不同的活动期风湿病表现．患者同时存在免疫功能紊乱和造血功能异常，可能由于免疫功能失调，如T、B细胞功能异常、高丙种球蛋白血症、出现自身抗体等引起不同的风湿性疾病表现，说明MDS与风湿性疾病之间存在内在的关联。     

Insights into myelodysplastic syndromes from current preclinical models

In recent years, there has been significant progress made in our understanding of the molecular genetics of myelodysplastic syndromes (MDS). Using massively parallel sequencing techniques, recurring mutations are identified in up to 80% of MDS cases, including many with a normal karyotype. The differential role of some of these mutations in the initiation and progression of MDS is starting to be elucidated. Engineering candidate genes in mice to model MDS has contributed to recent insights into this complex disease. In this review, we examine currently available mouse models, with detailed discussion of selected models. Finally, we highlight some advances made in our understanding of MDS biology, and conclude with discussions of questions that remain unanswered.     

Aggressive chemotherapy in adult primary myelodysplastic syndromes

Summary Twenty-nine adult patients with primary myelodysplastic syndromes (MDS) and an excess of marrow blasts were treated by aggressive chemotherapy while still in MDS phase (20 cases) or after progression to ANLL (9 cases). Median age was 47.5 (range 18–68). Twenty-eight patients received a combination of Rubidazone and Ara C and 1 received High dose Ara C. Fourteen patients (48%) achieved complete remission (CR), 5 (17%) were treatment failures (F) and 10 (35%) died during therapy induced aplasia (DA). Median disease free survival was 8.5 months. Median survival of the whole population was 6 months from the onset of treatment, and 17 months in patients achieving CR. These results were significantly less favorable than those obtained at our institution in de novo ANLL with the same chemotherapy regimens. No statistically significant prognostic factors of treatment outcome emerged but patients with normal cytogenetic findings seemed to have both a higher CR rate and longer remissions than patients with abnormal karyotypes. Patients under 50 did not have higher CR rates than older patients, although they had longer remissions (with 3 out of 6 CRs exceeding 2 years). Finally, treatment outcome and survival were identical in patients treated in the MDS phase and in those treated after progression to ANLL. Combination chemotherapy is a highly toxic approach in MDS and essentially seems to benefit younger patients with a normal karyotype, in whom some long remissions can be obtained.     

Phase II clinical study of erlotinib for treatment of myelodysplastic syndromes

Outcome in patients with myelodysplastic syndrome (MDS) after azanucleoside failure is poor with unmet need for active novel agents. Preclinical data have suggested that erlotinib has in vivo and in vitro off epidermal growth factor receptor (EGFR)‐target activity in MDS. We conducted a phase II study with single‐agent erlotinib 150 mg/day orally in MDS patients following azanucleoside failure. All intermediate‐2 or high‐risk MDS patients by International Prognostic Scoring System and only those low/intermediate‐1 patients with transfusion‐dependent anemia or platelet counts <50 × 10⁹/L or a significant clinical hemorrhage requiring platelet transfusion or ANC <1 × 10⁹/L were eligible, with most of our patients being at high risk. In 35 eligible patients, overall best response was 14% (3 patients having marrow complete response and 2 hematological improvement). Four deaths occurred on study (sepsis, intracranial hemorrhage, sudden death, and acute myeloid leukemia (AML)). The most common observed grade 3/4 toxicities according to CTCAE v3 were diarrhea (17.1%), rash (17.1%), and infection (11.6%), accompanied by fatigue, thrombocytopenia, and anorexia at 5.7% each. Median overall survival was 6.8 months (95% CI 4.9–13.2), and leukemia‐free survival was 5 months (95% CI 3.4–7.3). Erlotinib was generally well tolerated, with modest single‐agent activity. Given these results and preclinical data suggesting synergistic effect with azanucleosides, the combination should be further explored. Am. J. Hematol. 89:809–812, 2014. © 2014 Wiley Periodicals, Inc.     

Prognostic relevance of histological findings on bone marrow biopsy in myelodysplastic syndromes

Summary Bone marrow biopsy (BMB) has aroused growing interest as a possible aid in the diagnostic and prognostic evaluation of myelodysplastic syndromes (MDS). Previous reports have pointed out that MDS patients with blastic aggregates or severe bone marrow (BM) fibrosis are characterized by a worse clinical outcome. BMBs of 106 MDS patients were retrospectively reviewed, and relationships among the different histological parameters as well as clinicopathological correlations were looked for. Three patterns of BM blastic infiltration (diffuse, cluster, and large) were recognized. Overt leukemic transformation and overall survival were selected as prognostic end points. BM infiltration was diffuse in 18, cluster in 48, and large in 40 cases. RAEB-t patients accounted for about half of the large cases, and none had a diffuse pattern (p<0.01). Nineteen patients showed extensive BM fibrosis; most of them were characterized by cluster blastic infiltration and megakaryocyte hyperplasia. Leukemic transformation occurred in 67% of large cases (p<0.001) and in none of the cluster cases with severe BM fibrosis (p<0.01); however, survival was equally poor in these two groups because of early leukemic transformation (large cases) and BM failure (cluster cases). The FAB classification did not significantly correlate with prognosis. Patients with cluster BM infiltration and severe fibrosis can be regarded as a true separate MDS subset characterized by unique clinicopathological and prognostic features. Because of the subacute clinical behavior of most cases, and the poor performance status of many elderly patients, there is still controversy as to the best therapeutic approach in MDS. Histological analysis allowed two groups of MDS patients to be identified, both characterized by poor life expectancy, who could benefit from early aggressive chemotherapy.     

Clonality in the myelodysplastic syndromes

The myelodysplastic syndromes (MDSs) comprise a heterogeneous group of stem cell disorders involving cytopenia and dysplastic changes in 3 hematopoietic lineages. Although it is accepted that MDS is a clonal disorder, the exact nature of the involvement of multipotent stem cells and progenitor cells has not been resolved. Most clonality studies of MDS support the proposal that the primary neoplastic event occurs, in most patients, at the level of a committed myeloid progenitor cell, capable of differentiation into multiple myeloid lineages. The extent of the involvement of T and B lymphocytes in MDS remains controversial. Much of the variation reported may result from disease heterogeneity and technical issues such as skewed methylation patterns occurring in studies analyzing X-chromosome inactivation patterns (XCIP) and possible impurities in lymphocyte preparation. A great deal of the evidence in support of T-lymphocyte involvement in MDS has been generated by XCIP studies, and some of these data need to be treated with caution, especially data from studies in which appropriate controls were omitted. In contrast, B-lymphocyte involvement in some patients with MDS is based on studies using more robust technology including combined immunophenotyping and fluorescence in situ hybridization. Clonality studies involving myeloid and lymphoid cells in MDS have yielded discrepant results with regard to the potential involvement of multipotent (lympho-myeloid) hematopoietic stem cells (HSCs). However, failure to detect a clonal marker in all cells of all lineages does not preclude multipotent-HSC involvement. Some recent studies have produced compelling evidence to show that, in some patients with MDS, the multipotent HSC is the target of the primary neoplastic event. It now seems probable that MDS arises in multipotent HSCs more commonly than previously recognized. Such data not only provide important new insights into the biology of MDS but also may have therapeutic implications. The determination of whether multipotent HSCs are involved in the MDS clone may be important for the use of autologous stem cell transplantation in these patients.