Pathogenesis And Treatment Of Aplastic Anemia

再生障碍性贫血的发病机制和治疗

• 批准号: 7321592
• 负责人: NEAL S YOUNG
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7321592
• 关键词: Pathogenesis; Treatment; Aplastic; Anemia

Aplastic anemia (AA) and other types of bone marrow failure have clinical and laboratory features consistent with an autoimmune pathophysiology, with a diversity of putative inciting antigens, including viruses, chemicals, medical drugs, and tumor antigens. Whatever it?s specific etiology, a majority of patients respond with hematologic improvement after immunosuppressive therapies. One important clinical feature of AA is its association with clonal hematologic diseases, especially paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplastic syndromes (MDS). In the clinic, studies have been directed towards more effective immunosuppression in aplastic anemia and the application of immunosuppressive regimens to related bone marrow failure syndromes. We have completed analysis of rabbit ATG, a more potent formulation antilymphocyte globulin recently licensed for use by the FDA in the US: about 1/3 of patients who have failed a course of horse ATG will respond hematologically to transfusion-independence on treatment with rabbit ATG; the majority of patients who relapse after horse ATG will respond to rabbit ATG. In patients with refractory aplastic anemia, unresponsive to horse ATG, we have also observed hematologic recovery using the monoclonal antibody CAMPATH, directed against CD52, a ubiquitous antigen on human lymphocytes. These clinical results ave led to the institution of a triple arm, randomized protocol for severe aplastic anemia on presentation; approximately 30 patients have been entered into this protocol, which compares horse ATG with a long course of cyclosporin; rabbit ATG with standard cyclosporin; and CAMPATH alone. Hematologic responses have been observed in all arms, including for CAMPATH , but more patients will be required for adequate comparison of the primary endpoints of hematologic response as well as relapse and clonal evolution. In studies in other diseases, we have established that the monoclonal antibody declizumab is effective not only in moderate aplastic anemia but also in pure red cell aplasia, leading to transfusion-independence in single lineage marrow failure in this format about 1/3 of patients, with no or minimal drug toxicity. In myelodysplasia, analysis of outcomes in a large number of patients who were treated with ATG with or without cyclosporin have shown a clear survival advantage, in comparison to historical data obtained in multi-center observations. Also in myelodysplasia, CAMPATH has been effective in several patients in a new protocol as a single agent, again with modest toxicity in comparison to ATG. The Hematology Branch has also participated in a large multi-center trial of Ecluzimab, a monoclonal antibody to the C5A component of complement; the results of this study will be published shortly. In the laboratory, efforts have concentrated on the mechanism of immune suppression, and the pathophysiology of late clonal events, including PNH, MDS, and acute leukemia. Studies of T-cell clonotypes continue to show a varied pattern in patients who respond, relapse, or are refractory to immunosuppressive therapy, although in general dominant clonal types recede with clinical improvement and recur, often accompanied by new clones of T-cells, on relapse. Current studies analyze the pattern of transcription of cytokines and immunomodulatory and other regulatory genes in isolated clones obtained by flow cytometric sorting based on the specific V-beta subfamily of the T-cell receptor. Other investigations have examined on the signal transduction pathways involved in T-cell polarization and the aberrant T-cell response. Results have including the finding of increased T-bet, a transcriptional regulator that is crucial for T-cell polarization to TH1 cells. New results, all consistent with an immune hypothesis for the pathogenesis of acquired aplastic anemia, include decreased PRF1 gene expression, low perforin, and low cytolytic activity in aplastic anemia, similar to that observed in familial hemophagocytic syndrome. Analogously, we have observed a high prevalence of decreased expression of SAP, a small immunomodulatory protein also important in the regulation of gamma interferon gene expression. In other studies relevant to the genetics of acquired aplastic anemia, we have described mutations in genes of the telomere repair complex, including for telomerase, TERT, and TERC in patients with apparently acquired aplastic anemia. In a large Mennonite kindred with a novel TERT mutation, the mutation has tracked with short telomeres and a spectrum of hematologic manifestations, including frank aplastic anemia, mild anemia, macrocytosis, and no hematologic abnormalities; the presence of the mutation can be inferred in six generations of this highly informative family; one patient with TERT mutation in this kindred suffered early liver failure, requiring liver transplantation. Studies of telomere repair in aplastic anemia have also led to the discovery that a likely mechanism of action of male hormones, historically utilized for the treatment of a variety of bone marrow failure conditions, is telomerase gene up-regulation. In primary lymphocytes and CD34 cells, different androgen preparations act through conversion by aromatase to estrodiol and binding to the estrogen receptor, leading to activation of TERT. Sex hormones can also up-regulate telomerase activity in cells from patients mutant in TERT. We are investigating the functional role in the Shwachman-Bodian-Diamond syndrome gene (SBDS), which we found to be mutated in heterozygosity in patients with apparently acquired aplastic anemia and in their mothers with life-long mild macrocytic anemia. The SBDS gene product does not associated physically with the telomere repair complex; heterogeneity of telomere length in DNA hybridization and other data suggests that SBDS may be related to an alternative pathway of telomere repair. In the area of clonal evolution, we have focused on two cytogenetically defined forms of myelodysplasia, trisomy 8 and monosomy 7 MDS. In trisomy 8 MDS, patients are frequently responsive to immunosuppressive therapy and their blood counts may be dependent on low level continued cyclosporin. In previous work, we demonstrated that trisomy MDS is similar to aplastic anemia in a high prevalence of oligoclonal T-cells, which appear to be specifically reactive to the cytogenetically aberrant clone. In more recent work, we have demonstrated a block in the apoptosis pathway: trisomy 8 cells, while annexin-positive, do not undergo DNA degradation and are capable of proliferation in vitro. This block has correlated with increased gene expression of c-myc, cyclin D1, and surviving. Most recently, the antigen in trisomy 8 has been shown to Wilms tumor-1 (WT-1), previously implicated in acute myelogenous leukemia and the subject of vaccine development in studies of malignant hematology. T-cells in a large proportion of patients with trisomy 8 are specific for WT1, as demonstrated as in tetramer methodology. In monosomy 7, the most frequent cytogenetic abnormality to evolve form aplastic anemia, clinical data have suggested an association with either the exogenous administration of granulocyte-colony stimulating factor (G-CSF) or persistent neutropenia and elevated endogenous G-CSF levels. We have shown that monosomy 7 does not arise de novo from normal bone marrow cells, but under conditions of high G-CSF stimulation, small clones of pre-existing monosomy 7 cells expand, as detected by fluorescent insitu hybridization (FISH), due to increased expression of a normal isoform of the G-CSF receptor which lacks the cytocellic signaling domain for differentiation. As a result, under conditions of high G-CSF in vivo, these cells likely are selected to undergo proliferation and not differentiation.

再生障碍性贫血（AA）和其他类型的骨髓衰竭具有与自身免疫病理生理学一致的临床和实验室特征，具有多种假定的刺激抗原，包括病毒、化学物质、药物和肿瘤抗原。无论其具体病因是什么，大多数患者在免疫抑制治疗后都会出现血液学改善的情况。 AA 的一项重要临床特征是其与克隆性血液疾病的相关性，尤其是阵发性睡眠性血红蛋白尿 (PNH) 和骨髓增生异常综合征 (MDS)。在临床上，研究主要针对再生障碍性贫血更有效的免疫抑制以及免疫抑制方案在相关骨髓衰竭综合征中的应用。我们已经完成了对兔 ATG 的分析，兔 ATG 是一种更有效的抗淋巴细胞球蛋白制剂，最近获得 FDA 许可在美国使用：大约 1/3 的马 ATG 疗程失败的患者会对兔 ATG 治疗的输血依赖性产生血液学反应；大多数在马 ATG 后复发的患者会对兔 ATG 产生反应。在对马 ATG 无反应的难治性再生障碍性贫血患者中，我们还使用针对 CD52（人类淋巴细胞上普遍存在的抗原）的单克隆抗体 CAMPATH 观察到血液学恢复。这些临床结果导致针对严重再生障碍性贫血制定了三组随机方案；大约 30 名患者已加入该方案，该方案将马 ATG 与长疗程环孢菌素进行比较；兔 ATG 与标准环孢菌素；和 CAMPATH 单独。包括 CAMPATH 在内的所有组均观察到了血液学反应，但需要更多患者来充分比较血液学反应的主要终点以及复发和克隆进化。在其他疾病的研究中，我们已经确定单克隆抗体declizumab不仅对中度再生障碍性贫血有效，而且对纯红细胞再生障碍性贫血有效，导致大约1/3的这种形式的单系骨髓衰竭患者出现输血依赖性，且无药物毒性或药物毒性极小。在骨髓增生异常中，与​​多中心观察中获得的历史数据相比，对大量接受 ATG 联合或不联合环孢菌素治疗的患者的结果分析显示出明显的生存优势。同样在骨髓增生异常方面，CAMPATH 作为单一药物在新方案中对数名患者有效，与 ATG 相比，毒性也较小。血液科还参与了 Ecluzimab 的大型多中心试验，Ecluzimab 是一种针对补体 C5A 成分的单克隆抗体；这项研究的结果将很快发表。在实验室，工作重点是免疫抑制机制和晚期克隆事件的病理生理学，包括 PNH、MDS 和急性白血病。 T 细胞克隆型的研究继续表明，对免疫抑制治疗有反应、复发或难治的患者存在不同的模式，尽管一般来说，主要克隆类型会随着临床改善而消退，并在复发时复发，通常伴随着新的 T 细胞克隆。目前的研究分析了基于 T 细胞受体的特定 V-β 亚家族通过流式细胞术分选获得的分离克隆中细胞因子和免疫调节及其他调节基因的转录模式。其他研究还研究了 T 细胞极化和异常 T 细胞反应所涉及的信号转导途径。结果包括发现 T-bet 增加，这是一种转录调节因子，对于 T 细胞极化为 TH1 细胞至关重要。新结果均与获得性再生障碍性贫血发病机制的免疫假说一致，包括再生障碍性贫血中 PRF1 基因表达降低、穿孔素水平降低和细胞溶解活性降低，与家族性噬血细胞综合征中观察到的情况类似。类似地，我们观察到 SAP 表达下降的比例很高，SAP 是一种小免疫调节蛋白，在调节 γ 干扰素基因表达中也很重要。在与获得性再生障碍性贫血遗传学相关的其他研究中，我们描述了明显获得性再生障碍性贫血患者的端粒修复复合物基因突变，包括端粒酶、TERT 和 TERC。在一个具有新的TERT突变的门诺派大家族中，该突变伴随着短端粒和一系列血液学表现，包括明显的再生障碍性贫血、轻度贫血、大红细胞增多症和无血液学异常；可以在这个信息丰富的家族的六代中推断出突变的存在；该家族中有一名TERT突变患者早期出现肝功能衰竭，需要进行肝移植。对再生障碍性贫血端粒修复的研究还发现，历史上用于治疗各种骨髓衰竭病症的雄性激素的一个可能作用机制是端粒酶基因上调。在原代淋巴细胞和 CD34 细胞中，不同的雄激素制剂通过芳香酶转化为雌二醇并与雌激素受体结合而发挥作用，导致 TERT 激活。性激素还可以上调 TERT 突变患者细胞中的端粒酶活性。我们正在研究 Shwachman-Bodian-Diamond 综合征基因 (SBDS) 的功能作用，我们发现该基因在明显获得性再生障碍性贫血的患者及其患有终生轻度大红细胞性贫血的母亲中发生杂合性突变。 SBDS基因产物与端粒修复复合体没有物理联系； DNA杂交和其他数据中端粒长度的异质性表明SBDS可能与端粒修复的替代途径有关。在克隆进化领域，我们重点关注两种细胞遗传学定义的骨髓增生异常形式：8 三体性和 7 单体性 MDS。在 8 三体性 MDS 中，患者经常对免疫抑制治疗有反应，他们的血细胞计数可能依赖于低水平的持续环孢菌素。在之前的工作中，我们证明三体性MDS与再生障碍性贫血相似，寡克隆T细胞的患病率很高，这些细胞似乎对细胞遗传学异常的克隆有特异性反应。在最近的工作中，我们证明了细胞凋亡途径的阻断：8 三体细胞虽然膜联蛋白呈阳性，但不会发生 DNA 降解，并且能够在体外增殖。该阻断与 c-myc、细胞周期蛋白 D1 和存活的基因表达增加相关。最近，8 三体中的抗原已被证明与肾母细胞瘤 1 (WT-1) 相关，该肿瘤先前与急性髓性白血病有关，也是恶性血液学研究中疫苗开发的主题。正如四聚体方法所证明的那样，大部分 8 三体性患者的 T 细胞对 WT1 具有特异性。 7号单体是再生障碍性贫血最常见的细胞遗传学异常，临床数据表明与外源性粒细胞集落刺激因子 (G-CSF) 或持续性中性粒细胞减少症和内源性 G-CSF 水平升高有关。我们已经证明，7号单体并不是从正常骨髓细胞中从头产生的，但在高G-CSF刺激的条件下，通过荧光原位杂交（FISH）检测到，由于缺乏用于分化的细胞信号传导结构域的G-CSF受体正常亚型的表达增加，预先存在的7号单体细胞的小克隆会扩增。因此，在体内高 G-CSF 的条件下，这些细胞可能被选择进行增殖而不是分化。