骨髓增生异常综合征患者Dickkopf-3基因启动子区甲基化状态研究

目的 了解骨髓增生异常综合征(MDS)患者WNT/β-catenin信号转导通路中拮抗基因Dickkopf-3(Dkk3)的甲基化状态,初步探索其甲基化状态与MDS发生及疾病进展的关系.方法 应用甲基化特异性PCR(MSP)法对43例MDS患者的骨髓或外周血Dkk3基因启动子区甲基化状况进行检测,并以70例门诊普通患者的外周血检测结果作为对照,同时对患者进行随访.结果 43例MDS患者中检出7例(16.3％)Dkk3甲基化,其中5例(11.6％)为半甲基化状态,2例(4.7％)为完全甲基化状态,70例正常对照中1例(1.4％)为Dkk3甲基化,组间DKK3甲基化率差异有统计学意义(x2=8.93,P=0.005).7例Dkk3甲基化的样本中,2例来自骨髓,5例来自外周血,其中难治性贫血(RA)2例,难治性细胞减少伴多系异常(RCMD)1例,难治性贫血伴原始细胞增多(RAEB)4例.单因素分析示不同性别、染色体核型、样本来源(骨髓/外周血)、危险度(WHO分类的预后评分系统≤2分及＞2分)及老年组与非老年组间甲基化率差异均无统计学意义(均P＞0.05).在35例长期随访患者中,9例患者转化为急性髓系白血病(AML)(6例DKK3甲基化者中3例,29例DKK3非甲基化者中6例),疾病进展组与稳定组间甲基化率差异无统计学意义(P＞0.05);死亡12例中3例为Dkk3甲基化阳性,但甲基化与非甲基化组间生存率差异无统计学意义(P＞0.05).结论 MDS患者中Dkk3基因存在较高的甲基化修饰,这可能是MDS发病的分子机制之一.本组患者例数较少,尚未发现甲基化与临床预后间的相关性.     

异常核型骨髓增生异常综合征64例预后分析

 【摘要】　目的　探讨骨髓增生异常综合征（MDS）患者染色体异常与预后的关系,对治疗效果进行分析。方法　回顾性分析122例MDS患者染色体核型,用吉姆萨显带法进行检测。难治性贫血（RA）、环形铁幼粒细胞难治性贫血（RAS）的治疗以诱导分化剂及刺激造血药物为主。原始细胞过多难治性贫血（RAEB）、转化型原始细胞过多难治性贫血（RAEB-t）、慢性粒-单核细胞白血病（CMML）的治疗以小剂量化疗和小剂量联合化疗方案为主。分析异常核型MDS患者疗效,以同期住院的正常核型MDS患者为对照。结果　检出异常核型MDS患者64例,治疗后完全缓解（CR）17例,CR率26.6 %。同期正常核型MDS患者58例,CR 30例,CR率51.7 %。正常和异常核型患者CR率差异有统计学意义（χ2＝8.13,P＝0.04）。复杂核型、-7、+8核型异常者易进展为急性白血病。结论　染色体核型分析在MDS的诊断与预后判断中有重要意义,不同的染色体核型改变进展为白血病的风险不同。     

骨髓增生异常综合征患者染色体核型分析

 目的　对骨髓增生异常综合征（MDS）染色体核型进行分析并结合血细胞计数、骨髓原始细胞数对其预后进行评估。方法　采用直接法、短期培养法和反带技术制备染色体,进行核型分析。结果　49例MDS患者中有22例（44.9 %）检出异常克隆。核型异常包括数目异常和结构异常,数目异常以-7,+8最常见。难治性贫血伴原始细胞增多（RAEB1和RAEB2）较难治性贫血（RA）和难治性贫血伴有环状铁粒幼细胞（RARS）检测到更高的异常核型比例。MDS染色体核型异常与原始细胞比例呈正相关。结论　染色体核型分析对MDS诊断、治疗及预后评估有重要价值。     

骨髓增生异常综合征各亚型染色体核型分布与预后的关系

目的 探讨骨髓增生异常综合征（MDS）各亚型中的染色体核型分布特点及其与预后的关系。方法 回顾分析151例原发性MDS患者的染色体核型,比较各亚型中的染色体核型分布特点、国际预后积分系统（IPSS）评分、白血病转化率及死亡率等,并比较其在汉族与维吾尔族MDS患者中有无民族差异性。结果 所有患者核型异常检出率为55.0 %（83／151）,其中简单异常占53.0 %（44／83）,复杂异常占47.0 %（39／83）。伴多系病态造血的难治性血细胞减少症（RCMD）、原始细胞过多的难治性贫血（RAEB）-Ⅰ、RAEB-Ⅱ亚型中复杂异常的检出率明显高于难治性贫血（RA）、环形铁粒幼细胞增多的RA（RARS）亚型。核型异常涉及各条染色体,发生频率较高的染色体畸变依次为-5／5q-、-7／7q-、＋8、-20／20q-、-X／-Y、i（17q）、9p-／9q-、＋21等。IPSS评分在各亚型中差异有统计学意义（χ2＝117.802,P＜0.01）;高危组的核型异常检出率明显高于低危组和中危组（均P＜0.05）。随访151例患者白血病转化率和死亡率分别为25.2 %（38／151）和43.7 %（66／151）,核型异常者白血病转化率和死亡率明显高于核型正常者（均P＜0.05）。核型异常者白血病转化中位时间和生存中位时间均短于核型正常者。汉族与维吾尔族MDS患者各亚型分布、核型异常特点及白血病转化率、死亡率等方面差异均无统计学意义（均P＞0.05）。结论 染色体核型异常在MDS不同亚型中存在差异且与预后密切相关,是影响MDS患者病情进展及预后的重要指标,对MDS的正确诊断、病情监测及预后评估有重要意义。     

骨髓增生异常综合征患者染色体核型分析

目的 对骨髓增生异常综合征(MDS)染色体核型进行分析并结合血细胞计数、骨髓原始细胞数对其预后进行评估.方法 采用直接法、短期培养法和反带技术制备染色体,进行核型分析.结果 49例MDS患者中有22例(44.9%)检出异常克隆.核型异常包括数日异常和结构异常,数目异常以-7,+8最常见.难治性贫血伴原始细胞增多(RAEB1和RAEB2)较难治性贫血(RA)和难治性贫血伴有环状铁粒幼细胞(RARS)检测到更高的异常核型比例.MDS染色体核型异常与原始细胞比例呈正相关.结论 染色体核型分析对MDS诊断、治疗及预后评估有重要价值.     

骨髓增生异常综合征患者染色体核型分析

目的 对骨髓增生异常综合征(MDS)染色体核型进行分析并结合血细胞计数、骨髓原始细胞数对其预后进行评估.方法 采用直接法、短期培养法和反带技术制备染色体,进行核型分析.结果 49例MDS患者中有22例(44.9%)检出异常克隆.核型异常包括数日异常和结构异常,数目异常以-7,+8最常见.难治性贫血伴原始细胞增多(RAEB1和RAEB2)较难治性贫血(RA)和难治性贫血伴有环状铁粒幼细胞(RARS)检测到更高的异常核型比例.MDS染色体核型异常与原始细胞比例呈正相关.结论 染色体核型分析对MDS诊断、治疗及预后评估有重要价值.     

骨髓增生异常综合征患者染色体核型分析

目的 对骨髓增生异常综合征(MDS)染色体核型进行分析并结合血细胞计数、骨髓原始细胞数对其预后进行评估.方法 采用直接法、短期培养法和反带技术制备染色体,进行核型分析.结果 49例MDS患者中有22例(44.9%)检出异常克隆.核型异常包括数日异常和结构异常,数目异常以-7,+8最常见.难治性贫血伴原始细胞增多(RAEB1和RAEB2)较难治性贫血(RA)和难治性贫血伴有环状铁粒幼细胞(RARS)检测到更高的异常核型比例.MDS染色体核型异常与原始细胞比例呈正相关.结论 染色体核型分析对MDS诊断、治疗及预后评估有重要价值.     

骨髓增生异常综合征患者染色体核型分析

目的 对骨髓增生异常综合征(MDS)染色体核型进行分析并结合血细胞计数、骨髓原始细胞数对其预后进行评估.方法 采用直接法、短期培养法和反带技术制备染色体,进行核型分析.结果 49例MDS患者中有22例(44.9%)检出异常克隆.核型异常包括数日异常和结构异常,数目异常以-7,+8最常见.难治性贫血伴原始细胞增多(RAEB1和RAEB2)较难治性贫血(RA)和难治性贫血伴有环状铁粒幼细胞(RARS)检测到更高的异常核型比例.MDS染色体核型异常与原始细胞比例呈正相关.结论 染色体核型分析对MDS诊断、治疗及预后评估有重要价值.     

骨髓增生异常综合征患者染色体核型分析

目的 对骨髓增生异常综合征(MDS)染色体核型进行分析并结合血细胞计数、骨髓原始细胞数对其预后进行评估.方法 采用直接法、短期培养法和反带技术制备染色体,进行核型分析.结果 49例MDS患者中有22例(44.9%)检出异常克隆.核型异常包括数日异常和结构异常,数目异常以-7,+8最常见.难治性贫血伴原始细胞增多(RAEB1和RAEB2)较难治性贫血(RA)和难治性贫血伴有环状铁粒幼细胞(RARS)检测到更高的异常核型比例.MDS染色体核型异常与原始细胞比例呈正相关.结论 染色体核型分析对MDS诊断、治疗及预后评估有重要价值.     

骨髓增生异常综合征患者染色体核型分析

目的 对骨髓增生异常综合征(MDS)染色体核型进行分析并结合血细胞计数、骨髓原始细胞数对其预后进行评估.方法 采用直接法、短期培养法和反带技术制备染色体,进行核型分析.结果 49例MDS患者中有22例(44.9%)检出异常克隆.核型异常包括数日异常和结构异常,数目异常以-7,+8最常见.难治性贫血伴原始细胞增多(RAEB1和RAEB2)较难治性贫血(RA)和难治性贫血伴有环状铁粒幼细胞(RARS)检测到更高的异常核型比例.MDS染色体核型异常与原始细胞比例呈正相关.结论 染色体核型分析对MDS诊断、治疗及预后评估有重要价值.     

JAK-STAT信号通路与骨髓增生异常综合征Th17/Treg细胞免疫失调相关性的研究

目的:研究骨髓增生异常综合征（myelodysplastic syndrome,MDS）患者外周血中Th17与Treg细胞比例及其相关细胞因子白细胞介素-2（IL-2）、白细胞介素-6（IL-6）和转化生长因子-β1（TGF-β1）的表达及意义。方法:采用流式细胞术对健康对照组、初诊MDS患者、初诊急性髓系白血病（acute myeloid leukemia,AML）患者的外周血Th17细胞和Treg细胞进行检测,采用双抗体夹心酶联免疫吸附法(ELISA)分别检测上述三组患者外周血IL-2、IL-6与TGF-β1分泌水平,应用RT-PCR和Western blot实验检测STAT3、STAT5 mRNA与蛋白表达水平。结果:与健康对照组相比,初诊MDS患者和AML患者外周血中Treg细胞比例增高（P＜0.05）,但MDS患者组与AML患者组之间没有明显差异（P＞0.05）,三组受试者之间Th17细胞比例无明显差异（P＞0.05）;与健康对照组相比,初诊MDS患者组IL-2表达明显升高（P＜0.05）,初诊AML患者组TGF-β1和IL-2表达均显著增加（P＜0.05）;而MDS患者组与AML患者组相比,TGF-β1、IL-6、IL-2表达水平均无明显差异（P＞0.05）;与健康对照组相比,初诊AML患者组、MDS患者组外周血中STAT5 mRNA与蛋白表达均明显增加（P＜0.05）,但两者之间无差异（P＞0.05）。结论:IL-2/STAT5信号通路可能调节初始Th细胞向Treg细胞转化的关键点,也支持Treg细胞数量增高与MDS的病情进展及其向白血病转化可能有关的论断。     

Telomerase activity in myelodysplastic syndromes

Myelodysplastic syndromes are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral cytopenias. Telomeres are thought to be critical in maintaining normal hematopoiesis. In this study, we assessed telomere dynamics in order to obtain further insight into the pathogenesis of MDS. We studied telomerase activity (TA) in mononuclear cells from peripheral blood (PB) and bone marrow (BM) from patients with myelodysplastic syndrome (MDS; n=24), acute myeloid leukemia (AML; n=14), chronic myeloid leukemia (CML; n=12) and 11 normal controls using a polymerase chain reaction-based telomeric repeat amplification assay. Telomerase activities (mean+/-S.D.) were found as 0.199+/-0.09, 0.414+/-0.55, 0.253+/-0.26 and 0.181+/-0.05 pg/ml in PB mononuclear cells, respectively (P>0.05). Comparison of TA of BM mononuclear cells from 19 MDS patients versus 10 BM samples from normal controls revealed no significant difference (P=0.3). There was no correlation between the levels of TA and clinical and prognostic parameters of the patients with MDS, such as degree of anemia, platelet counts on presentation, gender, presence of organomegaly, bone marrow fibrosis and BM blast percentages. Patients who had higher TA had significantly inferior survival compared with patients who had lower TA (P=0.005). Consistent with previous data, our results suggest that in patients with MDS, telomerase activity might be insufficient to compensate for the telomere shortening. Furthermore, TA might be prognostically important in patients with MDS. Measurements of enzymatic activity in association with telomere length studies may help to understand the prognostic role of telomere dynamics in patients with myelodysplastic syndromes more reliably.     

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.     

急性白血病p27基因表达的临床意义

 目的　探讨急性白血病（AL）p27基因mRNA的表达及临床意义。方法　采用RT-PCR检测65例初治AL和41例造血系统非恶性肿瘤患者（对照组）的骨髓单个核细胞中p27 基因mRNA的表达水平,分析p27 mRNA表达与AL的年龄、性别、外周血白细胞计数和髓外浸润临床特征的相关性。结果　AL中p27基因mRNA表达的阳性率为36.9 %（24／65）,显著低于对照组的87.8 %（36／41）,差异有统计学意义（P＜0.05）,急性髓细胞白血病（AML）组和急性淋巴细胞白血病（ALL）组分别为34.2 %（13／38）和37.5 %（9／24）,均低于对照组,差异有统计学意义（均P＜0.05）,但AML组和ALL组比较,差异无统计学意义（P＞0.05）。年龄≥14岁组（52例）和＜14岁组（13例）比较,p27 mRNA表达差异无统计学意义（P＞0.05）,男性组（34例）和女性组（31例）比较,差异无统计学意义（P＞0.05）,外周血白细胞计数≥20×109／L组（31例）和＜20×109／L组（34例）比较,与白细胞计数呈负相关（r＝-0.284,P＜0.05）,有髓外浸润组（37例）和无髓外浸润组（28例）比较,与髓外浸润呈负相关（r＝-0.300,P＜0.05）。结论　AL存在p27基因 mRNA的表达缺失,缺失同时存在于AML和ALL中,缺失与患者外周血白细胞计数、髓外浸润呈负相关,这可能是AL发生、发展的机制之一。     

多发性骨髓瘤患者恶性克隆及其前体细胞检测

 为了探讨多发性骨髓瘤(MM)克隆起源, 病情判断, 以克隆性免疫球蛋白重链(IgH)基因重排为骨髓瘤基因标志, 采用聚合酶链反应(PCR)技术分析MM患者骨髓和外周血克隆性IgH基因重排。42例MM的骨髓标本34例阳性, 阳性率80.95%, 阳性率与临床分期及免疫分型不相关(P＞0.05)。10例反应性浆细胞增多症(RP)及12例正常人骨髓均阴性。24例形态学检查正常的MM外周血16例阳性, 阳性率66.67%, Ⅱ、Ⅲ期患者阳性率明显高于Ⅰ期(P＞0.025), Ⅱ、Ⅲ期之间无差异(P＞0.05)。与免疫分型不相关(P＞0.05)。外周血与骨髓均阳性患者, 克隆性IgH基因重排带位于同一电泳位置, 不同患者重排带呈多态性。研究结果表明：克隆性IgH基因重排可作为骨髓瘤克隆基因标志, MM外周血存在克隆性B细胞, 且与骨髓瘤细胞起源于同一克隆。 MM骨髓和外周血克隆性IgH基因重排检测对MM诊断, 骨髓瘤细胞克隆起源研究, 病情判断均有一定价值。     

Factors influencing survival in myelodysplastic syndromes in a Brazilian population: comparison of FAB and WHO classifications

The WHO classification for myelodysplastic syndromes (MDS) has introduced new categories with prognostic relevance. Our aim was to examine the predictive value of the WHO and the FAB classification compared to parameters of peripheral blood, bone marrow and IPSS. Clinical data, peripheral blood counts, bone marrow (BM) cytology and histology and survival were analyzed in consecutive newly diagnosed adult patients with MDS. All cases were diagnosed according to FAB criteria and reclassified by the WHO proposal. Among 150 patients entering the study median age was 58 years (12-90). According to FAB, 90 patients had refractory anemia (RA), 18 sideroblastic anemia, 34 refractory anemia with excess of blasts (RAEB), three RAEB-t and five chronic myelomonocytic leukemia. Using the WHO proposal, one half of the patients with RA changed category. One patient had the 5q-syndrome. There were 25 cases with refractory cytopenias with multilineage dysplasia (RCMD) and 23 WHO "unclassified". These last patients presented few cell atypias, favorable IPSS and a good survival as has been described for refractory cytopenias in pediatric MDS. Hypocellular BM was found in 24% of the patients. Karyotype was available in only 85 cases. In the univariate analysis, both classifications, hemoglobin values, hypercellular bone marrow and IPSS had an influence on survival. Using the bootstrap resampling as stability test for the model created by the multivariate analysis, the WHO classification entered the model in 73%, FAB in 38% and IPSS in only 7%. Therefore, in a setting with a high number of low-risk MDS, the WHO classification is the best predictor of survival of the patients.     

原发性骨髓纤维化患者骨髓病理特点及JAK2 V617F基因突变研究

目的 探讨原发性骨髓纤维化(PMF)患者的骨髓病理特点,检测JAK2 V617F突变的发生率,分析JAK2 V617F突变对PMF患者血细胞及骨髓纤维组织增生的影响.方法 37例PMF患者的骨髓切片经苏木精-吉姆萨-伊红(HGE)染色及Gomori染色后进行形态学研究,采用荧光定量PCR技术检测患者骨髓中JAK2 V617F基因突变情况.结果 37例PMF患者中JAK2 V617F基因突变阳性率为54％(20/37).突变阳性组患者白细胞计数和血小板计数分别为(12.79±3.54)×109/L和(312.47±203.81)×109/L,高于突变阴性组的(4.22±2.07)×109/L和(136.43±104.87)×109/L,差异均有统计学意义(t=3.158,P=0.025;t=2.623,P=0.046),但两组患者血红蛋白数和纤维组织增生程度比较,差异均无统计学意义(均P＞0.05).结论 JAK2 V617F基因突变检测对PMF,特别是白细胞计数和血小板计数增高的PMF诊断有一定意义.     

Aberrant methylation in pediatric myelodysplastic syndrome

Background: Aberrant methylation of gene promoter region is responsible for inappropriate gene silencing, and it has been associated to initiation and progression of cancer. Aberrant promoter methylation is frequently observed in adult patients with myelodysplastic syndrome (MDS), but in pediatric patients it has been poorly investigated. Methods: We examined the promoter methylation status of 13 genes in bone marrow cells collected at diagnosis of 21 pediatric patients with MDS (subtype RAEB or RAEB-t). For this analysis, we performed sodium bisulfite treatment of genomic DNA, followed by methylation specific PCR (MSP). Results: In pediatric MDS samples, we observed two genes frequently methylated: CALCA was methylated in 85.7% (18/21) of the analyzed samples and CDKN2B in 50% (6/12). Conclusions: Our findings indicate that CALCA and CDKN2B are frequently methylated in pediatric MDS. It suggests that aberrant methylation in pediatric MDS seems to be similar to adult MDS, thus pediatric patients could be also benefited with treatment using demethylating agents.     

Induction of gene expression by 5-Aza-2'-deoxycytidine in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) but not epithelial cells by DNA-methylation-dependent and -independent mechanisms.

The methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR, decitabine) has therapeutic efficacy in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Using microarray analysis, we investigated global changes in gene expression after 5-Aza-CdR treatment in AML. In the AML cell line OCI-AML2, Aza-CdR induced the expression of 81 out of 22 000 genes; 96 genes were downregulated (> or =2-fold change in expression). RT-PCR analysis of 10 randomly selected genes confirmed the changes of expression in AML cells. Similar results were obtained with primary AML and MDS cells after treatment with 5-Aza-CdR ex vivo and in vivo, respectively. In contrast, significantly fewer changes in gene expression and cytotoxicity were detected in normal peripheral blood mononuclear and bone marrow cells or transformed epithelial cells treated with 5-Aza-CdR. Interestingly, only 50.6% of the induced genes contain putative CpG islands in the 5' region. To further investigate the significance of promoter methylation in the induced genes, we analyzed the actual methylation status of randomly selected 5-Aza-CdR-inducible genes. We detected hypermethylation exclusively in the 5' region of the myeloperoxidase (MPO) gene. DNA methylation inversely correlated with MPO expression in newly diagnosed untreated AML patients (P< or =0.004). In contrast, all other analyzed 5-Aza-CdR-inducible genes revealed no CpG methylation in the promoter region, suggesting a methylation-independent effect of 5-Aza-CdR.     

Prognostic Relevance of SFRP1 Gene Promoter Methylation in Colorectal Carcinoma

Background: The development of colorectal carcinoma (CRC) involves many genetic and epigenetic alterations andmethylation being an important epigenetic event has been described as a diagnostic and prognostic biomarker. SecretedFrizzled- Related Protein 1 (SFRP1) gene regulates diverse physiological processes via the Wnt signaling. Promoterhypermethylation of SFRP1 gene is an epigenetic regulation mechanism that downregulates SFRP1 protein level in thetumor, and happens to be one of the significant events in colorectal carcinogenesis. We studied the clinicopathologicalrelationship of CRC including survival outcomes with SFRP1 gene promoter methylation. Methods: We evaluatedpromoter methylation status of SFRP1 gene by methylation-specific PCR (MS-PCR) in the tumor tissue in 54 casesof stage II-III CRC patients in north India. The MS-PCR result was further validated by bisulfite sequencing. Results:SFRP1 gene was methylated in 72.2% cases and un-methylated in 27.8%. We found, that SFRP1 gene methylation intumor was associated with lymph node invasion (p=0.05). The mean overall survival was 22.318 months and 45.173months respectively for patients with methylated and unmethylated SFRP1 gene (p= 0.010, log rank test), (HR = 17.313,95% CI: 2.021-148.290 P=0.009). Conclusion: Study indicates that promoter methylation of SFRP1 gene is associatedwith lymph-node metastasis and poor mean overall survival and it can be a prognostic marker in CRC.