骨髓增生异常综合征的分层诊断及去甲基化分段治疗
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摘要
第一部分
Th22细胞和Th17细胞在骨髓增生异常综合征中的差异化表达及其调控机制研究
研究背景：
骨髓增生异常综合征(MDS)是一组克隆性造血干/祖细胞疾病,主要表现为病态造血和高风险性向急性白血病进展。在MDS的发生、发展过程中,自身免疫介导的骨髓抑制及具有清除恶性克隆功能的免疫监视系统起重要作用。T淋巴细胞克隆性扩增,临床上免疫调节治疗效果可,均提示T辅助细胞和细胞毒性T细胞参与构成MDS的异常免疫系统。
近来,一种新的能够产生IL-17并特异性表达转录因子RORC的T辅助细胞被人们发现,随即被命名为Th17细胞。Th17细胞在炎症免疫反应中的研究日益深入。IL-17A是Th17细胞的主要效应因子,被认为是多种免疫异常疾病如类风湿性关节炎、系统性红斑狼疮和多发性骨髓瘤等的致病因素之一。有研究显示低危MDS患者外周血中Thl7细胞表达升高,但其异常细胞免疫的发生机制仍不明朗。
Th22细胞是新近发现的一种新的CD4+T淋巴细胞亚群,其特点是分泌IL-22和TNF-α,而非IL-17或IFN-γ,亦不表达转录因子T-bet或RORC。在IL-6和TNF-α的促进作用下,芳香烃受体活化,初始性CD4+T细胞可以向Th22表型方向分化。有研究表明在炎症性皮肤异常病人中Th22细胞能够渗透进入表皮组织并参与调节表皮的炎症反应。Th22细胞表达水平亦被证实与胃癌疾病进展密切相关。综上所述,Th22细胞在炎症免疫和肿瘤免疫逃逸机制中均发挥重要作用。
IL-22是一种IL-10家族细胞因子,可由活化的Th22和Th17细胞分泌。IL-22在炎症免疫反应和恶性肿瘤中的表达趋势不尽相同：它既可以与IL-17发挥协同作用,也可以独立发挥免疫调节作用。在炎症性皮肤病和间变性淋巴瘤激酶阳性的间变性大细胞淋巴瘤患者中IL-22表达上调,定向诱导免疫系统攻击自身的靶组织细胞。系统性红斑狼疮患者的IL-22表达下调。在炎症性肠病中,经由未致敏细胞或记忆-效应细胞的诱导,在不同微环境下IL-22既能够抑制炎症反应又可以促进炎症发生发展。
迄今为止,尚未出现MDS中Th22细胞亚群相关的报道。为探究Th22细胞在MDS疾病发生发展中的致病机制,我们检测了早期MDS患者、晚期MDS患者和健康志愿者外周Th22、Th17及Thl细胞的表达情况；同时检测了外周血单个核细胞中转录因子RORC和调节因子IL-6、TNF-α及IL-23mRNA的表达水平；并且检测了外周血血浆和骨髓上清中IL-22和IL-17的含量。此外,我们将不同的T细胞亚群进行了相关性分析,探讨Th细胞分化与MDS疾病预后分期的关系。
研究目的：
通过检测MDS患者外周血中Th22、Th17细胞所占的比例及转录因子RORC,调节因子IL-6、TNF-α及IL-23mRNA的表达水平,研究调节性因子对早期MDS、晚期MDS患者外周血辅助性T细胞亚群Th22/Th17表达量的影响,探讨不同临床分期MDS患者的炎症免疫或免疫逃逸状态,从预后分期的角度寻找可以协助早期、晚期MDS临床诊断的免疫学指标。
研究方法：
1.收集17例早期MDS患者、20例晚期MDS患者及20例健康志愿者的外周血,同时采集25例MDS患者及10例肿瘤学炎症免疫学方面无异常的外科患者的骨髓。
2.通过流式细胞术检测外周血Th22、Th17和Thl细胞在辅助性T细胞亚群中所占的比例。
3.采用酶联免疫吸附测定法(enzyme-linked immunoadsorbent assay, ELISA)检测外周血血浆和骨髓上清中细胞因子IL-22和IL-17的浓度。
4.分离早期MDS、晚期MDS患者及健康志愿者的外周血单个核细胞,提取总RNA,采用实时荧光定量PCR技术测定调节因子RORC. IL-6、TNF-α、IL-23mRNA的表达水平。
5.统计分析：根据是否服从正态分布,结果数据以均数土标准差(mean土SD)或者中位数(数值区间)表示。比较两组数值变量之间的差异性时,根据数值变量是否服从正态分布,选择独立样本t检验或非参数检验进行分析。早期MDS、晚期MDS以及健康对照组三组之间比较差异性时,使用one-way ANOVA进行分析统计,其中任意两组之间比较差异性时用Newman-Keuls检验(q检验)两两比较法进行分析统计；当任何一组数值变量不符合正态分布时,应用Kruskal-Wallis检验(H检验)和Nemenyi检验进行分析统计。进行变量之间的相关性分析时,根据双变量是否均服从正态分布采用Pearson相关或Spearman等级相关方法进行分析统计。所有统计分析均使用SAS9.1软件进行。P<0.05定为具有统计学差异。
研究结果：
1.早期MDS患者、晚期MDS患者和健康志愿者外周Th22细胞的表达情况与正常对照组(0.71±0.17%)相比,Th22(CD4+IFN-γ-IL-17-IL-22+)细胞亚群在所有分期MDS患者外周血中所占比例显著增加(1.55±0.74%,P<0.0001)；Th22细胞在早期MDS患者外周血中所占的比例显著高于对照组(1.27±0.50%vs.0.71±0.17%,P=0.002)；与早期MDS患者相比,晚期MDS患者Th22细胞的表达量明显升高(1.77±0.84%vs.1.27±0.50%,P=0.03)。
2.早期MDS患者、晚期MDS患者和健康志愿者外周Th17细胞的表达情况Th17(CD4+IL17+)细胞亚群在早期MDS患者外周血中所占的比例(1.90%；0.58-6.01%)显著高于晚期MDS组(1.16%；0.15-1.86%；P=0.002)以及对照组(0.97%；0.55-1.69%；P=0.002)。
3.MDS患者和健康志愿者外周Thl细胞的表达情况外周血中Th1(CD4+IFN-γ+)细胞亚群在外周血中占辅助性T细胞的比例在MDS组以及对照组之间无明显差异。
4.MDS组和对照组血浆和骨髓上清中IL-22和IL-17的水平血浆中IL-22的浓度在MDS组和健康对照组之间无显著性差异。骨髓上清中IL-22的浓度在两组之间亦无显著性差异。MDS组及健康对照组血浆中IL-17的浓度无统计学差异,骨髓上清中IL-17的浓度亦无统计学差异。
5.早期MDS组、晚期MDS组和对照组外周血单个核细胞中转录因子RORC和调节因子IL-6、TNF-α及IL-23mRNA的相对定量表达在早期MDS组、晚期MDS组和健康对照组三组中,外周血单个核细胞转录因子RORC基因仅在早期MDS组表达明显升高(P=0.0007；P=0.002),外周血单个核细胞中调节性细胞因子IL-6及TNF-α基因仅在晚期MDS组表达显著增高(P<0.05；P<0.001)；外周血单个核细胞中IL-23基因表达水平在三组之间均无统计学差异。
6.MDS患者外周T细胞亚群Th22、Th17及Thl细胞之间的关联性在早期MDS患者中,外周Th22与Th17细胞亚群的数目比例呈正相关(r=0.675,P=0.004)；然而,在晚期MDS患者中,外周Th22细胞与Th17细胞亚群的分化表达量无相关性(r=0.138,P=0.610)。在总体所有分期的MDS患者中,外周Th22亚群与Thl亚群的表达无相关性(P=0.053)。
结论：
早期MDS患者外周血中辅助性T细胞向Thl7亚群方向的分化量、转录因子RORC mRNA表达量显著增加；晚期MDS患者外周血中辅助性T细胞向Th22亚群方向的分化量、IL-6mRNA和]TNF-α mRNA表达量显著增加,提示Th22/Th17细胞参与调节早期MDS炎症免疫和晚期MDS免疫逃逸的异常免疫状态。就疾病分期而言,外周血中Th22/Th17比率对于早期、晚期MDS的分层诊断具有重要的免疫学指示意义。
第二部分
地西他滨分段治疗骨髓增生异常综合征及疗效评估体系
研究背景：
表观遗传学是指在不涉及核苷酸序列变化的情形下基因表达发生了改变,包括DNA甲基化、RNA干扰、组蛋白修饰等。细胞遗传学变化是MDS主要的发病机制,此外,单倍剂量不足、次级突变及表观遗传学改变等亦是MDS重要的致病机理。研究表明MDS表观遗传学改变主要表现为造血祖细胞内基因组水平上广泛的DNA甲基化和组蛋白乙酰化、甲基化,集中发生在抑癌基因及其他有丝分裂抑制因子的转录表达区域。高危型MDS异常甲基化位点的数目比低危型MDS异常甲基化位点多。转录启动子区DNA甲基转移酶DNMT3A和TET2异常甲基化比较普遍。DNMTs可以催化CpG岛中胞嘧啶甲基化为5-甲基胞嘧啶,是MDS治疗的关键靶点。
研究表明大剂量地西他滨具有细胞毒性,小剂量地西他滨则可以抑制DNMTs的活性,发挥去甲基化功能,使沉默的抑癌基因如E-cadherin, P16, hMLH1, VHL, P15,P21等恢复正常去甲基化状态而重新活化表达。
美国食品药品监督管理局推荐DNMTs抑制剂地西他滨在临床上适用于治疗IPSS-R预后中危/高危/极高危MDS及其他治疗无效的较低危MDS。多项回顾性临床研究和前瞻性队列研究表明地西他滨可以使部分较高危MDS患者获得细胞遗传学改善,降低MDS向急性髓系白血病转化的风险,延长总生存期。但在地西他滨实际应用过程中,骨髓抑制发生率较高且多为III或IV级,主要表现为血小板减少、中性粒细胞减少和贫血。
鉴于地西他滨传统治疗体系中常发生延迟给药或提前中止疗程,为进一步提高地西他滨的疗效,我们探索出一套分段治疗及疗效评估体系,现对该体系做一详细说明,并同传统地西他滨治疗体系进行比较分析。
研究目的：
论证研究地西他滨分段治疗及疗效评估体系的合理性、可行性及安全性,分析比较地西他滨传统治疗体系与分段治疗评估体系治疗骨髓增生异常综合征的有效性。
研究方法：
研究对象选择山东大学齐鲁医院2012年2月至2013年12月确诊为MDS的57例患者。24例患者接受传统小剂量地西他滨治疗方案：应用地西他滨20m/平方米/天,静脉输注,连续5天,4周一疗程。在地西他滨治疗第2疗程后复查骨髓细胞学和外周血象,评价骨髓和血液学缓解情况,评估疗效。33位MDS患者接受小剂量地西他滨分段治疗及疗效评估体系治疗：用药剂量同前；首次应用地西他滨前根据MDS患者基线特征评估情况,加强支持治疗,并同时进行抗微生物“三联”预防。治疗第2疗程完后监测血小板计数变化情况,第3疗程后复查骨髓原始细胞数、外周血象,评估疗效。第4疗程后进行巩固治疗,第6疗程后进入维持治疗。
第1疗程后,统计分析传统治疗组和分段治疗评估组两组之间在3-4级血液学副反应及感染发生率方面有无差异,分析比较两组之间在延迟给药发生率、提前中止治疗发生率等方面有无差异；第2疗程结束后重点关注血小板变化情况,动态监测血小板数目,分析比较两组中血小板改善情况；第3疗程后分段治疗评估组进行骨髓原始细胞数和外周血象复查,传统治疗组已在第2疗程后复查骨髓原始细胞数和外周血象,统计分析两组中地西他滨治疗的总有效率(overall response rate, ORR)是否存在统计学差异,ORR评价指标包括完全缓解(complete remission, CR)率和部分缓解(partial remission, PR)率。比较两组输血依赖情况及地西他滨中位疗程数有无统计学差异。
研究结果：
1.地西他滨传统治疗体系和分段治疗评估体系第一疗程后发生感染的情况传统治疗组地西他滨应用第一疗程后发生感染的机率为66.7%,分段治疗组第一疗程后发生感染的机率为39.4%,P=0.042<0.05,传统治疗组与分段治疗组MDS患者并发感染的机率具有统计学差异。
2.地西他滨传统治疗体系和分段治疗评估体系第一疗程后发生3～4级中性粒细胞减少的情况传统治疗组不良反应-3-4级中性粒细胞减少的发生率为33.3%,分段治疗组3-4级中性粒细胞减少的发生率为9.1%,P＝0.039<0.05,分段治疗组和传统治疗组3-4级中性粒细胞减少发生率具有显著统计学差异。
3.地西他滨传统治疗体系和分段治疗评估体系第一疗程后发生3-4级血小板减少的情况传统治疗组不良反应3-4级血小板减少的发生率为50.0%,分段治疗组3-4级血小板减少的发生率为24.2%,P=0.044<0.05,说明分段治疗组和传统治疗组3～4级血小板减少发生率具有显著统计学差异。
4.地西他滨传统治疗体系和分段治疗评估体系第一疗程后发生3-4级贫血的情况传统治疗组不良反应3～4级贫血的发生率为29.2%,分段治疗组3-4级贫血的发生率为6.1%,P=0.027<0.05,说明分段治疗组和传统治疗组3～4级贫血发生率具有统计学差异。
5.地西他滨传统治疗体系和分段治疗评估体系一疗程后延迟给药和中止治疗的发生情况传统治疗组地西他滨应用一疗程后延迟给药发生率为66.7%,分段治疗组治疗过程中延迟给药发生率为36.4%,P=0.024<0.05,说明分段治疗组和传统治疗组地西他滨应用一疗程后延迟给药发生率具有统计学差异。一疗程后中止治疗的发生率没有统计学差异。
6.地西他滨传统治疗体系和分段治疗评估体系治疗反应的对比传统治疗组及分段治疗组地西他滨的总有效率ORR(CR%+PR%)分别为12.5%和36.4%,P=0.043<0.05,分段治疗及疗效评估体系可以显著提高地西他滨治疗的有效率。传统治疗组血小板改善率为41.7%,分段治疗组血小板改善率为69.7%,P=0.034<0.05,分段治疗组和传统治疗组MDS患者获得血小板改善的机率具有显著统计学差异,分段治疗及疗效评估体系中MDS患者的血小板反应更佳。传统治疗组和分段治疗组两组中MDS患者的血小板数目均比其自身的中性粒细胞数目和血红蛋白含量恢复得早。分段治疗组获得血小板改善的MDS患者其血小板计数水平多在第2-3疗程开始回升,而传统治疗组获得血小板改善的MDS患者其血小板计数水平常在第3、4疗程开始回升。在地西他滨传统治疗组中,治疗前输血依赖者13例,治疗后输血依赖者9例,脱离输血依赖患者的比例为30.8%。在地西他滨分段治疗组中,治疗前输血依赖者24例,治疗后输血依赖者8例,脱离输血依赖患者的比例为66.7%,P=0.036,分段治疗评估体系中更高比例的患者脱离输血依赖,输血频次或输血量较大幅度地减低。
7.传统治疗体系和分段治疗评估体系地西他滨中位疗程数对比传统治疗组MDS患者使用地西他滨治疗的疗程数是1-5,中位疗程数是4；分段治疗组MDS患者使用地西他滨治疗的疗程数是2-8,中位疗程数是7。比较两组之间中位疗程数的差异,P<0.01,提示分段治疗及疗效评估体系可以确保地西他滨疗程的衔接性。
结论：与地西他滨传统治疗MDS相比,在分段治疗及疗效评估体系的各个环节中
我们进行了“三联”预防微生物感染,降低了感染率；及时应用造血生长因子或成
分输血促进血液学恢复,降低了延迟或减量给药的发生率；分时段科学监测患者
外周血和骨髓的耐受情况及恢复水平。地西他滨分段治疗骨髓增生异常综合征及
疗效评估体系可以确保去甲基化治疗的连续性及足够的疗程数,减少血液学不良
反应及继发感染的机会,减少延迟给药和输血依赖的发生,最终提高初治或其他
治疗无效的MDS患者的总体反应率。
Section I
Th22cells as well as Th17cells expand differentially in patients with early-stage and late-stage myelodysplastic syndrome
Background:
Myelodysplastic syndrome (MDS) encompasses a heterogeneous group of clonal hematopoietic stem-cell disorders, characterized by ineffective hematopoiesis and an increased probability of developing acute leukemia. Autoimmune-mediated myelosuppression and immune surveillance of malignant clone play recognized crucial roles in the process of MDS. Findings of expanded clonal T lymphocytes and clinical responses to immunoregulatory therapy have led to the speculation that both T helper and cytotoxic T cells are involved in the immunological pathophysiology of MDS.
Recently, a separate Th cell subset which secrets the cytokine interleukin(IL)-17and expresses the lineage-specific transcription factor retinoic acid receptor-related orphan receptor C (RORC) was identified and named T-helper cell type17(Th17). Since then, Th17cells have entered the limelight because of their comprehensive involvement in inflammations. IL-17A, a representative Th17cytokine, has been described in various models of immune-mediated tissue injury, such as rheumatoid arthritis, lupus, myeloma and so on. Although one study reported an increased number of peripheral Th17cells in low risk MDS lately, the mechanism of cellular immune abnormalities still remains undiscovered.
Th22subset, which has the ability of secreting IL-22and tumor necrosis factor alpha (TNF-a), has become a new subset clearly separated from other Th cells. This subset does not express any of cytokine interferon (IFN)-y, IL-17or their associated transcription factors T-bet and RORC. Aryl hydrocarbon receptor (AHR) activation participates in priming naive CD4+T cells to Th22subset plus the promotion by IL-6and TNF-a. Th22cells have been found regulating epidermal responses within the epidermal layer prominently in inflammatory skin diseases. In addition, a preferential expansion of Th22cells contributing to gastric cancer progression has been described. These data above support that Th22cells are involved in the pathophysiology of inflammatory immune reactions and immune evasion of neoplasms.
IL-22belongs to the IL-10cytokine family and is primarily secreted by Th22cells on activation. The expression trend of IL-22in cancers and autoimmune disorders is diverse, with IL-17as siblings but not twins based on the local biological characteristics. The expression level of IL-22was up-regulated in skin inflammatory pathology and anaplastic lymphoma kinase positive anaplastic large cell lymphoma, inducing directional signal linking the immune system to the targeted tissue-resident cells. Meanwhile, it was down-regulated in systemic lupus erythematosus. Within disorders such as inflammatory bowel disease (IBD), once inducted by the naive or memory/effector cells, IL-22has access to inhibit or accelerate the inflammatory reactions regarding the idiographic microenvironment.
To date, no report exist with regard to Th22cell subset or their association with Th17or Thl subset among MDS patients. To investigate their possible roles in the immunological pathophysiology of MDS, we measured the percentages of circulating Th22, Th17, Th1and mRNA expression levels of RORC, IL-6, TNF-a and IL-23in peripheral blood mononuclear cells (PBMCs) as well as the cytokine expression levels of IL-22or IL-17in peripheral blood (PB) and bone marrow (BM). To explore the potential relationship between Th polarization and different stages of MDS, the correlations of various Th subsets were evaluated at the same time.
Objective:
Measure the frequencies of periphery Th22, Th17subsets and determine the mRNA expression of transcription factor RORC, cytokines such as IL-6, TNF-a and IL-23. Explore the regulation effects of transcription factor and cytokines on Th22/Th17polarization in different stages of myelodysplastic syndrome. Attempt to elucidate the relevance between the switched immune status and clinical prognosis of myelodysplastic syndrome as well as search for peripheral blood immunological index to guide the diagnosis of different disease stages.
Materials and methods:
1. PB was collected from seventeen patients with early-stage MDS (E-MDS), twenty patients with late-stage MDS (L-MDS) and twenty age-matched healthy volunteers. BM was obtained from twenty-five MDS patients and ten hematologically normal surgical patients without any oncological or immunological issues.
2. The percentages of circulating Th22, Th17, Thl subset were measured in E-MDS patients, L-MDS patients and healthy controls by flow cytometry.
3. The cytokine levels of IL-22and IL-17both in PB and BM plasma were examined by enzyme-linked immunosorbent assay.
4. For RNA isolation, PBMCs were isolated from the anticoagulated blood donated by E-MDS patients, L-MDS patients and healthy controls. The mRNA expression levels of RORC, IL-6, TNF-a and IL-23were determined by real-time quantitative polymerase chain reaction.
5. Results were expressed as mean±SD or median (range). Comparisons between two groups were assessed by the non-paired t-test or the Wilcoxon rank-sum test to compare parametric and non-parametric data respectively. Statistical significance among E-MDS, L-MDS and healthy control three groups was determined by ANOVA, and difference between any two groups was determined by Newman-Keuls multiple comparison test (q test) unless the data were not normally distributed, in which case Kruskal-Wallis test (H test) and Nemenyi test were used. The Pearson or Spearman correlation test was used for correlation analysis depending on data distribution. All tests were performed by SAS9.1system. P value less than0.05was considered statistically significant.
Results:
1. The percentage of circulating Th22subset in E-MDS patients, L-MDS patients and healthy controls Compared with healthy controls, the percentage of peripheral Th22cells was significantly higher in total MDS patients (0.71±0.17%vs.1.55±0.74%, P<0.0001). The number of peripheral Th22cells in E-MDS is higher than that in controls (1.27±0.50%vs.0.71±0.17%, P=0.002). Also a significant increase was shown in L-MDS compared with E-MDS patients (1.77±0.84%vs.1.27±0.50%, P=0.03).
2. The percentage of circulating Th17subset in E-MDS patients, L-MDS patients and healthy controls The percentage of peripheral Th17cells was significantly elevated in E-MDS (median,1.90%; range,0.58-6.01%) when compared with L-MDS (median,1.16%; range,0.15-1.86%)(P=0.002) or healthy control group (0.97±0.29%; P=0.002).
3. The percentage of circulating Th1subset in MDS patients and healthy controls For peripheral Thl cells, no significant difference was observed between MDS patients and healthy controls.
4. The cytokine levels of IL-22and IL-17in PB and BM plasma from MDS patients and healthy controls No significant difference of PB IL-22or IL-17level between MDS patients (median,22.64pg/ml; range,16.02-54.66) and healthy controls (median,23.86pg/ml; range,14.05-36.49) was observed, consistent with BM findings.
5. mRNA expression levels of RORC, IL-6, TNF-a and IL-23in E-MDS, L-MDS cohort and controls
The relative amount of RORC mRNA in E-MDS patients was markedly increased compared with healthy controls and L-MDS patients (P=0.0007; P=0.002). The relative amount of mRNA of IL-6in L-MDS patients was much higher than that in E-MDS patients (P<0.05) and healthy controls(P<0.001). TNF-a mRNA level was also present on higher levels in L-MDS compared with E-MDS and controls (P<0.05; P<0.005). There was no significant difference in IL-23p19mRNA expression levels among E-MDS, L-MDS and healthy controls.
6. Correlation between Th22, Thl7, and Thl Cells in MDS Patients In E-MDS patients, there existed a statistically positive correlation between peripheral Th22cells and Thl7cells (r=0.675, P=0.004) while no statistical correlation was shown among L-MDS patients (r=0.138, P=0.610). Peripheral Th22subset showed no significant correlation with peripheral Thl subset (P=0.053).
Conclusion:
E-MDS cohort demonstrated the dominance of Thl7subset and its specific transcription factor RORC. On the other hand, L-MDS cohort showed an increased frequency of Th22cells in peripheral blood, along with higher mRNA expression levels of IL-6and TNF-a. Our study indicates that Th22cells along with Th17cells or not are involved in regulating the autoimmune reaction of E-MDS and immune evasion of L-MDS. As for the disease stages, peripheral Th22/Th17ratio is important to the stratified diagnosis of E-MDS and L-MDS.
section Ⅱ
Clinical study of decitabine in the segmented treatment and evaluation system for myelodysplastic syndrome
Background:
Epigenetics refers to alteration in gene expression that does not involve changes to the corresponding DNA sequence. The common epigenetic events include DNA methylation, RNA interference, histone modification and so on. Despite that cytogenetic heterogeneity constitutes the principal pathophysiological mechanism of MDS, haploinsufficiency, secondary mutation as well as epigenetic modification also contribute to the disease phenotype. What top the ratings of epigenetic modifications in MDS are DNA methylation and histone modification on genomic level, but in fact these modifications are focally increased around the promoter region of tumor suppressor gene and other mitosis inhibitors. It is noted that there are more hypermethylation sites in high risk MDS compared with low risk MDS. DNA methyltransferase3A (DNMT3A) and TET2mutations in the promoter region have been recognized to be involved in DNA methylation. In the CpG dinucleotides enriched site, DNMT functions in the methylation of cytimidine converting to5-methylcytosine, which is thought to mediate much of the increased methylation process and has therefore become a crucial treatment target in MDS.
Researchers have proved that high-dose decitabine has direct cytotoxicity to clonal cells whereas low-dose decitabine induces hypomethylation through inhibiting the activity of DNMTs, which is distinct from immediate cytotoxicity. The normal demethylated state of former silencing tumor suppressor genes such as E-cadherin, p16, hMLHl, VHL, p15and P21shall be restored, followed with reactivation and gene transcription.
Decitabine was recently approved by the US Food and Drug Administration (FDA) for treating patients with intermediate risk/high risk/very high risk MDS or nonresponsive MDS. A number of retrospective and prospective studies have revealed that decitabine favors to cytogenetic improvements, decreases the probability of developing acute leukemia and prolongs the overall survival in the majority of patients with higher risk MDS. However, during the clinical practice of decitabine, myelosuppression often occurs, resulting in thrombocytopenia, neutropenia or anemia. What's worse, higher grade3/4myelosuppression happens more frequently.
Given that the current clinical scheme of decitabine often goes wrong due to dose delay or early discontinuance, we designed a novel segmented treatment and evaluation system to improve the long-term efficacy of low-dose decitabine. At present, we describe this novel segmented treatment and evaluation system in detail and make comparison with the original prevailing protocol.
Objective:
Expound the rationality, feasibility and drug safety of the segmented treatment and evaluation system of decitabine for treating MDS; compare and analyze the clinical effectiveness of decitabine used in the segmented system and conventional system.
Methods:
A total of57patients with MDS were recruited in this study. Enrollment took place between February2012and December2013in the Department of Hematology of Qilu Hospital, Shandong University.24patients were treated with decitabine in the conventional scheme, i.e.20mg/m2/d, intravenous (IV) infused for5days and repeated every4weeks. The myelogram and hemogram changes were reexamined after the second course to evaluate the marrow remission and hematologic remission.33MDS patients were incorporated into the segmented treatment and evaluation system with the same dose of decitabine as the conventional scheme. However, before starting the segmented treatment, the baseline characteristics of the subjects should be assessed. Antibiotics, antivirals and antifungals were preventively applied. Best supportive care was supposed to be aggressive. After the second course, only platelet count was monitored whereas after the third course, complete blood count and blasts in bone marrow were tested. Intensification therapy was carried on after the forth course and maintainance therapy after the sixth course.
The incidence of grades3to4hematological side effects and infections were analyzed in the conventional remedy system and segmented remedy system after the first course, as well as the incidence of dose delay and early discontinuance. The platelet counts were highlightly concerned in the second course and were dynamicly monitored until achieving platelet hematologic improvement (HI-P). The myelogram and hemogram changes were reexamined after the third course in the segmented remedy system whereas these had been reexamined after the second course in the conventional remedy system. The overall response rate (ORR), transfusion-dependence and median course in these two different systems were compared using chi-square test and non-parametric test via SPSS17.0software.
Results:
1. Infections in the conventional and segmented remedy systems after the first decitabine course
The infection rate in the conventional treatment group after the first decitabine course was66.7%versus39.4%in the segmented treatment and evaluation group (P=0.042<0.05). The infection rate in the segmented treatment and evaluation group was significantly lower compared with that in the conventional treatment group.
2. Grades3to4neutropenia in the conventional and segmented remedy systems after the first decitabine course
Compared with the conventional remedy group, the incidence rate of Grades3to4neutropenia was significantly lower in the segmented remedy group (9.1%vs.33.3%, P=0.039<0.05).
3. Grades3to4thrombocytopenia in the conventional and segmented remedy systems after the first decitabine course
The incidence rate of Grades3to4thrombocytopenia in the segmented remedy group was obviously reduced when compared with the conventional remedy group (24.2%vs.50.0%, P=0.044<0.05).
4. Grades3to4anemia in the conventional and segmented remedy systems after the first decitabine course
The incidence rate of Grades3to4anemia in the segmented remedy group was markedly reduced compared with that in the conventional remedy group (6.1%vs.29.2%, P=0.027<0.05).
5. Dose delay or early discontinuance in the conventional and segmented remedy systems after the first decitabine course
Significant difference in the incidence of dose delay between the segmented remedy group and the conventional remedy group was observed (36.4%vs.66.7%, P=0.024<0.05). No difference in the incidence of early discontinuance after the first course was found between the two groups.
6. Comparision of responses to decitabine in the conventional and segmented remedy systems
Significant increment of ORR (CR%plus PR%) was obtained in the segmented remedy group when compared with the index in the conventional remedy group (36.4%vs.12.5%, P=0.043<0.05).
The incidence rate of platelet hematologic improvement (HI-P) in the conventional and segmented remedy groups was41.7%and69.7%respectively (P=0.034), showing a much better platelet response in the latter group.
The platelet counts of MDS patients who finally achieved HI-P in the segmented group usually began to rebound during the second to third course whereas it was the third to forth course during which platelet counts began to rebound in the conventional group.
In the conventional group,13cases were transfusion-dependent at baseline,4cases (30.8%) became transfusion-independent during the trial. In the segmented group,24cases were transfusion-dependent at baseline,16cases (66.7%) became transfusion-independent during the trial, P=0.036, indicating that the segmented treatment and evaluation system can alleviate transfusion-dependence.
7. Comparision of the median courses of decitabine in the conventional and segmented remedy systems
The number of medication courses in the conventional scheme reached to5as the upper limit with a median of4while the courses in the segmented scheme reached to8at most, with a median of7. The therapeutic courses were noteworthyly increased in the segmented remedy group so as to guarantee the consecutive demethylation therapy.
Conclusion:
Different from the therapeutic strategy in the conventional system, prophylactic antibiotics, antivirals and antifungals were simultaneously applied in the segmented system, resulting in reduced infection; patients incorporated in the segmented system were given best supportive care such as hematopoietic growth factors or blood-component transfusion in time to promoting the hematological recovery, leading to diminished dose delay or dose reduction; the tolerance and recovery situation in peripheral blood and bone marrow were detected in different time periods in the segmented system. The segmented system of decitabine turned out to have more access to guaranteeing the consecutive and sufficient medication courses, reducing the opportunities for hematologic toxicity and secondary infection, dose delay or blood transfusion, and ultimately improving the overall response rate of MDS patients who were newly diagnosed or nonresponsive to previous therapies.
Th22细胞和Th17细胞在骨髓增生异常综合征中的差异化表达及其调控机制研究
研究背景：
骨髓增生异常综合征(MDS)是一组克隆性造血干/祖细胞疾病,主要表现为病态造血和高风险性向急性白血病进展。在MDS的发生、发展过程中,自身免疫介导的骨髓抑制及具有清除恶性克隆功能的免疫监视系统起重要作用。T淋巴细胞克隆性扩增,临床上免疫调节治疗效果可,均提示T辅助细胞和细胞毒性T细胞参与构成MDS的异常免疫系统。
近来,一种新的能够产生IL-17并特异性表达转录因子RORC的T辅助细胞被人们发现,随即被命名为Th17细胞。Th17细胞在炎症免疫反应中的研究日益深入。IL-17A是Th17细胞的主要效应因子,被认为是多种免疫异常疾病如类风湿性关节炎、系统性红斑狼疮和多发性骨髓瘤等的致病因素之一。有研究显示低危MDS患者外周血中Thl7细胞表达升高,但其异常细胞免疫的发生机制仍不明朗。
Th22细胞是新近发现的一种新的CD4+T淋巴细胞亚群,其特点是分泌IL-22和TNF-α,而非IL-17或IFN-γ,亦不表达转录因子T-bet或RORC。在IL-6和TNF-α的促进作用下,芳香烃受体活化,初始性CD4+T细胞可以向Th22表型方向分化。有研究表明在炎症性皮肤异常病人中Th22细胞能够渗透进入表皮组织并参与调节表皮的炎症反应。Th22细胞表达水平亦被证实与胃癌疾病进展密切相关。综上所述,Th22细胞在炎症免疫和肿瘤免疫逃逸机制中均发挥重要作用。
IL-22是一种IL-10家族细胞因子,可由活化的Th22和Th17细胞分泌。IL-22在炎症免疫反应和恶性肿瘤中的表达趋势不尽相同：它既可以与IL-17发挥协同作用,也可以独立发挥免疫调节作用。在炎症性皮肤病和间变性淋巴瘤激酶阳性的间变性大细胞淋巴瘤患者中IL-22表达上调,定向诱导免疫系统攻击自身的靶组织细胞。系统性红斑狼疮患者的IL-22表达下调。在炎症性肠病中,经由未致敏细胞或记忆-效应细胞的诱导,在不同微环境下IL-22既能够抑制炎症反应又可以促进炎症发生发展。
迄今为止,尚未出现MDS中Th22细胞亚群相关的报道。为探究Th22细胞在MDS疾病发生发展中的致病机制,我们检测了早期MDS患者、晚期MDS患者和健康志愿者外周Th22、Th17及Thl细胞的表达情况；同时检测了外周血单个核细胞中转录因子RORC和调节因子IL-6、TNF-α及IL-23mRNA的表达水平；并且检测了外周血血浆和骨髓上清中IL-22和IL-17的含量。此外,我们将不同的T细胞亚群进行了相关性分析,探讨Th细胞分化与MDS疾病预后分期的关系。
研究目的：
通过检测MDS患者外周血中Th22、Th17细胞所占的比例及转录因子RORC,调节因子IL-6、TNF-α及IL-23mRNA的表达水平,研究调节性因子对早期MDS、晚期MDS患者外周血辅助性T细胞亚群Th22/Th17表达量的影响,探讨不同临床分期MDS患者的炎症免疫或免疫逃逸状态,从预后分期的角度寻找可以协助早期、晚期MDS临床诊断的免疫学指标。
研究方法：
1.收集17例早期MDS患者、20例晚期MDS患者及20例健康志愿者的外周血,同时采集25例MDS患者及10例肿瘤学炎症免疫学方面无异常的外科患者的骨髓。
2.通过流式细胞术检测外周血Th22、Th17和Thl细胞在辅助性T细胞亚群中所占的比例。
3.采用酶联免疫吸附测定法(enzyme-linked immunoadsorbent assay, ELISA)检测外周血血浆和骨髓上清中细胞因子IL-22和IL-17的浓度。
4.分离早期MDS、晚期MDS患者及健康志愿者的外周血单个核细胞,提取总RNA,采用实时荧光定量PCR技术测定调节因子RORC. IL-6、TNF-α、IL-23mRNA的表达水平。
5.统计分析：根据是否服从正态分布,结果数据以均数土标准差(mean土SD)或者中位数(数值区间)表示。比较两组数值变量之间的差异性时,根据数值变量是否服从正态分布,选择独立样本t检验或非参数检验进行分析。早期MDS、晚期MDS以及健康对照组三组之间比较差异性时,使用one-way ANOVA进行分析统计,其中任意两组之间比较差异性时用Newman-Keuls检验(q检验)两两比较法进行分析统计；当任何一组数值变量不符合正态分布时,应用Kruskal-Wallis检验(H检验)和Nemenyi检验进行分析统计。进行变量之间的相关性分析时,根据双变量是否均服从正态分布采用Pearson相关或Spearman等级相关方法进行分析统计。所有统计分析均使用SAS9.1软件进行。P<0.05定为具有统计学差异。
研究结果：
1.早期MDS患者、晚期MDS患者和健康志愿者外周Th22细胞的表达情况与正常对照组(0.71±0.17%)相比,Th22(CD4+IFN-γ-IL-17-IL-22+)细胞亚群在所有分期MDS患者外周血中所占比例显著增加(1.55±0.74%,P<0.0001)；Th22细胞在早期MDS患者外周血中所占的比例显著高于对照组(1.27±0.50%vs.0.71±0.17%,P=0.002)；与早期MDS患者相比,晚期MDS患者Th22细胞的表达量明显升高(1.77±0.84%vs.1.27±0.50%,P=0.03)。
2.早期MDS患者、晚期MDS患者和健康志愿者外周Th17细胞的表达情况Th17(CD4+IL17+)细胞亚群在早期MDS患者外周血中所占的比例(1.90%；0.58-6.01%)显著高于晚期MDS组(1.16%；0.15-1.86%；P=0.002)以及对照组(0.97%；0.55-1.69%；P=0.002)。
3.MDS患者和健康志愿者外周Thl细胞的表达情况外周血中Th1(CD4+IFN-γ+)细胞亚群在外周血中占辅助性T细胞的比例在MDS组以及对照组之间无明显差异。
4.MDS组和对照组血浆和骨髓上清中IL-22和IL-17的水平血浆中IL-22的浓度在MDS组和健康对照组之间无显著性差异。骨髓上清中IL-22的浓度在两组之间亦无显著性差异。MDS组及健康对照组血浆中IL-17的浓度无统计学差异,骨髓上清中IL-17的浓度亦无统计学差异。
5.早期MDS组、晚期MDS组和对照组外周血单个核细胞中转录因子RORC和调节因子IL-6、TNF-α及IL-23mRNA的相对定量表达在早期MDS组、晚期MDS组和健康对照组三组中,外周血单个核细胞转录因子RORC基因仅在早期MDS组表达明显升高(P=0.0007；P=0.002),外周血单个核细胞中调节性细胞因子IL-6及TNF-α基因仅在晚期MDS组表达显著增高(P<0.05；P<0.001)；外周血单个核细胞中IL-23基因表达水平在三组之间均无统计学差异。
6.MDS患者外周T细胞亚群Th22、Th17及Thl细胞之间的关联性在早期MDS患者中,外周Th22与Th17细胞亚群的数目比例呈正相关(r=0.675,P=0.004)；然而,在晚期MDS患者中,外周Th22细胞与Th17细胞亚群的分化表达量无相关性(r=0.138,P=0.610)。在总体所有分期的MDS患者中,外周Th22亚群与Thl亚群的表达无相关性(P=0.053)。
结论：
早期MDS患者外周血中辅助性T细胞向Thl7亚群方向的分化量、转录因子RORC mRNA表达量显著增加；晚期MDS患者外周血中辅助性T细胞向Th22亚群方向的分化量、IL-6mRNA和]TNF-α mRNA表达量显著增加,提示Th22/Th17细胞参与调节早期MDS炎症免疫和晚期MDS免疫逃逸的异常免疫状态。就疾病分期而言,外周血中Th22/Th17比率对于早期、晚期MDS的分层诊断具有重要的免疫学指示意义。
第二部分
地西他滨分段治疗骨髓增生异常综合征及疗效评估体系
研究背景：
表观遗传学是指在不涉及核苷酸序列变化的情形下基因表达发生了改变,包括DNA甲基化、RNA干扰、组蛋白修饰等。细胞遗传学变化是MDS主要的发病机制,此外,单倍剂量不足、次级突变及表观遗传学改变等亦是MDS重要的致病机理。研究表明MDS表观遗传学改变主要表现为造血祖细胞内基因组水平上广泛的DNA甲基化和组蛋白乙酰化、甲基化,集中发生在抑癌基因及其他有丝分裂抑制因子的转录表达区域。高危型MDS异常甲基化位点的数目比低危型MDS异常甲基化位点多。转录启动子区DNA甲基转移酶DNMT3A和TET2异常甲基化比较普遍。DNMTs可以催化CpG岛中胞嘧啶甲基化为5-甲基胞嘧啶,是MDS治疗的关键靶点。
研究表明大剂量地西他滨具有细胞毒性,小剂量地西他滨则可以抑制DNMTs的活性,发挥去甲基化功能,使沉默的抑癌基因如E-cadherin, P16, hMLH1, VHL, P15,P21等恢复正常去甲基化状态而重新活化表达。
美国食品药品监督管理局推荐DNMTs抑制剂地西他滨在临床上适用于治疗IPSS-R预后中危/高危/极高危MDS及其他治疗无效的较低危MDS。多项回顾性临床研究和前瞻性队列研究表明地西他滨可以使部分较高危MDS患者获得细胞遗传学改善,降低MDS向急性髓系白血病转化的风险,延长总生存期。但在地西他滨实际应用过程中,骨髓抑制发生率较高且多为III或IV级,主要表现为血小板减少、中性粒细胞减少和贫血。
鉴于地西他滨传统治疗体系中常发生延迟给药或提前中止疗程,为进一步提高地西他滨的疗效,我们探索出一套分段治疗及疗效评估体系,现对该体系做一详细说明,并同传统地西他滨治疗体系进行比较分析。
研究目的：
论证研究地西他滨分段治疗及疗效评估体系的合理性、可行性及安全性,分析比较地西他滨传统治疗体系与分段治疗评估体系治疗骨髓增生异常综合征的有效性。
研究方法：
研究对象选择山东大学齐鲁医院2012年2月至2013年12月确诊为MDS的57例患者。24例患者接受传统小剂量地西他滨治疗方案：应用地西他滨20m/平方米/天,静脉输注,连续5天,4周一疗程。在地西他滨治疗第2疗程后复查骨髓细胞学和外周血象,评价骨髓和血液学缓解情况,评估疗效。33位MDS患者接受小剂量地西他滨分段治疗及疗效评估体系治疗：用药剂量同前；首次应用地西他滨前根据MDS患者基线特征评估情况,加强支持治疗,并同时进行抗微生物“三联”预防。治疗第2疗程完后监测血小板计数变化情况,第3疗程后复查骨髓原始细胞数、外周血象,评估疗效。第4疗程后进行巩固治疗,第6疗程后进入维持治疗。
第1疗程后,统计分析传统治疗组和分段治疗评估组两组之间在3-4级血液学副反应及感染发生率方面有无差异,分析比较两组之间在延迟给药发生率、提前中止治疗发生率等方面有无差异；第2疗程结束后重点关注血小板变化情况,动态监测血小板数目,分析比较两组中血小板改善情况；第3疗程后分段治疗评估组进行骨髓原始细胞数和外周血象复查,传统治疗组已在第2疗程后复查骨髓原始细胞数和外周血象,统计分析两组中地西他滨治疗的总有效率(overall response rate, ORR)是否存在统计学差异,ORR评价指标包括完全缓解(complete remission, CR)率和部分缓解(partial remission, PR)率。比较两组输血依赖情况及地西他滨中位疗程数有无统计学差异。
研究结果：
1.地西他滨传统治疗体系和分段治疗评估体系第一疗程后发生感染的情况传统治疗组地西他滨应用第一疗程后发生感染的机率为66.7%,分段治疗组第一疗程后发生感染的机率为39.4%,P=0.042<0.05,传统治疗组与分段治疗组MDS患者并发感染的机率具有统计学差异。
2.地西他滨传统治疗体系和分段治疗评估体系第一疗程后发生3～4级中性粒细胞减少的情况传统治疗组不良反应-3-4级中性粒细胞减少的发生率为33.3%,分段治疗组3-4级中性粒细胞减少的发生率为9.1%,P＝0.039<0.05,分段治疗组和传统治疗组3-4级中性粒细胞减少发生率具有显著统计学差异。
3.地西他滨传统治疗体系和分段治疗评估体系第一疗程后发生3-4级血小板减少的情况传统治疗组不良反应3-4级血小板减少的发生率为50.0%,分段治疗组3-4级血小板减少的发生率为24.2%,P=0.044<0.05,说明分段治疗组和传统治疗组3～4级血小板减少发生率具有显著统计学差异。
4.地西他滨传统治疗体系和分段治疗评估体系第一疗程后发生3-4级贫血的情况传统治疗组不良反应3～4级贫血的发生率为29.2%,分段治疗组3-4级贫血的发生率为6.1%,P=0.027<0.05,说明分段治疗组和传统治疗组3～4级贫血发生率具有统计学差异。
5.地西他滨传统治疗体系和分段治疗评估体系一疗程后延迟给药和中止治疗的发生情况传统治疗组地西他滨应用一疗程后延迟给药发生率为66.7%,分段治疗组治疗过程中延迟给药发生率为36.4%,P=0.024<0.05,说明分段治疗组和传统治疗组地西他滨应用一疗程后延迟给药发生率具有统计学差异。一疗程后中止治疗的发生率没有统计学差异。
6.地西他滨传统治疗体系和分段治疗评估体系治疗反应的对比传统治疗组及分段治疗组地西他滨的总有效率ORR(CR%+PR%)分别为12.5%和36.4%,P=0.043<0.05,分段治疗及疗效评估体系可以显著提高地西他滨治疗的有效率。传统治疗组血小板改善率为41.7%,分段治疗组血小板改善率为69.7%,P=0.034<0.05,分段治疗组和传统治疗组MDS患者获得血小板改善的机率具有显著统计学差异,分段治疗及疗效评估体系中MDS患者的血小板反应更佳。传统治疗组和分段治疗组两组中MDS患者的血小板数目均比其自身的中性粒细胞数目和血红蛋白含量恢复得早。分段治疗组获得血小板改善的MDS患者其血小板计数水平多在第2-3疗程开始回升,而传统治疗组获得血小板改善的MDS患者其血小板计数水平常在第3、4疗程开始回升。在地西他滨传统治疗组中,治疗前输血依赖者13例,治疗后输血依赖者9例,脱离输血依赖患者的比例为30.8%。在地西他滨分段治疗组中,治疗前输血依赖者24例,治疗后输血依赖者8例,脱离输血依赖患者的比例为66.7%,P=0.036,分段治疗评估体系中更高比例的患者脱离输血依赖,输血频次或输血量较大幅度地减低。
7.传统治疗体系和分段治疗评估体系地西他滨中位疗程数对比传统治疗组MDS患者使用地西他滨治疗的疗程数是1-5,中位疗程数是4；分段治疗组MDS患者使用地西他滨治疗的疗程数是2-8,中位疗程数是7。比较两组之间中位疗程数的差异,P<0.01,提示分段治疗及疗效评估体系可以确保地西他滨疗程的衔接性。
结论：与地西他滨传统治疗MDS相比,在分段治疗及疗效评估体系的各个环节中
我们进行了“三联”预防微生物感染,降低了感染率；及时应用造血生长因子或成
分输血促进血液学恢复,降低了延迟或减量给药的发生率；分时段科学监测患者
外周血和骨髓的耐受情况及恢复水平。地西他滨分段治疗骨髓增生异常综合征及
疗效评估体系可以确保去甲基化治疗的连续性及足够的疗程数,减少血液学不良
反应及继发感染的机会,减少延迟给药和输血依赖的发生,最终提高初治或其他
治疗无效的MDS患者的总体反应率。
Section I
Th22cells as well as Th17cells expand differentially in patients with early-stage and late-stage myelodysplastic syndrome
Background:
Myelodysplastic syndrome (MDS) encompasses a heterogeneous group of clonal hematopoietic stem-cell disorders, characterized by ineffective hematopoiesis and an increased probability of developing acute leukemia. Autoimmune-mediated myelosuppression and immune surveillance of malignant clone play recognized crucial roles in the process of MDS. Findings of expanded clonal T lymphocytes and clinical responses to immunoregulatory therapy have led to the speculation that both T helper and cytotoxic T cells are involved in the immunological pathophysiology of MDS.
Recently, a separate Th cell subset which secrets the cytokine interleukin(IL)-17and expresses the lineage-specific transcription factor retinoic acid receptor-related orphan receptor C (RORC) was identified and named T-helper cell type17(Th17). Since then, Th17cells have entered the limelight because of their comprehensive involvement in inflammations. IL-17A, a representative Th17cytokine, has been described in various models of immune-mediated tissue injury, such as rheumatoid arthritis, lupus, myeloma and so on. Although one study reported an increased number of peripheral Th17cells in low risk MDS lately, the mechanism of cellular immune abnormalities still remains undiscovered.
Th22subset, which has the ability of secreting IL-22and tumor necrosis factor alpha (TNF-a), has become a new subset clearly separated from other Th cells. This subset does not express any of cytokine interferon (IFN)-y, IL-17or their associated transcription factors T-bet and RORC. Aryl hydrocarbon receptor (AHR) activation participates in priming naive CD4+T cells to Th22subset plus the promotion by IL-6and TNF-a. Th22cells have been found regulating epidermal responses within the epidermal layer prominently in inflammatory skin diseases. In addition, a preferential expansion of Th22cells contributing to gastric cancer progression has been described. These data above support that Th22cells are involved in the pathophysiology of inflammatory immune reactions and immune evasion of neoplasms.
IL-22belongs to the IL-10cytokine family and is primarily secreted by Th22cells on activation. The expression trend of IL-22in cancers and autoimmune disorders is diverse, with IL-17as siblings but not twins based on the local biological characteristics. The expression level of IL-22was up-regulated in skin inflammatory pathology and anaplastic lymphoma kinase positive anaplastic large cell lymphoma, inducing directional signal linking the immune system to the targeted tissue-resident cells. Meanwhile, it was down-regulated in systemic lupus erythematosus. Within disorders such as inflammatory bowel disease (IBD), once inducted by the naive or memory/effector cells, IL-22has access to inhibit or accelerate the inflammatory reactions regarding the idiographic microenvironment.
To date, no report exist with regard to Th22cell subset or their association with Th17or Thl subset among MDS patients. To investigate their possible roles in the immunological pathophysiology of MDS, we measured the percentages of circulating Th22, Th17, Th1and mRNA expression levels of RORC, IL-6, TNF-a and IL-23in peripheral blood mononuclear cells (PBMCs) as well as the cytokine expression levels of IL-22or IL-17in peripheral blood (PB) and bone marrow (BM). To explore the potential relationship between Th polarization and different stages of MDS, the correlations of various Th subsets were evaluated at the same time.
Objective:
Measure the frequencies of periphery Th22, Th17subsets and determine the mRNA expression of transcription factor RORC, cytokines such as IL-6, TNF-a and IL-23. Explore the regulation effects of transcription factor and cytokines on Th22/Th17polarization in different stages of myelodysplastic syndrome. Attempt to elucidate the relevance between the switched immune status and clinical prognosis of myelodysplastic syndrome as well as search for peripheral blood immunological index to guide the diagnosis of different disease stages.
Materials and methods:
1. PB was collected from seventeen patients with early-stage MDS (E-MDS), twenty patients with late-stage MDS (L-MDS) and twenty age-matched healthy volunteers. BM was obtained from twenty-five MDS patients and ten hematologically normal surgical patients without any oncological or immunological issues.
2. The percentages of circulating Th22, Th17, Thl subset were measured in E-MDS patients, L-MDS patients and healthy controls by flow cytometry.
3. The cytokine levels of IL-22and IL-17both in PB and BM plasma were examined by enzyme-linked immunosorbent assay.
4. For RNA isolation, PBMCs were isolated from the anticoagulated blood donated by E-MDS patients, L-MDS patients and healthy controls. The mRNA expression levels of RORC, IL-6, TNF-a and IL-23were determined by real-time quantitative polymerase chain reaction.
5. Results were expressed as mean±SD or median (range). Comparisons between two groups were assessed by the non-paired t-test or the Wilcoxon rank-sum test to compare parametric and non-parametric data respectively. Statistical significance among E-MDS, L-MDS and healthy control three groups was determined by ANOVA, and difference between any two groups was determined by Newman-Keuls multiple comparison test (q test) unless the data were not normally distributed, in which case Kruskal-Wallis test (H test) and Nemenyi test were used. The Pearson or Spearman correlation test was used for correlation analysis depending on data distribution. All tests were performed by SAS9.1system. P value less than0.05was considered statistically significant.
Results:
1. The percentage of circulating Th22subset in E-MDS patients, L-MDS patients and healthy controls Compared with healthy controls, the percentage of peripheral Th22cells was significantly higher in total MDS patients (0.71±0.17%vs.1.55±0.74%, P<0.0001). The number of peripheral Th22cells in E-MDS is higher than that in controls (1.27±0.50%vs.0.71±0.17%, P=0.002). Also a significant increase was shown in L-MDS compared with E-MDS patients (1.77±0.84%vs.1.27±0.50%, P=0.03).
2. The percentage of circulating Th17subset in E-MDS patients, L-MDS patients and healthy controls The percentage of peripheral Th17cells was significantly elevated in E-MDS (median,1.90%; range,0.58-6.01%) when compared with L-MDS (median,1.16%; range,0.15-1.86%)(P=0.002) or healthy control group (0.97±0.29%; P=0.002).
3. The percentage of circulating Th1subset in MDS patients and healthy controls For peripheral Thl cells, no significant difference was observed between MDS patients and healthy controls.
4. The cytokine levels of IL-22and IL-17in PB and BM plasma from MDS patients and healthy controls No significant difference of PB IL-22or IL-17level between MDS patients (median,22.64pg/ml; range,16.02-54.66) and healthy controls (median,23.86pg/ml; range,14.05-36.49) was observed, consistent with BM findings.
5. mRNA expression levels of RORC, IL-6, TNF-a and IL-23in E-MDS, L-MDS cohort and controls
The relative amount of RORC mRNA in E-MDS patients was markedly increased compared with healthy controls and L-MDS patients (P=0.0007; P=0.002). The relative amount of mRNA of IL-6in L-MDS patients was much higher than that in E-MDS patients (P<0.05) and healthy controls(P<0.001). TNF-a mRNA level was also present on higher levels in L-MDS compared with E-MDS and controls (P<0.05; P<0.005). There was no significant difference in IL-23p19mRNA expression levels among E-MDS, L-MDS and healthy controls.
6. Correlation between Th22, Thl7, and Thl Cells in MDS Patients In E-MDS patients, there existed a statistically positive correlation between peripheral Th22cells and Thl7cells (r=0.675, P=0.004) while no statistical correlation was shown among L-MDS patients (r=0.138, P=0.610). Peripheral Th22subset showed no significant correlation with peripheral Thl subset (P=0.053).
Conclusion:
E-MDS cohort demonstrated the dominance of Thl7subset and its specific transcription factor RORC. On the other hand, L-MDS cohort showed an increased frequency of Th22cells in peripheral blood, along with higher mRNA expression levels of IL-6and TNF-a. Our study indicates that Th22cells along with Th17cells or not are involved in regulating the autoimmune reaction of E-MDS and immune evasion of L-MDS. As for the disease stages, peripheral Th22/Th17ratio is important to the stratified diagnosis of E-MDS and L-MDS.
section Ⅱ
Clinical study of decitabine in the segmented treatment and evaluation system for myelodysplastic syndrome
Background:
Epigenetics refers to alteration in gene expression that does not involve changes to the corresponding DNA sequence. The common epigenetic events include DNA methylation, RNA interference, histone modification and so on. Despite that cytogenetic heterogeneity constitutes the principal pathophysiological mechanism of MDS, haploinsufficiency, secondary mutation as well as epigenetic modification also contribute to the disease phenotype. What top the ratings of epigenetic modifications in MDS are DNA methylation and histone modification on genomic level, but in fact these modifications are focally increased around the promoter region of tumor suppressor gene and other mitosis inhibitors. It is noted that there are more hypermethylation sites in high risk MDS compared with low risk MDS. DNA methyltransferase3A (DNMT3A) and TET2mutations in the promoter region have been recognized to be involved in DNA methylation. In the CpG dinucleotides enriched site, DNMT functions in the methylation of cytimidine converting to5-methylcytosine, which is thought to mediate much of the increased methylation process and has therefore become a crucial treatment target in MDS.
Researchers have proved that high-dose decitabine has direct cytotoxicity to clonal cells whereas low-dose decitabine induces hypomethylation through inhibiting the activity of DNMTs, which is distinct from immediate cytotoxicity. The normal demethylated state of former silencing tumor suppressor genes such as E-cadherin, p16, hMLHl, VHL, p15and P21shall be restored, followed with reactivation and gene transcription.
Decitabine was recently approved by the US Food and Drug Administration (FDA) for treating patients with intermediate risk/high risk/very high risk MDS or nonresponsive MDS. A number of retrospective and prospective studies have revealed that decitabine favors to cytogenetic improvements, decreases the probability of developing acute leukemia and prolongs the overall survival in the majority of patients with higher risk MDS. However, during the clinical practice of decitabine, myelosuppression often occurs, resulting in thrombocytopenia, neutropenia or anemia. What's worse, higher grade3/4myelosuppression happens more frequently.
Given that the current clinical scheme of decitabine often goes wrong due to dose delay or early discontinuance, we designed a novel segmented treatment and evaluation system to improve the long-term efficacy of low-dose decitabine. At present, we describe this novel segmented treatment and evaluation system in detail and make comparison with the original prevailing protocol.
Objective:
Expound the rationality, feasibility and drug safety of the segmented treatment and evaluation system of decitabine for treating MDS; compare and analyze the clinical effectiveness of decitabine used in the segmented system and conventional system.
Methods:
A total of57patients with MDS were recruited in this study. Enrollment took place between February2012and December2013in the Department of Hematology of Qilu Hospital, Shandong University.24patients were treated with decitabine in the conventional scheme, i.e.20mg/m2/d, intravenous (IV) infused for5days and repeated every4weeks. The myelogram and hemogram changes were reexamined after the second course to evaluate the marrow remission and hematologic remission.33MDS patients were incorporated into the segmented treatment and evaluation system with the same dose of decitabine as the conventional scheme. However, before starting the segmented treatment, the baseline characteristics of the subjects should be assessed. Antibiotics, antivirals and antifungals were preventively applied. Best supportive care was supposed to be aggressive. After the second course, only platelet count was monitored whereas after the third course, complete blood count and blasts in bone marrow were tested. Intensification therapy was carried on after the forth course and maintainance therapy after the sixth course.
The incidence of grades3to4hematological side effects and infections were analyzed in the conventional remedy system and segmented remedy system after the first course, as well as the incidence of dose delay and early discontinuance. The platelet counts were highlightly concerned in the second course and were dynamicly monitored until achieving platelet hematologic improvement (HI-P). The myelogram and hemogram changes were reexamined after the third course in the segmented remedy system whereas these had been reexamined after the second course in the conventional remedy system. The overall response rate (ORR), transfusion-dependence and median course in these two different systems were compared using chi-square test and non-parametric test via SPSS17.0software.
Results:
1. Infections in the conventional and segmented remedy systems after the first decitabine course
The infection rate in the conventional treatment group after the first decitabine course was66.7%versus39.4%in the segmented treatment and evaluation group (P=0.042<0.05). The infection rate in the segmented treatment and evaluation group was significantly lower compared with that in the conventional treatment group.
2. Grades3to4neutropenia in the conventional and segmented remedy systems after the first decitabine course
Compared with the conventional remedy group, the incidence rate of Grades3to4neutropenia was significantly lower in the segmented remedy group (9.1%vs.33.3%, P=0.039<0.05).
3. Grades3to4thrombocytopenia in the conventional and segmented remedy systems after the first decitabine course
The incidence rate of Grades3to4thrombocytopenia in the segmented remedy group was obviously reduced when compared with the conventional remedy group (24.2%vs.50.0%, P=0.044<0.05).
4. Grades3to4anemia in the conventional and segmented remedy systems after the first decitabine course
The incidence rate of Grades3to4anemia in the segmented remedy group was markedly reduced compared with that in the conventional remedy group (6.1%vs.29.2%, P=0.027<0.05).
5. Dose delay or early discontinuance in the conventional and segmented remedy systems after the first decitabine course
Significant difference in the incidence of dose delay between the segmented remedy group and the conventional remedy group was observed (36.4%vs.66.7%, P=0.024<0.05). No difference in the incidence of early discontinuance after the first course was found between the two groups.
6. Comparision of responses to decitabine in the conventional and segmented remedy systems
Significant increment of ORR (CR%plus PR%) was obtained in the segmented remedy group when compared with the index in the conventional remedy group (36.4%vs.12.5%, P=0.043<0.05).
The incidence rate of platelet hematologic improvement (HI-P) in the conventional and segmented remedy groups was41.7%and69.7%respectively (P=0.034), showing a much better platelet response in the latter group.
The platelet counts of MDS patients who finally achieved HI-P in the segmented group usually began to rebound during the second to third course whereas it was the third to forth course during which platelet counts began to rebound in the conventional group.
In the conventional group,13cases were transfusion-dependent at baseline,4cases (30.8%) became transfusion-independent during the trial. In the segmented group,24cases were transfusion-dependent at baseline,16cases (66.7%) became transfusion-independent during the trial, P=0.036, indicating that the segmented treatment and evaluation system can alleviate transfusion-dependence.
7. Comparision of the median courses of decitabine in the conventional and segmented remedy systems
The number of medication courses in the conventional scheme reached to5as the upper limit with a median of4while the courses in the segmented scheme reached to8at most, with a median of7. The therapeutic courses were noteworthyly increased in the segmented remedy group so as to guarantee the consecutive demethylation therapy.
Conclusion:
Different from the therapeutic strategy in the conventional system, prophylactic antibiotics, antivirals and antifungals were simultaneously applied in the segmented system, resulting in reduced infection; patients incorporated in the segmented system were given best supportive care such as hematopoietic growth factors or blood-component transfusion in time to promoting the hematological recovery, leading to diminished dose delay or dose reduction; the tolerance and recovery situation in peripheral blood and bone marrow were detected in different time periods in the segmented system. The segmented system of decitabine turned out to have more access to guaranteeing the consecutive and sufficient medication courses, reducing the opportunities for hematologic toxicity and secondary infection, dose delay or blood transfusion, and ultimately improving the overall response rate of MDS patients who were newly diagnosed or nonresponsive to previous therapies.
引文
1. Tefferi A, Vardiman JW. Myelodysplastic syndromes. The New England journal of medicine 2009;361:1872-1885.
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6. Aggarwal S, van de Loosdrecht AA, Alhan C, et al. Role of immune responses in the pathogenesis of low-risk MDS and high-risk MDS:implications for immunotherapy. British journal of haematology 2011;153:568-581.
7. Giannouli S, Kanellopoulou T, Voulgarelis M. Myelodysplasia and autoimmunity. Current opinion in rheumatology 2012;24:97-102.
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9. Fozza C, Longinotti M. The role of T-cells in the pathogenesis of myelodysplastic syndromes:passengers and drivers. Leukemia research 2013;37:201-203.
10. Molldrem JJ, Jiang YZ, Stetler-Stevenson M, et al. Haematological response of patients with myelodysplastic syndrome to antithymocyte globulin is associated with a loss of lymphocyte-mediated inhibition of CFU-GM and alterations in T-cell receptor Vbeta profiles. British journal of haematology 1998;102:1314-1322.
11. Kochenderfer JN, Kobayashi S, Wieder ED, et al. Loss of T-lymphocyte clonal dominance in patients with myelodysplastic syndrome responsive to immunosuppression. Blood 2002;100:3639-3645.
12. Steinman L. A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of T cell-mediated tissue damage. Nature medicine 2007;13:139-145.
13. Ivanov, II, McKenzie BS, Zhou L, et al. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+T helper cells. Cell 2006;126:1121-1133.
14. Ciofani M, Madar A, Galan C, et al. A validated regulatory network for Th17 cell specification. Cell 2012;151:289-303.
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20. Eyerich S, Eyerich K, Pennino D, et al. Th22 cells represent a distinct human T cell subset involved in epidermal immunity and remodeling. The Journal of clinical investigation 2009;119:3573-3585.
21.Duhen T, Geiger R, Jarrossay D, et al. Production of interleukin 22 but not interleukin 17 by a subset of human skin-homing memory T cells. Nature immunology 2009;10:857-863.
22. Ramirez JM, Brembilla NC, Sorg O, et al. Activation of the aryl hydrocarbon receptor reveals distinct requirements for IL-22 and IL-17 production by human T helper cells. European journal of immunology 2010;40:2450-2459.
23. Zhuang Y, Peng LS, Zhao YL, et al. Increased intratumoral IL-22-producing CD4(+) T cells and Th22 cells correlate with gastric cancer progression and predict poor patient survival. Cancer Immunol Immunother 2012 Apr 13. [Epub ahead of print].
24. Trifari S, Kaplan CD, Tran EH, et al. Identification of a human helper T cell population that has abundant production of interleukin 22 and is distinct from T(H)-17, T(H)1 and T(H)2 cells. Nature immunology 2009;10:864-871.
25. Cella M, Otero K, Colonna M. Expansion of human NK-22 cells with IL-7, IL-2, and IL-lbeta reveals intrinsic functional plasticity. Proceedings of the National Academy of Sciences of the United States of America 2010;107:10961-10966.
26. Korn T, Bettelli E, Oukka M, et al. IL-17 and Th17 Cells. Annu Rev Immunol 2009;27:485-517.
27. Volpe E, Servant N, Zollinger R, et al. A critical function for transforming growth factor-beta, interleukin 23 and proinflammatory cytokines in driving and modulating human T(H)-17 responses. Nat Immunol 2008;9:650-657.
28. Zheng Y, Danilenko DM, Valdez P, et al. Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis. Nature 2007; 445:648-651.
29. Bard JD, Gelebart P, Anand M, et al. Aberrant expression of IL-22 receptor 1 and autocrine IL-22 stimulation contribute to tumorigenicity in ALK+anaplastic large cell lymphoma. Leukemia 2008;22:1595-1603.
30. Cheng F, Guo Z, Xu H, et al. Decreased plasma IL22 levels, but not increased IL17 and IL23 levels, correlate with disease activity in patients with systemic lupus erythematosus. Annals of the rheumatic diseases 2009;68:604-606.
31. Andoh A, Zhang Z, Inatomi O, et al. Interleukin-22, a member of the IL-10 subfamily, induces inflammatory responses in colonic subepithelial myofibroblasts. Gastroenterology 2005; 129:969-984.
32. Sugimoto K, Ogawa A, Mizoguchi E, et al. IL-22 ameliorates intestinal inflammation in a mouse model of ulcerative colitis. The Journal of clinical investigation 2008; 118:534-544.
33. Vardiman JW, Thiele J, Arber DA, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 2009;114:937-951.
34. Greenberg PL, Tuechler H, Schanz J, et al. Revised international prognostic scoring system for myelodysplastic syndromes. Blood 2012;120:2454-2465.
35. Cheson BD, Bennett JM, Kantarjian H, et al. Report of an international working group to standardize response criteria for myelodysplastic syndromes. Blood 2000;96:3671-3674.
36. Bouchliou I, Miltiades P, Nakou E, et al. Th17 and Foxp3(+) T regulatory cell dynamics and distribution in myelodysplastic syndromes. Clinical immunology 2011;139:350-359.
37. Zhang L, Li JM, Liu XG, et al. Elevated Th22 cells correlated with Th17 cells in patients with rheumatoid arthritis. Journal of clinical immunology 2011;31:606-614.
38. Kondo A, Yamashita T, Tamura H, et al. Interferon-gamma and tumor necrosis factor-alpha induce an immunoinhibitory molecule, B7-H1, via nuclear factor-kappaB activation in blasts in myelodysplastic syndromes. Blood 2010;116:1124-1131.
39. Parker JE, Mufti GJ, Rasool F, et al. The role of apoptosis, proliferation, and the Bcl-2-related proteins in the myelodysplastic syndromes and acute myeloid leukemia secondary to MDS. Blood 2000;96:3932-3938.
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41. Chamuleau ME, Westers TM, van Dreunen L, et al. Immune mediated autologous cytotoxicity against hematopoietic precursor cells in patients with myelodysplastic syndrome. Haematologica 2009;94:496-506.
42. Kordasti SY, Ingram W, Hayden J, et al. CD4+CD25high Foxp3+regulatory T cells in myelodysplastic syndrome (MDS). Blood 2007;110:847-850.
43. Epling-Burnette PK, Bai F, Painter JS, et al. Reduced natural killer (NK) function associated with high-risk myelodysplastic syndrome (MDS) and reduced expression of activating NK receptors. Blood 2007; 109:4816-4824.
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45. Alexandrakis M, Coulocheri S, Xylouri I, et al. Elevated serum TNF-alpha concentrations are predictive of shortened survival in patients with high-risk myelodysplastic syndromes. Haematologia 1998;29:13-24.
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1. Molldrem JJ, Jiang YZ, Stetler-Stevenson M, Mavroudis D, Hensel N, et al. (1998) Haematological response of patients with myelodysplastic syndrome to antithymocyte globulin is associated with a loss of lymphocyte-mediated inhibition of CFU-GM and alterations in T-cell receptor Vbeta profiles. Br J Haematol 102:1314-1322.
2. Kochenderfer JN, Kobayashi S, Wieder ED, Su C, Molldrem JJ (2002) Loss of T-lymphocyte clonal dominance in patients with myelodysplastic syndrome responsive to immunosuppression. Blood 100:3639-3645.
3. Fozza C, Contini S, Galleu A, Simula MP, Virdis P, et al. (2009) Patients with myelodysplastic syndromes display several T-cell expansions, which are mostly polyclonal in the CD4(+) subset and oligoclonal in the CD8(+) subset. Exp Hematol 37:947-955.
4. Steinman L (2007) A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of T cell-mediated tissue damage. Nat Med 13:139-145.
5. Ivanov, II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, et al. (2006) The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+T helper cells. Cell 126:1121-1133.
6. McGeachy MJ, Cua DJ (2008) Th17 cell differentiation:the long and winding road. Immunity 28:445-453.
7. Kordasti SY, Afzali B, Lim Z, Ingram W, Hayden J, et al. (2009) IL-17-producing CD4(+) T cells, pro-inflammatory cytokines and apoptosis are increased in low risk myelodysplastic syndrome. Br J Haematol 145:64-72.
8. Eyerich S, Eyerich K, Pennino D, Carbone T, Nasorri F, et al. (2009) Th22 cells represent a distinct human T cell subset involved in epidermal immunity and remodeling. J Clin Invest 119:3573-3585.
9. Duhen T, Geiger R, Jarrossay D, Lanzavecchia A, Sallusto F (2009) Production of interleukin 22 but not interleukin 17 by a subset of human skin-homing memory T cells. Nat Immunol 10:857-863.
10. Chizzolini C, Ramirez JM, Brembilla NC, Sorg O, Chicheportiche R, et al. (2010) Activation of the aryl hydrocarbon receptor reveals distinct requirements for IL-22 and IL-17 production by human T helper cells. European Journal of Immunology 40:2450-2459.
11. Zhuang Y, Peng LS, Zhao YL, Shi Y, Mao XH, et al. (2012 Apr 13. [Epub ahead of print]) Increased intratumoral IL-22-producing CD4(+) T cells and Th22 cells correlate with gastric cancer progression and predict poor patient survival. Cancer Immunol Immunother.
12. Trifari S, Kaplan CD, Tran EH, Crellin NK, Spits H (2009) Identification of a human helper T cell population that has abundant production of interleukin 22 and is distinct from T(H)-17, T(H)1 and T(H)2 cells. Nat Immunol 10:864-871.
13. Zheng Y, Danilenko DM, Valdez P, Kasman I, Eastham-Anderson J, et al. (2007) Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis. Nature 445:648-651.
14. Bard JD, Gelebart P, Anand M, Amin HM, Lai R (2008) Aberrant expression of IL-22 receptor 1 and autocrine IL-22 stimulation contribute to tumorigenicity in ALK+anaplastic large cell lymphoma. Leukemia 22:1595-1603.
15. Cheng F, Guo Z, Xu H, Yan D, Li Q (2009) Decreased plasma IL22 levels, but not increased IL17 and IL23 levels, correlate with disease activity in patients with systemic lupus erythematosus. Ann Rheum Dis 68:604-606.
16. Andoh A, Zhang Z, Inatomi O, Fujino S, Deguchi Y, et al. (2005) Interleukin-22, a member of the IL-10 subfamily, induces inflammatory responses in colonic subepithelial myofibroblasts. Gastroenterology 129:969-984.
17. Sugimoto K, Ogawa A, Mizoguchi E, Shimomura Y, Andoh A, et al. (2008) IL-22 ameliorates intestinal inflammation in a mouse model of ulcerative colitis. J Clin Invest 118:534-544.
18. Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, et al. (2009) The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia:rationale and important changes. Blood 114: 937-951.
19. Cheson BD, Bennett JM, Kantarjian H, Pinto A, Schiffer CA, et al. (2000) Report of an international working group to standardize response criteria for myelodysplastic syndromes. Blood 96:3671-3674.
20. Bouchliou I, Miltiades P, Nakou E, Spanoudakis E, Goutzouvelidis A, et al. (2011) Th17 and Foxp3(+) T regulatory cell dynamics and distribution in myelodysplastic syndromes. Clin Immunol 139:350-359.
21. Zhang L, Li JM, Liu XG, Ma DX, Hu NW, et al. (2011) Elevated Th22 Cells Correlated with Th17 Cells in Patients with Rheumatoid Arthritis. J Clin Immunol 31:606-614.
22. Kondo A, Yamashita T, Tamura H, Zhao W, Tsuji T, et al. (2010) Interferon-gamma and tumor necrosis factor-alpha induce an immunoinhibitory molecule, B7-H1, via nuclear factor-kappaB activation in blasts in myelodysplastic syndromes. Blood 116:1124-1131.
23. Aggarwal S, van de Loosdrecht AA, Alhan C, Ossenkoppele GJ, Westers TM, et al. (2011) Role of immune responses in the pathogenesis of low-risk MDS and high-risk MDS:implications for immunotherapy. Br J Haematol 153:568-581.
24. Korn T, Bettelli E, Oukka M, Kuchroo VK (2009) IL-17 and Th17 Cells. Annu Rev Immunol 27:485-517.
25. Parker JE, Mufti GJ, Rasool F, Mijovic A, Devereux S, et al. (2000) The role of apoptosis, proliferation, and the Bcl-2-related proteins in the myelodysplastic syndromes and acute myeloid leukemia secondary to MDS. Blood 96:3932-3938.
26. Kotsianidis I, Bouchliou I, Nakou E, Spanoudakis E, Margaritis D, et al. (2009) Kinetics, function and bone marrow trafficking of CD4+CD25+FOXP3+ regulatory T cells in myelodysplastic syndromes (MDS). Leukemia 23:510-518.
27. Chamuleau MED, Westers TM, van Dreunen L, Groenland J, Zevenbergen A, et al. (2009) Immune mediated autologous cytotoxicity against hematopoietic precursor cells in patients with myelodysplastic syndrome. Haematologica-the Hematology Journal 94:496-506.
28. Kordasti SY, Ingram W, Hayden J, Darling D, Barber L, et al. (2007) CD4+CD25high Foxp3+regulatory T cells in myelodysplastic syndrome (MDS). Blood 110:847-850.
29. Epling-Burnette PK, Bai F, Painter JS, Rollison DE, Salih HR, et al. (2007) Reduced natural killer (NK) function associated with high-risk myelodysplastic syndrome (MDS) and reduced expression of activating NK receptors. Blood 109: 4816-4824.
30. Ye ZJ, Zhou Q, Yuan ML, Du RH, Yang WB, et al. (2012) Differentiation and Recruitment of IL-22-Producing Helper T Cells Stimulated by Pleural Mesothelial Cells in Tuberculous Pleurisy. Am J Respir Crit Care Med 185:660-669.
31. Alexandrakis M, Coulocheri S, Xylouri I, Ganotakis E, Eliakis P, et al. (1998) Elevated serum TNF-alpha concentrations are predictive of shortened survival in patients with high-risk myelodysplastic syndromes. Haematologia (Budap) 29: 13-24.
32. Wolk K, Kunz S, Witte E, Friedrich M, Asadullah K, et al. (2004) IL-22 increases the innate immunity of tissues. Immunity 21:241-254.
33. Xie MH, Aggarwal S, Ho WH, Foster J, Zhang Z, et al. (2000) Interleukin (IL)-22, a novel human cytokine that signals through the interferon receptor-related proteins CRF2-4 and IL-22R. J Biol Chem 275:31335-31339.
34. Dhiman R, Indramohan M, Barnes PF, Nayak RC, Paidipally P, et al. (2009) IL-22 produced by human NK cells inhibits growth of Mycobacterium tuberculosis by enhancing phagolysosomal fusion. J Immunol 183:6639-6645.
35. Schnyder B, Lima C, Schnyder-Candrian S (2010) Interleukin-22 is a negative regulator of the allergic response. Cytokine 50:220-227.
36. Kitagawa M, Saito I, Kuwata T, Yoshida S, Yamaguchi S, et al. (1997) Overexpression of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma by bone marrow cells from patients with myelodysplastic syndromes. Leukemia 11:2049-2054.
37. Prabhala RH, Pelluru D, Fulciniti M, Prabhala HK, Nanjappa P, et al. (2010) Elevated IL-17 produced by TH17 cells promotes myeloma cell growth and inhibits immune function in multiple myeloma. Blood 115:5385-5392.
38. Sabat R, Wolk K, Kunz S, Asadullah K (2002) Cutting edge:Immune cells as sources and targets of the IL-10 family members? Journal of Immunology 168: 5397-5402.
39. Wolk K, Witte E, Wallace E, Docke WD, Kunz S, et al. (2006) IL-22 regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes:a potential role in psoriasis. Eur J Immunol 36: 1309-1323.
40. Kiladjian JJ, Bourgeois E, Lobe I, Braun T, Visentin G, et al. (2006) Cytolytic function and survival of natural killer cells are severely altered in myelodysplastic syndromes. Leukemia 20:463-470.
41. Volpe E, Servant N, Zollinger R, Bogiatzi SI, Hupe P, et al. (2008) A critical function for transforming growth factor-beta, interleukin 23 and proinflammatory cytokines in driving and modulating human T(H)-17 responses. Nat Immunol 9: 650-657.
42. Pardanani A, Finke C, Lasho TL, Al-Kali A, Begna KH, et al. (2012) IPSS-independent prognostic value of plasma CXCL10, IL-7 and IL-6 levels in myelodysplastic syndromes. Leukemia 26:693-699.
43. Zhou L, McMahon C, Bhagat T, Alencar C, Yu Y, et al. (2011) Reduced SMAD7 leads to overactivation of TGF-beta signaling in MDS that can be reversed by a specific inhibitor of TGF-beta receptor I kinase. Cancer Res 71:955-963.
1. Bird A. DNA methylation patterns and epigenetic memory. Genes & development 2002; 16:6-21.
2. Lippman Z, Martienssen R. The role of RNA interference in heterochromatic silencing. Nature 2004;431:364-370.
3. Iizuka M, Smith MM. Functional consequences of histone modifications. Current opinion in genetics & development 2003; 13:154-160.
4. Figueroa ME, Skrabanek L, Li Y, et al. MDS and secondary AML display unique patterns and abundance of aberrant DNA methylation. Blood 2009; 114:3448-3458.
5. Das PM, Singal R. DNA methylation and cancer. Journal of clinical oncology official journal of the American Society of Clinical Oncology 2004;22:4632-4642.
6. Walter MJ, Ding L, Shen D, et al. Recurrent DNMT3A mutations in patients with myelodysplastic syndromes. Leukemia 2011;25:1153-1158.
7. Jankowska AM, Szpurka H, Tiu RV, et al. Loss of heterozygosity 4q24 and TET2 mutations associated with myelodysplastic/myeloproliferative neoplasms. Blood 2009; 113:6403-6410.
8. Jiang Y, Dunbar A, Gondek LP, et al. Aberrant DNA methylation is a dominant mechanism in MDS progression to AML. Blood 2009;113:1315-1325.
9. Ball MP, Li JB, Gao Y, et al. Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells. Nature biotechnology 2009;27:361-368.
10. Greenberg PL, Attar E, Bennett JM, et al. Myelodysplastic syndromes:clinical practice guidelines in oncology. Journal of the National Comprehensive Cancer Network:JNCCN 2013;11:838-874.
11. Pliml J, Sorm F. Synthesis of 2'deoxy-D-ribofuranosyl-5-azacytosine. Coll Czeck Chem Commun 1964;29:2576-2577.
12. Juttermann R, Li E, Jaenisch R. Toxicity of 5-aza-2'-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation. Proceedings of the National Academy of Sciences of the United States of America 1994;91:11797-11801.
13. Ferguson AT, Vertino PM, Spitzner JR, et al. Role of estrogen receptor gene demethylation and DNA methyltransferase.DNA adduct formation in 5-aza-2'deoxycytidine-induced cytotoxicity in human breast cancer cells. The Journal of biological chemistry 1997;272:32260-32266.
14. Attadia V. Effects of 5-aza-2'-deoxycytidine on differentiation and oncogene expression in the human monoblastic leukemia cell line U-937. Leukemia 1993;7 Suppl 1:9-16.
15. Gonzalez-Zulueta M, Bender CM, Yang AS, et al. Methylation of the 5'CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing. Cancer research 1995;55:4531-4535.
16. Yoshiura K, Kanai Y, Ochiai A, et al. Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human carcinomas. Proceedings of the National Academy of Sciences of the United States of America 1995;92:7416-7419.
17. Herman JG, Umar A, Polyak K, et al. Incidence and functional consequences of hMLHl promoter hypermethylation in colorectal carcinoma. Proceedings of the National Academy of Sciences of the United States of America 1998;95:6870-6875.
18. Daskalakis M, Nguyen TT, Nguyen C, et al. Demethylation of a hypermethylated P15/INK4B gene in patients with myelodysplastic syndrome by 5-Aza-2'-deoxycytidine (decitabine) treatment. Blood 2002; 100:2957-2964.
19. Mund C, Hackanson B, Stresemann C, et al. Characterization of DNA demethylation effects induced by 5-Aza-2'-deoxycytidine in patients with myelodysplastic syndrome. Cancer research 2005;65:7086-7090.
20. Kantarjian H, Issa JP, Rosenfeld CS, et al. Decitabine improves patient outcomes in myelodysplastic syndromes:results of a phase III randomized study. Cancer 2006;106:1794-1803.
21. Lubbert M, Suciu S, Baila L, et al. Low-dose decitabine versus best supportive care in elderly patients with intermediate-or high-risk myelodysplastic syndrome (MDS) ineligible for intensive chemotherapy:final results of the randomized phase III study of the European Organisation for Research and Treatment of Cancer Leukemia Group and the German MDS Study Group. Journal of clinical oncology:official journal of the American Society of Clinical Oncology 2011;29:1987-1996.
22. Lubbert M, Wijermans P, Kunzmann R, et al. Cytogenetic responses in high-risk myelodysplastic syndrome following low-dose treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine. British journal of haematology 2001;114:349-357.
23. Wijermans P, Lubbert M, Verhoef G, et al. Low-dose 5-aza-2'-deoxycytidine, a DNA hypomethylating agent, for the treatment of high-risk myelodysplastic syndrome:a multicenter phase II study in elderly patients. Journal of clinical oncology:official journal of the American Society of Clinical Oncology 2000; 18:956-962.
24. Kantarjian H, Oki Y, Garcia-Manero G, et al. Results of a randomized study of 3 schedules of low-dose decitabine in higher-risk myelodysplastic syndrome and chronic myelomonocytic leukemia. Blood 2007; 109:52-57.
25. Kantarjian HM, O'Brien S, Shan J, et al. Update of the decitabine experience in higher risk myelodysplastic syndrome and analysis of prognostic factors associated with outcome. Cancer 2007; 109:265-273.
26. Steensma DP, Baer MR, Slack JL, et al. Multicenter study of decitabine administered daily for 5 days every 4 weeks to adults with myelodysplastic syndromes:the alternative dosing for outpatient treatment (ADOPT) trial. Journal of clinical oncology:official journal of the American Society of Clinical Oncology 2009;27:3842-3848.
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29. Bard JD, Gelebart P, Anand M, et al. Aberrant expression of IL-22 receptor 1 and autocrine IL-22 stimulation contribute to tumorigenicity in ALK+anaplastic large cell lymphoma. Leukemia 2008;22:1595-1603.
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46. Wolk K, Kunz S, Witte E, et al. IL-22 increases the innate immunity of tissues. Immunity 2004;21:241-254.
47. Xie MH, Aggarwal S, Ho WH, et al. Interleukin (IL)-22, a novel human cytokine that signals through the interferon receptor-related proteins CRF2-4 and IL-22R. The Journal of biological chemistry 2000;275:31335-31339.
48. Dhiman R, Indramohan M, Barnes PF, et al. IL-22 produced by human NK cells inhibits growth of Mycobacterium tuberculosis by enhancing phagolysosomal fusion. Journal of immunology 2009;183:6639-6645.
49. Schnyder B, Lima C, Schnyder-Candrian S. Interleukin-22 is a negative regulator of the allergic response. Cytokine 2010;50:220-227.
50. Kitagawa M, Saito I, Kuwata T, et al. Overexpression of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma by bone marrow cells from patients with myelodysplastic syndromes. Leukemia 1997; 11:2049-2054.
51. Prabhala RH, Pelluru D, Fulciniti M, et al. Elevated IL-17 produced by TH17 cells promotes myeloma cell growth and inhibits immune function in multiple myeloma. Blood 2010;115:5385-5392.
52. Wolk K, Kunz S, Asadullah K, et al. Cutting edge:immune cells as sources and targets of the IL-10 family members? Journal of immunology 2002;168:5397-5402.
53. Wolk K, Witte E, Wallace E, et al. IL-22 regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes:a potential role in psoriasis. European journal of immunology 2006;36:1309-1323.
54. Kiladjian JJ, Bourgeois E, Lobe I, et al. Cytolytic function and survival of natural killer cells are severely altered in myelodysplastic syndromes. Leukemia 2006;20:463-470.
55. Pardanani A, Finke C, Lasho TL, et al. IPSS-independent prognostic value of plasma CXCL10, IL-7 and IL-6 levels in myelodysplastic syndromes. Leukemia 2012;26:693-699.
56. Zhou L, McMahon C, Bhagat T, et al. Reduced SMAD7 leads to overactivation of TGF-beta signaling in MDS that can be reversed by a specific inhibitor of TGF-beta receptor I kinase. Cancer research 2011;71:955-963.
1. Bird A. DNA methylation patterns and epigenetic memory. Genes & development 2002;16:6-21.
2. Lippman Z, Martienssen R. The role of RNA interference in heterochromatic silencing. Nature 2004;431:364-370.
3. Iizuka M, Smith MM. Functional consequences of histone modifications. Current opinion in genetics & development 2003; 13:154-160.
4. Figueroa ME, Skrabanek L, Li Y, et al. MDS and secondary AML display unique patterns and abundance of aberrant DNA methylation. Blood 2009;114:3448-3458.
5. Das PM, Singal R. DNA methylation and cancer. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 2004;22:4632-4642.
6. Walter MJ, Ding L, Shen D, et al. Recurrent DNMT3A mutations in patients with myelodysplastic syndromes. Leukemia 2011;25:1153-1158.
7. Jankowska AM, Szpurka H, Tiu RV, et al. Loss of heterozygosity 4q24 and TET2 mutations associated with myelodysplastic/myeloproliferative neoplasms. Blood 2009;113:6403-6410.
8. Jiang Y, Dunbar A, Gondek LP, et al. Aberrant DNA methylation is a dominant mechanism in MDS progression to AML. Blood 2009; 113:1315-1325.
9. Ball MP, Li JB, Gao Y, et al. Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells. Nature biotechnology 2009;27:361-368.
10. Greenberg PL, Attar E, Bennett JM, et al. Myelodysplastic syndromes:clinical practice guidelines in oncology. Journal of the National Comprehensive Cancer Network:JNCCN 2013;11:838-874.
11. Pliml J, Sorm F. Synthesis of 2'deoxy-D-ribofuranosyl-5-azacytosine. Coll Czeck Chem Commun 1964;29:2576-2577.
12. Juttermann R, Li E, Jaenisch R. Toxicity of 5-aza-2'-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation. Proceedings of the National Academy of Sciences of the United States of America 1994;91:11797-11801.
13. Ferguson AT, Vertino PM, Spitzner JR, et al. Role of estrogen receptor gene demethylation and DNA methyltransferase.DNA adduct formation in 5-aza-2'deoxycytidine-induced cytotoxicity in human breast cancer cells. The Journal of biological chemistry 1997;272:32260-32266.
14. Attadia V. Effects of 5-aza-2'-deoxycytidine on differentiation and oncogene expression in the human monoblastic leukemia cell line U-937. Leukemia 1993;7 Suppl 1:9-16.
15. Gonzalez-Zulueta M, Bender CM, Yang AS, et al. Methylation of the 5'CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing. Cancer research 1995;55:4531-4535.
16. Yoshiura K, Kanai Y, Ochiai A, et al. Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human carcinomas. Proceedings of the National Academy of Sciences of the United States of America 1995;92:7416-7419.
17. Herman JG, Umar A, Polyak K, et al. Incidence and functional consequences of hMLHl promoter hypermethylation in colorectal carcinoma. Proceedings of the National Academy of Sciences of the United States of America 1998;95:6870-6875.
18. Daskalakis M, Nguyen TT, Nguyen C, et al. Demethylation of a hypermethylated P15/INK4B gene in patients with myelodysplastic syndrome by 5-Aza-2'-deoxycytidine (decitabine) treatment. Blood 2002;100:2957-2964.
19. Mund C, Hackanson B, Stresemann C, et al. Characterization of DNA demethylation effects induced by 5-Aza-2'-deoxycytidine in patients with myelodysplastic syndrome. Cancer research 2005;65:7086-7090.
20. Kantarjian H, Issa JP, Rosenfeld CS, et al. Decitabine improves patient outcomes in myelodysplastic syndromes:results of a phase Ⅲ randomized study. Cancer 2006;106:1794-1803.
21. Lubbert M, Suciu S, Baila L, et al. Low-dose decitabine versus best supportive care in elderly patients with intermediate-or high-risk myelodysplastic syndrome (MDS) ineligible for intensive chemotherapy:final results of the randomized phase III study of the European Organisation for Research and Treatment of Cancer Leukemia Group and the German MDS Study Group. Journal of clinical oncology:official journal of the American Society of Clinical Oncology 201129:1987-1996.
22. Lubbert M, Wijermans P, Kunzmann R, et al. Cytogenetic responses in high-risk myelodysplastic syndrome following low-dose treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine. British journal of haematology 2001;114:349-357.
23. Wijermans P, Lubbert M, Verhoef G, et al. Low-dose 5-aza-2'-deoxycytidine, a DNA hypomethylating agent, for the treatment of high-risk myelodysplastic syndrome:a multicenter phase II study in elderly patients. Journal of clinical oncology:official journal of the American Society of Clinical Oncology 2000; 18:956-962.
24. Kantarjian H, Oki Y, Garcia-Manero G, et al. Results of a randomized study of 3 schedules of low-dose decitabine in higher-risk myelodysplastic syndrome and chronic myelomonocytic leukemia. Blood 2007; 109:52-57.
25. Kantarjian HM, O'Brien S, Shan J, et al. Update of the decitabine experience in higher risk myelodysplastic syndrome and analysis of prognostic factors associated with outcome. Cancer 2007;109:265-273.
26. Steensma DP, Baer MR, Slack JL, et al. Multicenter study of decitabine administered daily for 5 days every 4 weeks to adults with myelodysplastic syndromes:the alternative dosing for outpatient treatment (ADOPT) trial. Journal of clinical oncology:official journal of the American Society of Clinical Oncology 2009;27:3842-3848.
27. Iastrebner M, Jang JH, Nucifora E, et al. Decitabine in myelodysplastic syndromes and chronic myelomonocytic leukemia:Argentinian/South Korean multi-institutional clinical experience. Leukemia & lymphoma 2010;51:2250-2257.
28. Lee JH, Jang JH, Park J, et al. A prospective multicenter observational study of decitabine treatment in Korean patients with myelodysplastic syndrome. Haematologica 2011;96:1441-1447.
29. Tan Z, Luo Z, X. Z. Effect of low dose Decitabine on myelodysplastic syndromes. Journal of Modern Oncology 2012;20:1682-1685.
30. Gao Y, Ping B, Zhou S. Decitabine for treatment of myelodysplastic syndrome in an elderly patient and review of literature. Journal of Southern Medical University 2012;32:280-282.
31. Lee JH, Lee KH, Lee JH, et al. Decreased incidence of febrile episodes with antibiotic prophylaxis in the treatment of decitabine for myelodysplastic syndrome. Leukemia research 2011;35:499-503.
32. Ruter B, Wijermans P, Claus R, et al. Preferential cytogenetic response to continuous intravenous low-dose decitabine (DAC) administration in myelodysplastic syndrome with monosomy 7. Blood 2007; 110:1080-1082; author reply 1083.
33. Vardiman JW, Thiele J, Arber DA, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 2009; 114:937-951.
34. Cheson BD, Greenberg PL, Bennett JM, et al. Clinical application and proposal for modification of the International Working Group (IWG) response criteria in myelodysplasia. Blood 2006; 108:419-425.
35. Ruter B, Wijermans PW, Lubbert M. Superiority of prolonged low-dose azanucleoside administration? Results of 5-aza-2'-deoxycytidine retreatment in high-risk myelodysplasia patients. Cancer 2006; 106:1744-1750.
36. Kirschbaum M, Gojo I, Goldberg S, et al. Vorinostat in combination with Decitabine for the treatment of relapsed or newly diagnosed acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS):A phase I, dose-escalation study.51st ASH Annual Meeting And Exposition 2009;poster session:poster board 11-66.
37. Uy GL, Abboud CN, Cashen AF, et al. Phase I study of panobinostat plus decitabine in elderly patients with advanced MDS or AML..53rd ASH Annual Meeting and Exposition 2011;poster session:poster board 1-40.
38. Greenberg PL, Tuechler H, Schanz J, et al. Revised international prognostic scoring system for myelodysplastic syndromes. Blood 2012;120:2454-2465.
39. Wang H, Wang XQ, Xu XP, et al. ID4 methylation predicts high risk of leukemic transformation in patients with myelodysplastic syndrome. Leukemia research 2010;34:598-604.
40. Claus R, Almstedt M, Lubbert M. Epigenetic treatment of hematopoietic malignancies:in vivo targets of demethylating agents. Seminars in oncology 2005;32:511-520.
41. Cornelison AM, Manero GG, Kantarjian H, et al. Decitabine (DAC) Can Be Safely Reduced After Achievement of Best Response In Patients (pts) with Myelodysplastic Syndrome (MDS).53rd ASH Annual Meeting and Exposition 2010;poster session:Poster Board 1-838.
42. Wang J, Yi Z, Wang S, et al. The effect of decitabine on megakaryocyte maturation and platelet release. Thrombosis and haemostasis 2011; 106:337-343.
43. van den Bosch J, Lubbert M, Verhoef G, et al. The effects of 5-aza-2'-deoxycytidine (Decitabine) on the platelet count in patients with intermediate and high-risk myelodysplastic syndromes. Leukemia research 2004;28:785-790.
44. Chabot GG, Momparler RL. Effects of 5-aza-2'-deoxycytidine on survival and cell cycle progression of L1210 leukemia cells. Leukemia research 1986; 10:533-537.
45. Gore SD. New ways to use DNA methyltransferase inhibitors for the treatment of myelodysplastic syndrome. Hematology/the Education Program of the American Society of Hematology American Society of Hematology Education Program 2011;2011:550-555.
46. Saba HI, Wijermans PW. Decitabine in myelodysplastic syndromes. Seminars in hematology 2005;42:S23-31.
47. Steensma DP. Decitabine treatment of patients with higher-risk myelodysplastic syndromes. Leukemia research 2009;33 Suppl 2:S12-17.
48. Yang AS, Doshi KD, Choi SW, et al. DNA methylation changes after 5-aza-2'-deoxycytidine therapy in patients with leukemia. Cancer research 2006;66:5495-5503.
49. Steensma DP, Baer MR, Slack JL, et al. Preliminary Results of a Phase II Study of Decitabine Administered Daily for 5 Days Every 4 Weeks to Adults with Myelodysplastic Syndrome (MDS). Blood (ASH Annual Meeting Abstracts) 2007;110:Abstract 1450.
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28. Lee JH, Jang JH, Park J, et al. A prospective multicenter observational study of decitabine treatment in Korean patients with myelodysplastic syndrome. Haematologica 2011;96:1441-1447.
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30. Gao Y, Ping B, Zhou S. Decitabine for treatment of myelodysplastic syndrome in an elderly patient and review of literature. Journal of Southern Medical University 2012;32:280-282.
31. Lee JH, Lee KH, Lee JH, et al. Decreased incidence of febrile episodes with antibiotic prophylaxis in the treatment of decitabine for myelodysplastic syndrome. Leukemia research 2011;35:499-503.
32. Ruter B, Wijermans P, Claus R, et al. Preferential cytogenetic response to continuous intravenous low-dose decitabine (DAC) administration in myelodysplastic syndrome with monosomy 7. Blood 2007;110:1080-1082; author reply 1083.
33. Vardiman JW, Thiele J, Arber DA, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 2009; 114:937-951.
34. Cheson BD, Greenberg PL, Bennett JM, et al. Clinical application and proposal for modification of the International Working Group (IWG) response criteria in myelodysplasia. Blood 2006; 108:419-425.
35. Ruter B, Wijermans PW, Lubbert M. Superiority of prolonged low-dose azanucleoside administration? Results of 5-aza-2'-deoxycytidine retreatment in high-risk myelodysplasia patients. Cancer 2006;106:1744-1750.
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37. Uy GL, Abboud CN, Cashen AF, et al. Phase I study of panobinostat plus decitabine in elderly patients with advanced MDS or AML..53rd ASH Annual Meeting and Exposition 2011;poster session.poster board 1-40.
38. Greenberg PL, Tuechler H, Schanz J, et al. Revised international prognostic scoring system for myelodysplastic syndromes. Blood 2012;120:2454-2465.
39. Wang H, Wang XQ, Xu XP, et al. ID4 methylation predicts high risk of leukemic transformation in patients with myelodysplastic syndrome. Leukemia research 2010;34:598-604.
40. Claus R, Almstedt M, Lubbert M. Epigenetic treatment of hematopoietic malignancies:in vivo targets of demethylating agents. Seminars in oncology 2005;32:511-520.
41. Cornelison AM, Manero GG, Kantarjian H, et al. Decitabine (DAC) Can Be Safely Reduced After Achievement of Best Response In Patients (pts) with Myelodysplastic Syndrome (MDS).53rd ASH Annual Meeting and Exposition 2010;poster session:Poster Board I-838.
42. Wang J, Yi Z, Wang S, et al. The effect of decitabine on megakaryocyte maturation and platelet release. Thrombosis and haemostasis 2011;106:337-343.
43. van den Bosch J, Lubbert M, Verhoef G, et al. The effects of 5-aza-2'-deoxycytidine (Decitabine) on the platelet count in patients with intermediate and high-risk myelodysplastic syndromes. Leukemia research 2004;28:785-790.
44. Chabot GG, Momparler RL. Effects of 5-aza-2'-deoxycytidine on survival and cell cycle progression of L1210 leukemia cells. Leukemia research 1986;10:533-537.
45. Gore SD. New ways to use DNA methyltransferase inhibitors for the treatment of myelodysplastic syndrome. Hematology/the Education Program of the American Society of Hematology American Society of Hematology Education Program 2011;2011:550-555.
46. Saba HI, Wijermans PW. Decitabine in myelodysplastic syndromes. Seminars in hematology 2005;42:S23-31.
47. Steensma DP. Decitabine treatment of patients with higher-risk myelodysplastic syndromes. Leukemia research 2009;33 Suppl 2:S12-17.
48. Yang AS, Doshi KD, Choi SW, et al. DNA methylation changes after 5-aza-2'-deoxycytidine therapy in patients with leukemia. Cancer research 2006;66:5495-5503.
49. Steensma DP, Baer MR, Slack JL, et al. Preliminary Results of a Phase Ⅱ Study of Decitabine Administered Daily for 5 Days Every 4 Weeks to Adults with Myelodysplastic Syndrome (MDS). Blood (ASH Annual Meeting Abstracts) 2007; 110:Abstract 1450.