非血缘脐血移植植入前综合征的临床与基础研究
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摘要
研究背景
异基因造血干细胞移植(allo-HSCT)是目前能够治愈血液系统恶性肿瘤的唯一方法。自1988年世界首例脐血移植(Cord Blood Transplantation, CBT)的成功和世界范围内脐血库的逐步建立及完善,脐血移植已被许多国家尤其是欧美及日本越来越多地用于多种疾病的治疗。世界上多中心大宗病例报道非血缘HLA1～2个位点不合的CBT与非血缘HLA全合的骨髓移植的长期疗效相当,从而确定了CBT在儿童和成人造血干细胞移植中的地位。CBT患者在脐血干细胞输注平均一周左右(即在中性粒细胞植入之前),部分患者会出现一系列免疫反应。1994年由Radford等首先提出植入综合征(engraftment syndrome, ES)这一概念,是指造血干细胞移植(HSCT)术后中性粒细胞恢复早期,部分患者出现发热、皮疹、腹泻、黄疸、非心源性的肺水肿、多器官功能衰竭等临床症状的统称,当时称之为毛细血管渗漏综合征(capillary leakage syndrome, CLS)。随后人们发现在CBT术后,部分患者在中性粒细胞植入前,甚至在未植入的患者中也可出现上述类似的临床症状。2005年日本学者将发生在CBT后中性粒细胞植入之前,出现的上述一系列免疫反应称之为植入前免疫反应(pre-immune response, PIR),2008年韩国学者称之为植入前综合征(pre-engraftment syndrome, PES)。文献报道PES的相关临床与基础文章较少,PES与脐血干细胞的植入、急性GVHD的发生、移植相关死亡率(TRM)、移植后复发、长期预后之间的关系,以及如何对重症PES进行干预治疗等,目前均尚不明确。
研究目的
本研究分为两部分即PES的临床研究和PES的发病机制研究。
第一部分,PES的临床研究：本中心自2000年4月至2013年12月共240例恶性血液病患者接受了非血缘CBT治疗,包括两个研究阶段,即第一阶段回顾性临床研究(自2000年4月至2012年2月,共有137例恶性血液病患者接受非血缘CBT)和第二阶段前瞻性临床研究(自2012年3月至2013年12月,共103例恶性血液病患者接受非血缘CBT)。研究内容包括：
第一,首先通过回顾性临床资料分析找出CBT后发生PES患者的临床特征。
第二,PES对脐血植入及原发性植入失败的影响,与移植相关死亡率及疾病复发的相关性,以及PES对预后的影响等。
第三,PES预后相关高危因素研究及病情危险程度分级,制定PES预后积分系统。
第四,通过前瞻性临床研究,根据PES预后危险程度分级,前瞻性进行分层干预治疗,以期改善重症PES的不良预后。
第二部分,PES发病机制的研究：检测PES患者NK、T细胞亚群的变化,PES伴有腹泻患者大便中细胞类型的鉴定,PES患者血清中单核巨噬细胞相关的细胞因子的检测,以及细胞因子在PES发生前、发生时及缓解后的流式检测结果,从细胞水平及分泌细胞因子的水平上来寻找引起PES发病的效应细胞。从PES患者血清中筛选出有差异表达的miRNA,在miRNA文库中寻找其编码的上游基因,了解其基因对PES发生所产生的调控作用,再寻找差异miRNA下游可能作用的靶基因及其相对应的信号传导通路,从分子水平上阐明PES的发病机理。
研究方法
1.非血缘CBT的病例选择
自2000年4月至2013年12月,共有240例高危或复发难治的恶性血液病患者在安徽省立医院接受非血缘CBT治疗。平均年龄17.9(2.0-48.0)岁,平均体重45.3(12.2-100.5)公斤；男性161例,女性79例。基础疾病包括：急性淋巴细胞白血病119例(其中ph阳性35例),急性髓细胞白血病67例,急性双表细胞白血病4例；慢性粒细胞白血病31例(其中慢性期12例,加速期／急变期19例)；骨髓增生异常综合征11例(其中MDS-RCMD5例,MDS-RAEB6例),非霍奇金淋巴瘤7例,青少年型粒单核细胞白血病(JMML)1例。接受CBT时处于完全缓解状态者198例,17例患者原发诱导耐药,25例复发后再诱导治疗未缓解。16例患者伴有中枢神经系统白血病。移植前巨细胞病毒血清学检测186例阳性(77.5%)。从疾病初诊至移植时间平均为9.8(2.6-67.2)月。
2.脐血的选择
单份脐血移植HLA≥4/6个位点相合(HLA≥5/6位点相合：冷冻前TNC＞2.5×107/kg,其中CD34细胞>1.0×105/kg,或TNC＞2.0×107/kg,其中CD34+细胞＞1.2×105/kg。HLA4/6位点相合：冷冻前TNC＞3.5×107/kg,其中CD34+细胞>1.2×105/kg,或TNC＞3.0×107/kg,其中CD34+细胞>1.5x105/kg)。如果达不到上述要求者则采用双份脐血移植,要求TNC＞3.5×107/kg或CD34+细胞＞1.5×105/kg。脐血的输注：采用中心静脉缓慢推注15-20分钟。留取标本检测每份脐血的TNC、CD34+细胞、CD3+细胞、NK细胞数量和干／祖细胞培养,脐血袋进行STR-PCR检测。
3.预处理方案
230例采用清髓性方案即BU/CY2或TBI/CY2为基础的预处理方案,10例采用减低强度(Flu/CY/TBI)为基础的方案。
成人AML或ALL患者：fTBI/Ara-C/CY:fTBI (3GY Bid×2d,-9～-8天,剂量率5-7cGY/min,肺剂量7GY)/Ara-C (2g/M2q12h×2d,-6～-5天,髓细胞白血病患者在使用Ara-c前加用G-CSF5ug/kg/d至Ara-c结束)/CY(60mg/kg×2d,-3～-2天)。本方案主要适用于年龄≥14岁,或者存在中枢神经系统白血病的患者。
儿童ALL患者：BU/CY/Flu:BU (0.8mg/kg q6h×4d,-7～-4天)/CY(60mg/kgx2d,-3--2天)/Flu (30mg/M2×4d,-9～-6天)。本方案主要适用年龄<14岁的ALL,或者之前曾经接受过中枢神经系统放疗的ALL患者。
儿童AML患者：Ara-c/BU/CY (AML):Ara-C (2g/M2q12h×2d,-9～-8天,使用Ara-c前加用G-CSF5ug/kg/d至Ara-c结束),BU (0.8mg/kg q6h×4d,-7～-4天)/CY (60mg/kg×2d,-3～-2天)。本方案主要适用年龄<14岁的AML,或者之前曾经接受过中枢神经系统放疗的AML患者。
4.GVHD的预防
预防GVHD采用CSA/MMF方案：CSA:2.5～3mg/kg/d,24小时持续输注,-1天开始,血清CSA平均浓度维持在250～300ug/ml至30-45天,胃肠道功能恢复后,按照静脉剂量的2倍改口服,谷浓度维持200ug/ml,以后根据是否存在急性GVHD适当减量,4-6月内停用。MMF:25mg/kg/d分三次口服,+1天至+30天,无GVHD者减量至停用。
5.植入及原发植入失败
中性粒细胞植入是指移植后连续3天中性粒细胞绝对值(ANC)≥0.5×109/L。血小板植入是指不依赖血小板悬液输注连续7天血小板≥20×109/L。原发植入失败是指+42天绝对中性粒细胞计数(ANC)＜0.5×109/L或骨髓<10%供者细胞嵌合(STR-PCR),继发植入失败是指植入后ANC逐渐降低至<0.5×109/L并且供者细胞比例逐渐减少从而丧失供者植入。
6.PES的定义是指在CBT后中性粒细胞植入之前,患者出现不明原因的非感染性发热(T≥-38.3℃,)同时伴有类似于急性GVHD的红斑皮疹。患者发热经过广谱抗生素治疗无效,无明确的感染部位,血液或分泌物培养阴性,需要排除感染性发热；患者皮疹需要排除药物过敏的可能；可以同时伴有体重增加(≥3%)、非感染性腹泻(每天2次,至少3天)、肝功能异常(转氨酶基值2倍以上或胆红素≥34mol/L)、肾功能损害(肌酐基值2倍以上)、弥漫性肺部浸润的非心源性肺水肿及缺氧症状、或者不能用其他原因解释的—过性脑病等。
7.统计学分析分类变量采用chi-square test,连续变量采用秩和检验进行分析；中性粒细胞或血小板植入、GVHD、感染、复发率、以及移植相关死亡率采用Kapla-Meier曲线分析,按照累计发生率表示。PES等的高危因素的筛选主要采用Cox regression进行回归分析。自体造血功能回复或植入前死亡与中性粒细胞植入、原发性植入失败或植入前死亡与GVHD、移植相关死亡率与复发等之间存在竞争风险。所有的资料分析均采用SPSS17.0,部分采用R软件进行分析(竞争风险)。p<0.05具有统计学意义。
8.PES的发病机制研究
检测PES患者NK、T细胞亚群的变化,PES伴有腹泻患者大便中细胞类型的鉴定,PES患者血清中NK、T细胞、补体水平、及单核巨噬细胞相关的细胞因子在PES发生前、发生时及缓解后的流式检测结果,从细胞水平及细胞因子水平上验证单核巨噬细胞在PES发病中的关键作用。从PES患者血清中筛选出有差异表达的miRNA,找出与单核巨噬细胞免疫相关的miRNA,在1miRNA文库中寻找其编码的上游基因,了解其基因对PES发生所产生的调控作用,再寻找差异miRNA下游可能作用的靶基因及其相对应的信号传导通路,从分子水平上阐明PES的发病机理。
研究结果
1.PES的高危因素本研究表明清髓性预处理方案及双份脐血移植是非血缘CBT患者发生PES的高危因素,而移植过程中未发生PES是原发性植入失败的高危因素。
2000年4月至2012年2月回顾性研究：共有137例恶性血液病患者接受非血缘CBT治疗,其中73例患者发生PES。PES的累计发生率为53.3%(95%CI,45.9-65.4%)。与未发生PES的患者相比较,清髓性预处理方案及双份脐血移植是PES发生的高危因素(p=0.008,0.016)。但是两组在年龄、性别、基础疾病、移植时的骨髓状态(CR与否)、HLA配型、ABO血型、预处理是否采用TBI或氟达拉滨的方案、细胞数(包括TNC、CD34、CD3、CD56计数)等方面均无明显统计学差异；在中性粒细胞植入和血小板植入的速率方面两者之间亦无明显统计学差异。本阶段研究表明原发性植入失败均出现在未发生PES组的患者(21.9%vs0%),表明未发生PES是原发性植入失败的高危因素(p<0.001)。
2.PES的临床症状充血性皮疹和非感染性发热是诊断PES最为特异性的症状。
临床症状平均出现在脐血移植后第7天(5-15),第一阶段发生PES的73例患者中,64例患者在14天内出现非感染性发热(87.6%),59例患者伴有红色充血性皮疹(80.8%),16例患者伴有非感染性腹泻(21.9%),11患者出现体重增加(≥3%)(15.1%),9例患者出现肝脏转氨酶升高(12.3%),5例伴有胆红素的升高(6.8%),4例患者出现肾功能的损害(5.5%),5例患者伴有低氧血症或肺水肿的表现(6.80%)。对发生PES与未发生PES患者的8项主要临床症状进行对比研究,发现充血性皮疹和非感染性发热是诊断PES最为特异性的症状,而体重增加、非感染性腹泻、肝肾功能损害等两组之间未见明显差异。
3.PES与急性GVHD的关系PES与中性粒细胞植入后急性GVHD的发生具有明显的正相关。
回顾性研究137例CBT患者,共有34例患者发生Ⅱ-Ⅳ急性GVHD(24.8%),Ⅲ-Ⅳ急性GVHD17例(12.4%)。100天Ⅱ-Ⅳ急性GVHD的累计发生率为25.2%(95%CI,15.6-32.6%)。PES患者发生急性GVHD的累计发生率明显高于未发生PES的患者。73例PES患者,其中25例(34.2%)患者出现Ⅱ-Ⅳ急性GVHD,100天累计发生率为35.6%(95%CI,29.3-43.8%)；64例未发生PES的患者,仅9例(14.1%)患者出现Ⅱ-Ⅳ急性GVHD,100天累计发生率为15.2%(95%CI,8.5-22.6%),两组之间具有明显统计学意义(p=0.019)。
4.PES与移植相关死亡率及复发PES的发生与移植相关死亡率及疾病复发之间无明显的相关性。
137例CBT患者,51例出现移植相关死亡(37.2%)。73例PES患者,其中23例出现移植相关死亡(31.5%),累计移植相关死亡率为30.6%(95%CI：20.3%-40.9%)；64例未发生PES组患者28例出现移植相关死亡(43.8%),累计移植相关死亡率为43.5%(95%CI：34.5%-50.2%)。尽管未发生PES组移植相关死亡率高于PES组,但是两组之间无明显统计学差异(p=0.10)。共20例患者出现疾病复发(14.5%)。PES组患者10例(13.7%)复发,未发生PES组10例(15.6%)复发；累计复发率分别为13.5%(95%CI：6.6%-19.2%)和15.8%(95%CI：9.5%-22.5%),两组之间无明显统计学差异(p=0.74)。
5.PES与长期生存的关系及预后不良的高危因素发生PES与未发生PES组患者之间的长期生存无明显差异。移植后＜7天内发生PES、超过3个以上临床症状、甲基泼尼松龙疗效差(一周内无效)是PES患者预后不良的高危因素。
生存分析发现,患者5-year OS(总生存)和5-year MFS(无肿瘤生存)率在PES组和未发生PES组患者之间均无明显统计学差异[63.9%(95%CI,58.6%-72.2%) vs59.2%(95%CI,52.1%-66.2%),59.2%(95%CI,50.8.5%-64.3%) vs56.8%(95%CI,48.6%-62.5%), respectively; p=0.65,0.82]。通过对PES发生时间、最高体温、临床症状、环孢素浓度、甲基泼尼松龙(MP)初始剂量、MP起效的天数、MP有效维持至减量的天数、MP治疗无效等8项因素进行多因素分析,发现：移植后<7天内发生PES,超过3个以上临床症状,MP疗效差(一周内无效)是PES患者预后不良的高危因素。
6.PES积分系统与预后根据上述高危因素制定的积分系统表明,发生PES患者的积分越高(重症PES),其移植相关死亡率就越高,长期生存就越差。
将上述多因素分析得出的三个高危因素进行积分。存在一项高危因素计1分,无高危因素为0分。研究表明具有高危因素(1分组+2分组+3分组)患者与0分组患者比较180天TRM明显升高,分别为(43.6%vs14.8%,p=0.021)；具有高危因素2分组+3分组患者与1分组患者比较180天TRM明显升高,两组分别为(60.8%vs19.4%,p=0.018)；具有高危因素3分组患者与2分组患者比较180天TRM明显升高,分别为(82.6%vs33.9%,p=0.015)；1年的OS四组之间有明显的差异(0分：82.8%；1分：79.2%；2分：48.7%；3分：0%)(p<0.001)。
7.PES的分层治疗前瞻性对发生PES患者进行高危分级后采用分层干预治疗,发现经过治疗之后重度PES患者的预后明显改善。
2012年3月至2013年12月前瞻性临床研究,共103例恶性血液病接受非血缘CBT(发生PES患者79例)。根据PES预后积分系统,采用甲基泼尼松龙(MP)为基础的干预治疗之后,0分组患者(无高危因素者)(n=26例)与至少有1项以上高危因素者(1分组+2分组+3分组)(n=53例)的移植相关死亡率、仅1项高危因素(1分组)(n=27例)与至少有2项以上高危因素者(2分组+3分组)的移植相关死亡率(n=26例)之间均无明显统计学差异(p=0.16；p=0.39)。对比分析发现,经过分层干预治疗之后,2分组+3分组患者1年OS由25.5%(2010年4月至2012年2月)上升至72.3%(2012年3月至2013年12月)(p=0.019),表明重症PES患者的预后明显改善。
8.PES发生效应细胞的鉴定及PES发生的细胞活化信号途径
实验研究表明,T淋巴细胞亚群、NK细胞及相关功能抗原的CD11b、CD27、 CD57表达在PES组与未发生PES组之间无统计学差异；细胞形态学及流式细胞仪检测(免疫表型)提示PES伴有腹泻患者大便中的细胞类型,以单核巨噬细胞占主导；PES患者血清中细胞因子的检测提示单核巨噬细胞相关的因子IL-1B、 MCP-1、IL-6、IL-18在PES发生时比移植前显著增加(p<0.01),也显著高于UCBT同期未发生PES的患者(p<0.01),并且当PES症状控制后细胞因子水平明显下降至移植前的水平。NK/T细胞相关细胞因子、及补体水平在PES发生前后,以及PES组患者与未发生PES的患者之间均无明显差别(p>0.05)
采用高通量测序的方法检测了3例CBT患者(9份标本)发生PES前、发生时、缓解时血清中的miRNA,结果显示差异表达的miRNA有hsa-let-7i-5p、 hsa-mir-98-5p、hsa-mir-152-3p、hsa-mir-223-3p等39个,通过靶基因预测+GO+Pathway分析,推测出PES的发生与单核巨噬细胞的激活相关的三条信号通路：Toll、NOD、RIG-I样受体信号通路相关。
结论及意义
截至目前,文献检索表明本研究是报道CBT相关PES例数最多、研究最为全面的临床研究,且首次提出根据PES高危因素的积分系统进行高危分级并分层干预治疗,国际目前尚未报道。
1.通过回顾性及前瞻性临床研究,发现清髓性预处理方案及双份脐血移植是PES发生的高危因素,两组在年龄、性别、基础疾病、移植时的骨髓状态(CR与否)、HLA配型、ABO血型、预处理是否采用TBI或氟达拉滨的方案、细胞数(包括TNC、CD34、CD3、CD56计数)等方面均无明显统计学差异。
2.本研究对发生PES患者的主要临床症状进行对比研究,表明充血性皮疹和发热是诊断PES最为特异性的症状。
3.PES与脐血干细胞能否植入密切相关,脐血移植过程中未发生PES是原发性植入失败的高危因素。多因素分析表明PES是发生急性GVHD的重要高危因素。
4.尽管发生PES的患者与未发生PES的患者相比较,患者的移植相关死亡率及复发率无明显统计学意义,但是相对于轻症PES来说,重症PES患者的移植相关死亡率明显增加。根据预后积分系统对PES患者进行分层治疗,能够明显改善重症PES患者的长期生存。
5.实验研究初步证实了单核巨噬细胞是引起PES发生的效应细胞,通过分子水平预测可能激活Toll、NOD、RIG-I样受体三条分子信号传导通路而发挥作用。
相对于其他移植类型,CBT最主要的问题是植入失败及较高的移植相关死亡率,也是CBT在国内开展较少的主要原因。本课题对于CBT后发生的PES做了较为全面的临床研究,特别是按照积分系统进行高危分级能够准确区分轻症PES和重症PES；高危分级后的分层干预治疗,一方面对于保留轻症PES促进脐血干细胞的植入,克服原发性植入失败,另一方面对于将重症PES转化为轻症PES,以减少重症PES所致的移植相关死亡率等,均具有重要的临床意义。
Introduction
Cord blood transplantation (CBT) is an effective and potential curative treatment for pediatric patients with hematological malignancies who lack HLA-identical related or unrelated donors. Over the last25years, the field of umbilical cord blood (UCB) banking and transplantation has grown exponentially. Over600000UCB units have been stored for transplantation worldwide, and＞30000CBTs have been performed. UCB serves as an alternative stem cell source; only30%of patients who require an allograft will have a human leukocyte antigen (HLA)-matched sibling donor. Despite＞20million adult volunteer donors in the National Marrow Donor Program and affiliated registries, many patients, particularly patients of diverse racial/ethnic backgrounds, will not have a suitably matched, unrelated volunteer donor identified in the required time period. UCB has extended access to transplantation, especially to patients of racial and ethnic minorities and is rapidly available. Post-transplantation immune disorders, including pre-engraftment syndrome (PES), engraftment syndrome, and acute graft-versus-host disease (GVHD) are problematic in CBT. The complex and intricate pathophysiology of post-transplantation immune disorders is a consequence of interactions between the donor and host innate and adaptive immune responses. PES, a clinical entity of unknown pathogenesis, has been described in patients receiving CBT. Although a uniform definition is lacking, PES is commonly characterized by noninfectious fever and various other clinical findings before neutrophil engraftment, including skin rash, pulmonary infiltrates, and diarrhea, jaundice, or weight gain. To date, PES is an entirely clinical entity with no known pathognomonic histopathologic changes or biochemical markers. Kishi et al were the first to report a pre-engraftment immune reaction, which occurred in35of45adult recipients of reduced-intensity conditioning CBT. However, PES still remains poorly characterized and its clinical significance and the prognostic impact after CBT are unclear. The first part of the research is to address these issues, we retrospectively analyzed the incidence, risk factors, and clinical outcomes of PES in unrelated CBT recipients enrolled in a multicenter CBT trial. The second part of the research, we investigated that donor monocyte-macrophages were the key effector cells which expressed high level cytokines in PES patients. By target gene prediction+GO+pathway analysis, we suspected that the occurrence of PES were activated by monocytes and macrophages, and associated with three possible Toll, NOD, and RIG-I-like receptor signaling pathways.
Methods and patients
Between April,2000and December,2013,240consecutive patients with hematologic malignancies underwent unrelated CBT in Anhui Provincial Hospital. All of the240hematologic malignancies patients referred for CBT had one of the features associated with poor outcomes:ⅰ) Patients were in first complete remission (CR1) but have high risk factors at first diagnosis:ALL with adverse cytogenetics or molecular abnormalities [ph+chromosome (BCR-ABL positive), hypodiploidy,11q23abnormalities (MLL rearrangements)], or with high level of minimal residual disease (MRD)(1%or more after completion of6weeks of induction therapy); AML with adverse cytogenetics or molecular abnormalities [inv(3)(q21q26.2) or t(3;3)(q21;q26.2), t(6;9)(p23;q34),11q23abnormalities (MLL rearrangements),-5or del(5q),-7or del(7q), complex karyotype, normal cytogenetics with FLT3-ITD mutation], or with a prior history of myelodysplastic syndrome (MDS). ⅱ) CR2(but the length of CR1≤12months), CR3or more. ⅲ) Primary induction failure, or no remission after relapse with salvage chemotherapy.
Cord blood units that were serologically matched for≥4of six HLA antigens and which contained at least3×107nucleated cells/kg and1.2×105CD34+/kg of recipient body weight before freezing were obtained from the cord blood bank at the China Cord Blood Bank Network. The units were not depleted of T lymphocytes. The choice to transplant one vs. two UCB units was based solely on cell dose criteria. If a UCB unit with the minimum cell dose was not available, the patient was transplanted with two partially HLA-matched UCB units, which needed to have a minimum cryopreserved dose of3.5×107nucleated cells/kg and2×105CD34+/kg, respectively. All patients received a myeloablative conditioning regimen of TBICY[total body irradiation (TBI, total12Gy,4fractions) and cyclophosphamide (CY,60mg/kg daily for2days)](age≥14years or primary induction failure or no remission after relapse) or BuCY2[busulfan (0.8mg/kg every6h for4days) and CY](age＜14years or prior radiotherapy which would presuppose a high risk of toxicity). Cytarabine (2.0g/m2every12h for2days) and/or carmustine (BCNU,250mg/m2) was added to the myeloablative conditioning regimen in order to penetrate the blood brain barrier to kill the CNS leukemia cells maximally (for some patients). Fludarabine (30mg/m2daily for4days) was combined with BuCY2to promote engraftment.
For GVHD prophylaxis, all patients were given a combination of cyclosporine (Novartis, Stein, Switzerland) and mycophenolate mofetil (Roche, Basel, Switzerland). Intravenous cyclosporine was started (2.5-3mg/kg/d) on day-1and continued until patients were able to take cyclosporine orally with target trough levels of200ng/mL at least1month. Mycophenolate mofetil (25mg/kg/d) was given on day+1. The rapidity of tapering was based on the presence or absence of GVHD, infectious disease, and relapse risks.
PES was defined as unexplained fever＞38.3℃not associated with documented infection and/or an unexplained erythematous skin rash resembling that of acute GVHD, with either the fever or the rash occurring before or at neutrophil recovery. Specifically, fever attributed to PES was not associated with any clinical evidence of infection, with patients having both a negative infectious disease workup and a continued lack of response to broad-spectrum antimicrobial agents. Erythematous skin rash attributed to PES was not associated with any clinical suspicion of drug allergy.Weight gainwas defined as a3%increase in body weight between the day of CBT and the onset of PES. Noninfectious diarrhea was defined as passage of watery stools more than twice a day for at least3consecutive days with no evidence of infectious etiology.
Variables related to the patient, the disease, and the transplantation procedures were compared with the use of the chi-squared test for categorical variables. The Cox regression model was used for multivariate analysis of clinical variables. The end-points were engraftment, graft failure, PES, acute and chronic GVHD, relapse, transplant-related mortality (TRM), malignancy-free survival (MFS), overall survival (OS). Time-to-event outcomes for neutrophil and platelet engraftment, PES, GVHD, TRM, and relapse were estimated using cumulative incidence curves. The difference in the cumulative incidence curves was based on Gray's test. The Kaplan-Meier method was used to estimate the probabilities of the survival (OS and MFS) which were compared using the log rank test. p<0.05was considered statistically significant. All analyses were performed with SPSS (version17.0).
The second part of the research, we investigated that donor monocyte-macrophages were the key effector cells which expressed high level cytokines in PES patients. Serum samples with known miRNA expression in patients with PES, using the difference in miRNA expression levels log2-ratio, Scatter plot diagram comparing the expressions. Difference between the two samples by miRNA expression was analyzed before and after PES. The detection of miRNA expression maybe found to play an important role in the pathogenesis of PES. This would also be helpful to find the molecular signaling pathways and the mechanism of PES.
Results
From2000to2012(the first period of the clinical research), a total of137patients received CBT, and73patients (53.3%) developed PES at a median of7days (range5-15days). The cumulative incidence of PES was53.3%(95%CI,45.9-65.4%). Comparing the patient demographics and graft characteristics of the PES patients and the non-PES patients, we found that myeloblative conditioning and double CBT were the high risks for the development of PES. But there were no significant differences between the2groups in terms of age, sex, weight, underlying malignancy, BM status at CBT, HLA match, ABO compatibility or conditioning approach. There also were no between-group differences in total infused TNC dose, total CD34+cell dose, total CD3cell dose, and total CD56cell dose. Total of14patients developed primary engraftment failure,2patients recovered with autologous hematopoisis, and the other12patients salvaged with haploid-transplantation. We found that patients with no PES was the high risk for primary engraftment failure (p＜0.001).
64patients had non-infectious fever (87.6%) within14days,59patients with red rash (80.8%),16patients with non-infectious diarrhea (21.9%),11patients with weight gain (≥3%)(15.1%),9patients with elevated liver transaminases (12.3%),5cases with elevated bilirubin (6.8%),4cases of renal damage (5.5%), and5patients with hypoxemia or pulmonary edema (6.8%). Rash and non-infectious fever are the most specific diagnostic symptoms, but weight gain, non-infectious diarrhea, liver and kidney dysfunction and other syndromes were not the specific characters of PES.
The incidence of acute GVHD in the PES group was higher than the non-PES group patients (p=0.019):Grade II-IV acute GVHD developed in25(34.2%) patients of the73PES group patients, and the100-day cumulative incidence of grade II-IV acute GVHD was35.6%(95%CI,29.3-43.8%); Only9patients developed II-IV acute GVHD in the64non-PES group patients, and100-day cumulative incidence of grade II-IV acute GVHD was15.2%(95%CI,8.5-22.6%).
The cumulative transplant-related mortality was30.6%(95%CI:20.3%-40.9%) in PES patients, and23patients (27%) died due to the transplant-related mortality (TRM). TRM was developed in28patients (43.8%) in the64non-PES group patients, and the cumulative transplant-related mortality was43.5%(95%CI:34.5%-50.2%) Although the transplant-related mortality was higher in the non-PES group than the PES group patients, but there was no significant difference (p=0.10). Total of20patients developed disease recurrence (14.5%). The cumulative recurrence rates were13.5%(95%CI:6.6%-19.2%) and15.8%(95%CI:9.5%-22.5%) in PES and non-PES group patients, and there was also no significant difference (p=0.74).
5-year OS and5-year MFS did not differ depending on the presence or absence of PES after CBT [63.9%(95%CI,58.6%-72.2%) vs59.2%(95%CI,52.1%-66.2%),59.2%(95%CI,50.8.5%-64.3%) vs56.8%(95%CI,48.6%-62.5%), respectively; p=0.65,0.82]. By the time of the occurrence of PES, the maximum temperature, clinical symptoms, cyclosporine concentrations, methylprednisolone (MP) initial dose, the days from the effectiveness of MP, the days of maintains of MP, ineffectiveness of MP, we found that PES occurred in7days (＜7days), over three or more clinical symptoms, poor effectiveness of MP (ineffectiveness within in one week) were the poor prognostic risk factors of PES.
We made a PES scoring system according to3high risk factors which had been described, and investigated the impact of high risk factors to the PES patients prognosis. The existence of one risk factor was defined as1point, and no risk factors with0score. Our study showed that the cumulative incidence of180-days transplant-related mortality (TRM) was higher in patients with at least one risk factors (43.6%vs14.8%, p=0.021); and180-days transplant-related mortality (TRM) was also higher in patients with more than one risk factors (2+3) than patients with one risk factor (60.8%vs19.4%, p=0.018);180-days TRM was also higher in patients with three risk factors (3scores) than patients with two risk factors (82.6%vs33.9%, p=0.015). For1-year OS, there were significant differences during the4groups patients (0point:82.8%;1point:79.2%;2points:48.7%;3points:0%)(p＜0.001).
From March2012to December2013((the second period of the clinical research)),103patients with hematologic malignancies underwent UBCT, and79patients developed PES. We used methylprednisolone (MP) intervention according to the different PES score system. Patients with1high risk factor were treated with MP1mg/kg/d, and Patients with2or more high risk factors were treated with MP2mg/kg/d. After the intervention of MP, there were no significant statistical differences between patients with0score (n=26patients) and with at least1score (1+2+3points)(n=53patients), and there were also no significant statistical differences between patients with1score (n=27patients) and with at least2scores (2+3points)(n=26patients)(p=0.16; p=0.39). After the treatment of MP according to the PES score system, the prognosis of severe PES was significant improved by1-year OS between the two different periods (April2010to February2012and March2012to December2013)(25.5%vs72.3%)(p=0.019).
According to our center experimental results and the clinical manifestations of PES, we found that the major effector cells of PES are donor monocyte-macrophages. We also found that donor monocyte-macrophages are the key effector cells which expressed high level cytokines in PES patients. Using HiSeq high-throughput sequencing technique of microRNA, we detected the serum miRNAs in3PES patients with periods of pre-PES, at the time of PES, and the remission of PES. About39obviously expressed miRNAs, such as hsa-let-7i-5p、hsa-mir-98-5p、 hsa-mir-152-3p、hsa-mir-223-3p, et al, were detected. By target gene prediction+GO+pathway analysis, we suspected that the occurrence of PES were activated by monocytes and macrophages, and associated with three possible signaling pathways: Toll, NOD, RIG-I-like receptor signaling pathways.
Conclusions
In conclusion, PES seems to be common after CBT and may be associated with enhanced engraftment, and patient with no PES was the high risk for primary engraftment failure. Rash and non-infectious fever are the most specific diagnostic symptoms. Although PES is closely associated with acute GVHD, PES after CBT is not associated with significant morbidity and is easily manageable with intravenous steroids. PES occurred within7days (<7days), over three or more clinical symptoms, poor effectiveness of MP (ineffectiveness within in one week) were the poor prognostic risk factors of PES. We made a PES scoring system according to these3high risk factors, and after the treatment of MP according to the PES score system, the prognosis of severe PES was significantly improved. We found that the major effector cells of PES are donor monocyte-macrophages. We also found that donor monocyte-macrophages are the key effector cells which expressed high level cytokines in PES patients. By target gene prediction+GO+pathway analysis, we suspected that the occurrence of PES were activated by monocytes and macrophages, and associated with three possible Toll, NOD, and RIG-I-like receptor signaling pathways. Because failure to recognize PES in CBT recipient risks unnecessary complications of this syndrome and unnecessarily long, empirical treatment, physicians should be aware of the possible occurrence of PES after CBT, especially in patients who have relevant risk factors for developing PES.
异基因造血干细胞移植(allo-HSCT)是目前能够治愈血液系统恶性肿瘤的唯一方法。自1988年世界首例脐血移植(Cord Blood Transplantation, CBT)的成功和世界范围内脐血库的逐步建立及完善,脐血移植已被许多国家尤其是欧美及日本越来越多地用于多种疾病的治疗。世界上多中心大宗病例报道非血缘HLA1～2个位点不合的CBT与非血缘HLA全合的骨髓移植的长期疗效相当,从而确定了CBT在儿童和成人造血干细胞移植中的地位。CBT患者在脐血干细胞输注平均一周左右(即在中性粒细胞植入之前),部分患者会出现一系列免疫反应。1994年由Radford等首先提出植入综合征(engraftment syndrome, ES)这一概念,是指造血干细胞移植(HSCT)术后中性粒细胞恢复早期,部分患者出现发热、皮疹、腹泻、黄疸、非心源性的肺水肿、多器官功能衰竭等临床症状的统称,当时称之为毛细血管渗漏综合征(capillary leakage syndrome, CLS)。随后人们发现在CBT术后,部分患者在中性粒细胞植入前,甚至在未植入的患者中也可出现上述类似的临床症状。2005年日本学者将发生在CBT后中性粒细胞植入之前,出现的上述一系列免疫反应称之为植入前免疫反应(pre-immune response, PIR),2008年韩国学者称之为植入前综合征(pre-engraftment syndrome, PES)。文献报道PES的相关临床与基础文章较少,PES与脐血干细胞的植入、急性GVHD的发生、移植相关死亡率(TRM)、移植后复发、长期预后之间的关系,以及如何对重症PES进行干预治疗等,目前均尚不明确。
研究目的
本研究分为两部分即PES的临床研究和PES的发病机制研究。
第一部分,PES的临床研究：本中心自2000年4月至2013年12月共240例恶性血液病患者接受了非血缘CBT治疗,包括两个研究阶段,即第一阶段回顾性临床研究(自2000年4月至2012年2月,共有137例恶性血液病患者接受非血缘CBT)和第二阶段前瞻性临床研究(自2012年3月至2013年12月,共103例恶性血液病患者接受非血缘CBT)。研究内容包括：
第一,首先通过回顾性临床资料分析找出CBT后发生PES患者的临床特征。
第二,PES对脐血植入及原发性植入失败的影响,与移植相关死亡率及疾病复发的相关性,以及PES对预后的影响等。
第三,PES预后相关高危因素研究及病情危险程度分级,制定PES预后积分系统。
第四,通过前瞻性临床研究,根据PES预后危险程度分级,前瞻性进行分层干预治疗,以期改善重症PES的不良预后。
第二部分,PES发病机制的研究：检测PES患者NK、T细胞亚群的变化,PES伴有腹泻患者大便中细胞类型的鉴定,PES患者血清中单核巨噬细胞相关的细胞因子的检测,以及细胞因子在PES发生前、发生时及缓解后的流式检测结果,从细胞水平及分泌细胞因子的水平上来寻找引起PES发病的效应细胞。从PES患者血清中筛选出有差异表达的miRNA,在miRNA文库中寻找其编码的上游基因,了解其基因对PES发生所产生的调控作用,再寻找差异miRNA下游可能作用的靶基因及其相对应的信号传导通路,从分子水平上阐明PES的发病机理。
研究方法
1.非血缘CBT的病例选择
自2000年4月至2013年12月,共有240例高危或复发难治的恶性血液病患者在安徽省立医院接受非血缘CBT治疗。平均年龄17.9(2.0-48.0)岁,平均体重45.3(12.2-100.5)公斤；男性161例,女性79例。基础疾病包括：急性淋巴细胞白血病119例(其中ph阳性35例),急性髓细胞白血病67例,急性双表细胞白血病4例；慢性粒细胞白血病31例(其中慢性期12例,加速期／急变期19例)；骨髓增生异常综合征11例(其中MDS-RCMD5例,MDS-RAEB6例),非霍奇金淋巴瘤7例,青少年型粒单核细胞白血病(JMML)1例。接受CBT时处于完全缓解状态者198例,17例患者原发诱导耐药,25例复发后再诱导治疗未缓解。16例患者伴有中枢神经系统白血病。移植前巨细胞病毒血清学检测186例阳性(77.5%)。从疾病初诊至移植时间平均为9.8(2.6-67.2)月。
2.脐血的选择
单份脐血移植HLA≥4/6个位点相合(HLA≥5/6位点相合：冷冻前TNC＞2.5×107/kg,其中CD34细胞>1.0×105/kg,或TNC＞2.0×107/kg,其中CD34+细胞＞1.2×105/kg。HLA4/6位点相合：冷冻前TNC＞3.5×107/kg,其中CD34+细胞>1.2×105/kg,或TNC＞3.0×107/kg,其中CD34+细胞>1.5x105/kg)。如果达不到上述要求者则采用双份脐血移植,要求TNC＞3.5×107/kg或CD34+细胞＞1.5×105/kg。脐血的输注：采用中心静脉缓慢推注15-20分钟。留取标本检测每份脐血的TNC、CD34+细胞、CD3+细胞、NK细胞数量和干／祖细胞培养,脐血袋进行STR-PCR检测。
3.预处理方案
230例采用清髓性方案即BU/CY2或TBI/CY2为基础的预处理方案,10例采用减低强度(Flu/CY/TBI)为基础的方案。
成人AML或ALL患者：fTBI/Ara-C/CY:fTBI (3GY Bid×2d,-9～-8天,剂量率5-7cGY/min,肺剂量7GY)/Ara-C (2g/M2q12h×2d,-6～-5天,髓细胞白血病患者在使用Ara-c前加用G-CSF5ug/kg/d至Ara-c结束)/CY(60mg/kg×2d,-3～-2天)。本方案主要适用于年龄≥14岁,或者存在中枢神经系统白血病的患者。
儿童ALL患者：BU/CY/Flu:BU (0.8mg/kg q6h×4d,-7～-4天)/CY(60mg/kgx2d,-3--2天)/Flu (30mg/M2×4d,-9～-6天)。本方案主要适用年龄<14岁的ALL,或者之前曾经接受过中枢神经系统放疗的ALL患者。
儿童AML患者：Ara-c/BU/CY (AML):Ara-C (2g/M2q12h×2d,-9～-8天,使用Ara-c前加用G-CSF5ug/kg/d至Ara-c结束),BU (0.8mg/kg q6h×4d,-7～-4天)/CY (60mg/kg×2d,-3～-2天)。本方案主要适用年龄<14岁的AML,或者之前曾经接受过中枢神经系统放疗的AML患者。
4.GVHD的预防
预防GVHD采用CSA/MMF方案：CSA:2.5～3mg/kg/d,24小时持续输注,-1天开始,血清CSA平均浓度维持在250～300ug/ml至30-45天,胃肠道功能恢复后,按照静脉剂量的2倍改口服,谷浓度维持200ug/ml,以后根据是否存在急性GVHD适当减量,4-6月内停用。MMF:25mg/kg/d分三次口服,+1天至+30天,无GVHD者减量至停用。
5.植入及原发植入失败
中性粒细胞植入是指移植后连续3天中性粒细胞绝对值(ANC)≥0.5×109/L。血小板植入是指不依赖血小板悬液输注连续7天血小板≥20×109/L。原发植入失败是指+42天绝对中性粒细胞计数(ANC)＜0.5×109/L或骨髓<10%供者细胞嵌合(STR-PCR),继发植入失败是指植入后ANC逐渐降低至<0.5×109/L并且供者细胞比例逐渐减少从而丧失供者植入。
6.PES的定义是指在CBT后中性粒细胞植入之前,患者出现不明原因的非感染性发热(T≥-38.3℃,)同时伴有类似于急性GVHD的红斑皮疹。患者发热经过广谱抗生素治疗无效,无明确的感染部位,血液或分泌物培养阴性,需要排除感染性发热；患者皮疹需要排除药物过敏的可能；可以同时伴有体重增加(≥3%)、非感染性腹泻(每天2次,至少3天)、肝功能异常(转氨酶基值2倍以上或胆红素≥34mol/L)、肾功能损害(肌酐基值2倍以上)、弥漫性肺部浸润的非心源性肺水肿及缺氧症状、或者不能用其他原因解释的—过性脑病等。
7.统计学分析分类变量采用chi-square test,连续变量采用秩和检验进行分析；中性粒细胞或血小板植入、GVHD、感染、复发率、以及移植相关死亡率采用Kapla-Meier曲线分析,按照累计发生率表示。PES等的高危因素的筛选主要采用Cox regression进行回归分析。自体造血功能回复或植入前死亡与中性粒细胞植入、原发性植入失败或植入前死亡与GVHD、移植相关死亡率与复发等之间存在竞争风险。所有的资料分析均采用SPSS17.0,部分采用R软件进行分析(竞争风险)。p<0.05具有统计学意义。
8.PES的发病机制研究
检测PES患者NK、T细胞亚群的变化,PES伴有腹泻患者大便中细胞类型的鉴定,PES患者血清中NK、T细胞、补体水平、及单核巨噬细胞相关的细胞因子在PES发生前、发生时及缓解后的流式检测结果,从细胞水平及细胞因子水平上验证单核巨噬细胞在PES发病中的关键作用。从PES患者血清中筛选出有差异表达的miRNA,找出与单核巨噬细胞免疫相关的miRNA,在1miRNA文库中寻找其编码的上游基因,了解其基因对PES发生所产生的调控作用,再寻找差异miRNA下游可能作用的靶基因及其相对应的信号传导通路,从分子水平上阐明PES的发病机理。
研究结果
1.PES的高危因素本研究表明清髓性预处理方案及双份脐血移植是非血缘CBT患者发生PES的高危因素,而移植过程中未发生PES是原发性植入失败的高危因素。
2000年4月至2012年2月回顾性研究：共有137例恶性血液病患者接受非血缘CBT治疗,其中73例患者发生PES。PES的累计发生率为53.3%(95%CI,45.9-65.4%)。与未发生PES的患者相比较,清髓性预处理方案及双份脐血移植是PES发生的高危因素(p=0.008,0.016)。但是两组在年龄、性别、基础疾病、移植时的骨髓状态(CR与否)、HLA配型、ABO血型、预处理是否采用TBI或氟达拉滨的方案、细胞数(包括TNC、CD34、CD3、CD56计数)等方面均无明显统计学差异；在中性粒细胞植入和血小板植入的速率方面两者之间亦无明显统计学差异。本阶段研究表明原发性植入失败均出现在未发生PES组的患者(21.9%vs0%),表明未发生PES是原发性植入失败的高危因素(p<0.001)。
2.PES的临床症状充血性皮疹和非感染性发热是诊断PES最为特异性的症状。
临床症状平均出现在脐血移植后第7天(5-15),第一阶段发生PES的73例患者中,64例患者在14天内出现非感染性发热(87.6%),59例患者伴有红色充血性皮疹(80.8%),16例患者伴有非感染性腹泻(21.9%),11患者出现体重增加(≥3%)(15.1%),9例患者出现肝脏转氨酶升高(12.3%),5例伴有胆红素的升高(6.8%),4例患者出现肾功能的损害(5.5%),5例患者伴有低氧血症或肺水肿的表现(6.80%)。对发生PES与未发生PES患者的8项主要临床症状进行对比研究,发现充血性皮疹和非感染性发热是诊断PES最为特异性的症状,而体重增加、非感染性腹泻、肝肾功能损害等两组之间未见明显差异。
3.PES与急性GVHD的关系PES与中性粒细胞植入后急性GVHD的发生具有明显的正相关。
回顾性研究137例CBT患者,共有34例患者发生Ⅱ-Ⅳ急性GVHD(24.8%),Ⅲ-Ⅳ急性GVHD17例(12.4%)。100天Ⅱ-Ⅳ急性GVHD的累计发生率为25.2%(95%CI,15.6-32.6%)。PES患者发生急性GVHD的累计发生率明显高于未发生PES的患者。73例PES患者,其中25例(34.2%)患者出现Ⅱ-Ⅳ急性GVHD,100天累计发生率为35.6%(95%CI,29.3-43.8%)；64例未发生PES的患者,仅9例(14.1%)患者出现Ⅱ-Ⅳ急性GVHD,100天累计发生率为15.2%(95%CI,8.5-22.6%),两组之间具有明显统计学意义(p=0.019)。
4.PES与移植相关死亡率及复发PES的发生与移植相关死亡率及疾病复发之间无明显的相关性。
137例CBT患者,51例出现移植相关死亡(37.2%)。73例PES患者,其中23例出现移植相关死亡(31.5%),累计移植相关死亡率为30.6%(95%CI：20.3%-40.9%)；64例未发生PES组患者28例出现移植相关死亡(43.8%),累计移植相关死亡率为43.5%(95%CI：34.5%-50.2%)。尽管未发生PES组移植相关死亡率高于PES组,但是两组之间无明显统计学差异(p=0.10)。共20例患者出现疾病复发(14.5%)。PES组患者10例(13.7%)复发,未发生PES组10例(15.6%)复发；累计复发率分别为13.5%(95%CI：6.6%-19.2%)和15.8%(95%CI：9.5%-22.5%),两组之间无明显统计学差异(p=0.74)。
5.PES与长期生存的关系及预后不良的高危因素发生PES与未发生PES组患者之间的长期生存无明显差异。移植后＜7天内发生PES、超过3个以上临床症状、甲基泼尼松龙疗效差(一周内无效)是PES患者预后不良的高危因素。
生存分析发现,患者5-year OS(总生存)和5-year MFS(无肿瘤生存)率在PES组和未发生PES组患者之间均无明显统计学差异[63.9%(95%CI,58.6%-72.2%) vs59.2%(95%CI,52.1%-66.2%),59.2%(95%CI,50.8.5%-64.3%) vs56.8%(95%CI,48.6%-62.5%), respectively; p=0.65,0.82]。通过对PES发生时间、最高体温、临床症状、环孢素浓度、甲基泼尼松龙(MP)初始剂量、MP起效的天数、MP有效维持至减量的天数、MP治疗无效等8项因素进行多因素分析,发现：移植后<7天内发生PES,超过3个以上临床症状,MP疗效差(一周内无效)是PES患者预后不良的高危因素。
6.PES积分系统与预后根据上述高危因素制定的积分系统表明,发生PES患者的积分越高(重症PES),其移植相关死亡率就越高,长期生存就越差。
将上述多因素分析得出的三个高危因素进行积分。存在一项高危因素计1分,无高危因素为0分。研究表明具有高危因素(1分组+2分组+3分组)患者与0分组患者比较180天TRM明显升高,分别为(43.6%vs14.8%,p=0.021)；具有高危因素2分组+3分组患者与1分组患者比较180天TRM明显升高,两组分别为(60.8%vs19.4%,p=0.018)；具有高危因素3分组患者与2分组患者比较180天TRM明显升高,分别为(82.6%vs33.9%,p=0.015)；1年的OS四组之间有明显的差异(0分：82.8%；1分：79.2%；2分：48.7%；3分：0%)(p<0.001)。
7.PES的分层治疗前瞻性对发生PES患者进行高危分级后采用分层干预治疗,发现经过治疗之后重度PES患者的预后明显改善。
2012年3月至2013年12月前瞻性临床研究,共103例恶性血液病接受非血缘CBT(发生PES患者79例)。根据PES预后积分系统,采用甲基泼尼松龙(MP)为基础的干预治疗之后,0分组患者(无高危因素者)(n=26例)与至少有1项以上高危因素者(1分组+2分组+3分组)(n=53例)的移植相关死亡率、仅1项高危因素(1分组)(n=27例)与至少有2项以上高危因素者(2分组+3分组)的移植相关死亡率(n=26例)之间均无明显统计学差异(p=0.16；p=0.39)。对比分析发现,经过分层干预治疗之后,2分组+3分组患者1年OS由25.5%(2010年4月至2012年2月)上升至72.3%(2012年3月至2013年12月)(p=0.019),表明重症PES患者的预后明显改善。
8.PES发生效应细胞的鉴定及PES发生的细胞活化信号途径
实验研究表明,T淋巴细胞亚群、NK细胞及相关功能抗原的CD11b、CD27、 CD57表达在PES组与未发生PES组之间无统计学差异；细胞形态学及流式细胞仪检测(免疫表型)提示PES伴有腹泻患者大便中的细胞类型,以单核巨噬细胞占主导；PES患者血清中细胞因子的检测提示单核巨噬细胞相关的因子IL-1B、 MCP-1、IL-6、IL-18在PES发生时比移植前显著增加(p<0.01),也显著高于UCBT同期未发生PES的患者(p<0.01),并且当PES症状控制后细胞因子水平明显下降至移植前的水平。NK/T细胞相关细胞因子、及补体水平在PES发生前后,以及PES组患者与未发生PES的患者之间均无明显差别(p>0.05)
采用高通量测序的方法检测了3例CBT患者(9份标本)发生PES前、发生时、缓解时血清中的miRNA,结果显示差异表达的miRNA有hsa-let-7i-5p、 hsa-mir-98-5p、hsa-mir-152-3p、hsa-mir-223-3p等39个,通过靶基因预测+GO+Pathway分析,推测出PES的发生与单核巨噬细胞的激活相关的三条信号通路：Toll、NOD、RIG-I样受体信号通路相关。
结论及意义
截至目前,文献检索表明本研究是报道CBT相关PES例数最多、研究最为全面的临床研究,且首次提出根据PES高危因素的积分系统进行高危分级并分层干预治疗,国际目前尚未报道。
1.通过回顾性及前瞻性临床研究,发现清髓性预处理方案及双份脐血移植是PES发生的高危因素,两组在年龄、性别、基础疾病、移植时的骨髓状态(CR与否)、HLA配型、ABO血型、预处理是否采用TBI或氟达拉滨的方案、细胞数(包括TNC、CD34、CD3、CD56计数)等方面均无明显统计学差异。
2.本研究对发生PES患者的主要临床症状进行对比研究,表明充血性皮疹和发热是诊断PES最为特异性的症状。
3.PES与脐血干细胞能否植入密切相关,脐血移植过程中未发生PES是原发性植入失败的高危因素。多因素分析表明PES是发生急性GVHD的重要高危因素。
4.尽管发生PES的患者与未发生PES的患者相比较,患者的移植相关死亡率及复发率无明显统计学意义,但是相对于轻症PES来说,重症PES患者的移植相关死亡率明显增加。根据预后积分系统对PES患者进行分层治疗,能够明显改善重症PES患者的长期生存。
5.实验研究初步证实了单核巨噬细胞是引起PES发生的效应细胞,通过分子水平预测可能激活Toll、NOD、RIG-I样受体三条分子信号传导通路而发挥作用。
相对于其他移植类型,CBT最主要的问题是植入失败及较高的移植相关死亡率,也是CBT在国内开展较少的主要原因。本课题对于CBT后发生的PES做了较为全面的临床研究,特别是按照积分系统进行高危分级能够准确区分轻症PES和重症PES；高危分级后的分层干预治疗,一方面对于保留轻症PES促进脐血干细胞的植入,克服原发性植入失败,另一方面对于将重症PES转化为轻症PES,以减少重症PES所致的移植相关死亡率等,均具有重要的临床意义。
Introduction
Cord blood transplantation (CBT) is an effective and potential curative treatment for pediatric patients with hematological malignancies who lack HLA-identical related or unrelated donors. Over the last25years, the field of umbilical cord blood (UCB) banking and transplantation has grown exponentially. Over600000UCB units have been stored for transplantation worldwide, and＞30000CBTs have been performed. UCB serves as an alternative stem cell source; only30%of patients who require an allograft will have a human leukocyte antigen (HLA)-matched sibling donor. Despite＞20million adult volunteer donors in the National Marrow Donor Program and affiliated registries, many patients, particularly patients of diverse racial/ethnic backgrounds, will not have a suitably matched, unrelated volunteer donor identified in the required time period. UCB has extended access to transplantation, especially to patients of racial and ethnic minorities and is rapidly available. Post-transplantation immune disorders, including pre-engraftment syndrome (PES), engraftment syndrome, and acute graft-versus-host disease (GVHD) are problematic in CBT. The complex and intricate pathophysiology of post-transplantation immune disorders is a consequence of interactions between the donor and host innate and adaptive immune responses. PES, a clinical entity of unknown pathogenesis, has been described in patients receiving CBT. Although a uniform definition is lacking, PES is commonly characterized by noninfectious fever and various other clinical findings before neutrophil engraftment, including skin rash, pulmonary infiltrates, and diarrhea, jaundice, or weight gain. To date, PES is an entirely clinical entity with no known pathognomonic histopathologic changes or biochemical markers. Kishi et al were the first to report a pre-engraftment immune reaction, which occurred in35of45adult recipients of reduced-intensity conditioning CBT. However, PES still remains poorly characterized and its clinical significance and the prognostic impact after CBT are unclear. The first part of the research is to address these issues, we retrospectively analyzed the incidence, risk factors, and clinical outcomes of PES in unrelated CBT recipients enrolled in a multicenter CBT trial. The second part of the research, we investigated that donor monocyte-macrophages were the key effector cells which expressed high level cytokines in PES patients. By target gene prediction+GO+pathway analysis, we suspected that the occurrence of PES were activated by monocytes and macrophages, and associated with three possible Toll, NOD, and RIG-I-like receptor signaling pathways.
Methods and patients
Between April,2000and December,2013,240consecutive patients with hematologic malignancies underwent unrelated CBT in Anhui Provincial Hospital. All of the240hematologic malignancies patients referred for CBT had one of the features associated with poor outcomes:ⅰ) Patients were in first complete remission (CR1) but have high risk factors at first diagnosis:ALL with adverse cytogenetics or molecular abnormalities [ph+chromosome (BCR-ABL positive), hypodiploidy,11q23abnormalities (MLL rearrangements)], or with high level of minimal residual disease (MRD)(1%or more after completion of6weeks of induction therapy); AML with adverse cytogenetics or molecular abnormalities [inv(3)(q21q26.2) or t(3;3)(q21;q26.2), t(6;9)(p23;q34),11q23abnormalities (MLL rearrangements),-5or del(5q),-7or del(7q), complex karyotype, normal cytogenetics with FLT3-ITD mutation], or with a prior history of myelodysplastic syndrome (MDS). ⅱ) CR2(but the length of CR1≤12months), CR3or more. ⅲ) Primary induction failure, or no remission after relapse with salvage chemotherapy.
Cord blood units that were serologically matched for≥4of six HLA antigens and which contained at least3×107nucleated cells/kg and1.2×105CD34+/kg of recipient body weight before freezing were obtained from the cord blood bank at the China Cord Blood Bank Network. The units were not depleted of T lymphocytes. The choice to transplant one vs. two UCB units was based solely on cell dose criteria. If a UCB unit with the minimum cell dose was not available, the patient was transplanted with two partially HLA-matched UCB units, which needed to have a minimum cryopreserved dose of3.5×107nucleated cells/kg and2×105CD34+/kg, respectively. All patients received a myeloablative conditioning regimen of TBICY[total body irradiation (TBI, total12Gy,4fractions) and cyclophosphamide (CY,60mg/kg daily for2days)](age≥14years or primary induction failure or no remission after relapse) or BuCY2[busulfan (0.8mg/kg every6h for4days) and CY](age＜14years or prior radiotherapy which would presuppose a high risk of toxicity). Cytarabine (2.0g/m2every12h for2days) and/or carmustine (BCNU,250mg/m2) was added to the myeloablative conditioning regimen in order to penetrate the blood brain barrier to kill the CNS leukemia cells maximally (for some patients). Fludarabine (30mg/m2daily for4days) was combined with BuCY2to promote engraftment.
For GVHD prophylaxis, all patients were given a combination of cyclosporine (Novartis, Stein, Switzerland) and mycophenolate mofetil (Roche, Basel, Switzerland). Intravenous cyclosporine was started (2.5-3mg/kg/d) on day-1and continued until patients were able to take cyclosporine orally with target trough levels of200ng/mL at least1month. Mycophenolate mofetil (25mg/kg/d) was given on day+1. The rapidity of tapering was based on the presence or absence of GVHD, infectious disease, and relapse risks.
PES was defined as unexplained fever＞38.3℃not associated with documented infection and/or an unexplained erythematous skin rash resembling that of acute GVHD, with either the fever or the rash occurring before or at neutrophil recovery. Specifically, fever attributed to PES was not associated with any clinical evidence of infection, with patients having both a negative infectious disease workup and a continued lack of response to broad-spectrum antimicrobial agents. Erythematous skin rash attributed to PES was not associated with any clinical suspicion of drug allergy.Weight gainwas defined as a3%increase in body weight between the day of CBT and the onset of PES. Noninfectious diarrhea was defined as passage of watery stools more than twice a day for at least3consecutive days with no evidence of infectious etiology.
Variables related to the patient, the disease, and the transplantation procedures were compared with the use of the chi-squared test for categorical variables. The Cox regression model was used for multivariate analysis of clinical variables. The end-points were engraftment, graft failure, PES, acute and chronic GVHD, relapse, transplant-related mortality (TRM), malignancy-free survival (MFS), overall survival (OS). Time-to-event outcomes for neutrophil and platelet engraftment, PES, GVHD, TRM, and relapse were estimated using cumulative incidence curves. The difference in the cumulative incidence curves was based on Gray's test. The Kaplan-Meier method was used to estimate the probabilities of the survival (OS and MFS) which were compared using the log rank test. p<0.05was considered statistically significant. All analyses were performed with SPSS (version17.0).
The second part of the research, we investigated that donor monocyte-macrophages were the key effector cells which expressed high level cytokines in PES patients. Serum samples with known miRNA expression in patients with PES, using the difference in miRNA expression levels log2-ratio, Scatter plot diagram comparing the expressions. Difference between the two samples by miRNA expression was analyzed before and after PES. The detection of miRNA expression maybe found to play an important role in the pathogenesis of PES. This would also be helpful to find the molecular signaling pathways and the mechanism of PES.
Results
From2000to2012(the first period of the clinical research), a total of137patients received CBT, and73patients (53.3%) developed PES at a median of7days (range5-15days). The cumulative incidence of PES was53.3%(95%CI,45.9-65.4%). Comparing the patient demographics and graft characteristics of the PES patients and the non-PES patients, we found that myeloblative conditioning and double CBT were the high risks for the development of PES. But there were no significant differences between the2groups in terms of age, sex, weight, underlying malignancy, BM status at CBT, HLA match, ABO compatibility or conditioning approach. There also were no between-group differences in total infused TNC dose, total CD34+cell dose, total CD3cell dose, and total CD56cell dose. Total of14patients developed primary engraftment failure,2patients recovered with autologous hematopoisis, and the other12patients salvaged with haploid-transplantation. We found that patients with no PES was the high risk for primary engraftment failure (p＜0.001).
64patients had non-infectious fever (87.6%) within14days,59patients with red rash (80.8%),16patients with non-infectious diarrhea (21.9%),11patients with weight gain (≥3%)(15.1%),9patients with elevated liver transaminases (12.3%),5cases with elevated bilirubin (6.8%),4cases of renal damage (5.5%), and5patients with hypoxemia or pulmonary edema (6.8%). Rash and non-infectious fever are the most specific diagnostic symptoms, but weight gain, non-infectious diarrhea, liver and kidney dysfunction and other syndromes were not the specific characters of PES.
The incidence of acute GVHD in the PES group was higher than the non-PES group patients (p=0.019):Grade II-IV acute GVHD developed in25(34.2%) patients of the73PES group patients, and the100-day cumulative incidence of grade II-IV acute GVHD was35.6%(95%CI,29.3-43.8%); Only9patients developed II-IV acute GVHD in the64non-PES group patients, and100-day cumulative incidence of grade II-IV acute GVHD was15.2%(95%CI,8.5-22.6%).
The cumulative transplant-related mortality was30.6%(95%CI:20.3%-40.9%) in PES patients, and23patients (27%) died due to the transplant-related mortality (TRM). TRM was developed in28patients (43.8%) in the64non-PES group patients, and the cumulative transplant-related mortality was43.5%(95%CI:34.5%-50.2%) Although the transplant-related mortality was higher in the non-PES group than the PES group patients, but there was no significant difference (p=0.10). Total of20patients developed disease recurrence (14.5%). The cumulative recurrence rates were13.5%(95%CI:6.6%-19.2%) and15.8%(95%CI:9.5%-22.5%) in PES and non-PES group patients, and there was also no significant difference (p=0.74).
5-year OS and5-year MFS did not differ depending on the presence or absence of PES after CBT [63.9%(95%CI,58.6%-72.2%) vs59.2%(95%CI,52.1%-66.2%),59.2%(95%CI,50.8.5%-64.3%) vs56.8%(95%CI,48.6%-62.5%), respectively; p=0.65,0.82]. By the time of the occurrence of PES, the maximum temperature, clinical symptoms, cyclosporine concentrations, methylprednisolone (MP) initial dose, the days from the effectiveness of MP, the days of maintains of MP, ineffectiveness of MP, we found that PES occurred in7days (＜7days), over three or more clinical symptoms, poor effectiveness of MP (ineffectiveness within in one week) were the poor prognostic risk factors of PES.
We made a PES scoring system according to3high risk factors which had been described, and investigated the impact of high risk factors to the PES patients prognosis. The existence of one risk factor was defined as1point, and no risk factors with0score. Our study showed that the cumulative incidence of180-days transplant-related mortality (TRM) was higher in patients with at least one risk factors (43.6%vs14.8%, p=0.021); and180-days transplant-related mortality (TRM) was also higher in patients with more than one risk factors (2+3) than patients with one risk factor (60.8%vs19.4%, p=0.018);180-days TRM was also higher in patients with three risk factors (3scores) than patients with two risk factors (82.6%vs33.9%, p=0.015). For1-year OS, there were significant differences during the4groups patients (0point:82.8%;1point:79.2%;2points:48.7%;3points:0%)(p＜0.001).
From March2012to December2013((the second period of the clinical research)),103patients with hematologic malignancies underwent UBCT, and79patients developed PES. We used methylprednisolone (MP) intervention according to the different PES score system. Patients with1high risk factor were treated with MP1mg/kg/d, and Patients with2or more high risk factors were treated with MP2mg/kg/d. After the intervention of MP, there were no significant statistical differences between patients with0score (n=26patients) and with at least1score (1+2+3points)(n=53patients), and there were also no significant statistical differences between patients with1score (n=27patients) and with at least2scores (2+3points)(n=26patients)(p=0.16; p=0.39). After the treatment of MP according to the PES score system, the prognosis of severe PES was significant improved by1-year OS between the two different periods (April2010to February2012and March2012to December2013)(25.5%vs72.3%)(p=0.019).
According to our center experimental results and the clinical manifestations of PES, we found that the major effector cells of PES are donor monocyte-macrophages. We also found that donor monocyte-macrophages are the key effector cells which expressed high level cytokines in PES patients. Using HiSeq high-throughput sequencing technique of microRNA, we detected the serum miRNAs in3PES patients with periods of pre-PES, at the time of PES, and the remission of PES. About39obviously expressed miRNAs, such as hsa-let-7i-5p、hsa-mir-98-5p、 hsa-mir-152-3p、hsa-mir-223-3p, et al, were detected. By target gene prediction+GO+pathway analysis, we suspected that the occurrence of PES were activated by monocytes and macrophages, and associated with three possible signaling pathways: Toll, NOD, RIG-I-like receptor signaling pathways.
Conclusions
In conclusion, PES seems to be common after CBT and may be associated with enhanced engraftment, and patient with no PES was the high risk for primary engraftment failure. Rash and non-infectious fever are the most specific diagnostic symptoms. Although PES is closely associated with acute GVHD, PES after CBT is not associated with significant morbidity and is easily manageable with intravenous steroids. PES occurred within7days (<7days), over three or more clinical symptoms, poor effectiveness of MP (ineffectiveness within in one week) were the poor prognostic risk factors of PES. We made a PES scoring system according to these3high risk factors, and after the treatment of MP according to the PES score system, the prognosis of severe PES was significantly improved. We found that the major effector cells of PES are donor monocyte-macrophages. We also found that donor monocyte-macrophages are the key effector cells which expressed high level cytokines in PES patients. By target gene prediction+GO+pathway analysis, we suspected that the occurrence of PES were activated by monocytes and macrophages, and associated with three possible Toll, NOD, and RIG-I-like receptor signaling pathways. Because failure to recognize PES in CBT recipient risks unnecessary complications of this syndrome and unnecessarily long, empirical treatment, physicians should be aware of the possible occurrence of PES after CBT, especially in patients who have relevant risk factors for developing PES.
引文
1. Giralt S, Bishop MR. Principles and overview of allogeneic hematopoietic stem cell transplantation. Cancer Treat Res 2009; 144:1-21.
2. Baxter-Lowe LA, Hurley CK. Advancement and clinical implications of HLA typing in allogeneic hematopoietic stem cell transplantation. Cancer Treat Res 2009;144:71-94.
3. Lv M, Huang XJ. Allogeneic hematopoietic stem cell transplantation in China: where we are and where to go. J Hematol Oncol 2012; 18:5-10.
4. Weisdorf D, Forman S. Allogeneic hematopoietic stem cell transplantation for adult acute lymphoblastic leukemia. Cancer Treat Res 2009; 144:441-54.
5. Terwey TH, Kim TD, Arnold R. Allogeneic hematopoietic stem cell transplantation for adult acute lymphocytic leukemia. Curr Hematol Malig Rep 2009;4(3):139-47.
6. Kebriaei P, Poon LM. The role of allogeneic hematopoietic stem cell transplantation in the therapy of patients with acute lymphoblastic leukemia. Curr Hematol Malig Rep 2012;7(2):144-52.
7. Phillips GL. Allogeneic hematopoietic stem cell transplantation (HSCT) for high-risk acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS):how can we improve outcomes in the near future? Leuk Res 2012;36(12):1490-5,
8. Norkin M, Hsu JW, Wingard JR. Quality of life, social challenges, and psychosocial support for long-term survivors after allogeneic hematopoietic stem-cell transplantation. Semin Hematol 2012;49(1):104-9.
9. Gluckman E, Broxmeyer HA, Auerbach AD, et al. Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med 1989;321(17):1174-1178.
10. Appelbaum FR. Pursuing the goal of a donor for everyone in need. N Engl J Med 2012;367(16):1555-1556.
11. Delaney C, Gutman JA, Appelbaum FR. Cord blood transplantation for haematological malignancies:conditioning regimens, double cord transplant and infectious complications. Br J Haematol 2009;147(2):207-16.
12. Moscardo F, Sanz GF, Sanz MA. Unrelated-donor cord blood transplantation for adult hematological malignancies. Leuk Lymphoma 2004;45(1):11-8.
13. Mogul MJ. Unrelated cord blood transplantation vs matched unrelated donor bone marrow transplantation:the risks and benefits of each choice. Bone Marrow Transplant 2000; 25 Suppl 2:S58-60.
14. Wadlow RC, Porter DL. Umbilical cord blood transplantation:where do we stand? Biol Blood Marrow Transplant 2002;8(12):637-47.
15. Anasetti C, Aversa F, Brunstein CG. Back to the future:mismatched unrelated donor, haploidentical related donor, or unrelated umbilical cord blood transplantation? Biol Blood Marrow Transplant 2012;18(1 Suppl):S161-5.
16. Atsuta Y, Morishima Y, Suzuki R, et al. Comparison of unrelated cord blood transplantation and HLA-mismatched unrelated bone marrow transplantation for adults with leukemia. Biol Blood Marrow Transplant 2012;18(5):780-7.
17. Atsuta Y, Suzuki R, Nagamura-Inoue T, et al. Disease-specific analyses of unrelated cord blood transplantation compared with unrelated bone marrow transplantation in adult patients with acute leukemia. Blood 2009; 113(8):1631-8.
18. Takahashi S, Iseki T, Ooi J, et al. Single-institute comparative analysis of unrelated bone marrow transplantation and cord blood transplantation for adult patients with hematologic malignancies. Blood 2004; 104(12):3813-20.
19. Ooi J, Iseki T, Takahashi S, et al. A clinical comparison of unrelated cord blood transplantation and unrelated bone marrow transplantation for adult patients with acute leukaemia in complete remission. Br J Haematol 2002;118(1):140-3.
20. Sanz GF, Saavedra S, Planelles D. Unrelated donor cord blood transplantation in adults with Hematologic malignancies. Blood 2001; 98:2332-8.
21. Kim DH, Sohn SK, Kim JG, Suh JS, Lee KS, Lee KB. Clinical impact of hyperacute graft-versus-host disease on Results of allogeneic stem cell transplantation. Bone Marrow Transplant 2004; 33:1025-30.
22. Lee CK, Gingrich RD, Hohl RJ, Ajram KA. Engraftment syndrome in autologous bone marrow and peripheral stem cell transplantation. Bone Marrow Transplant 1995;16(1):175-82.
23. Ravoet C, Feremans W, Husson B, et al. Clinical evidence for an engraftment syndrome associated with early and steep neutrophil recovery after autologous blood stem cell transplantation. Bone Marrow Transplant 1996;18(5):943-7.
24. Kawano C, Muroi K, Kuribara R, et al. Engraftment syndrome after autologous peripheral blood stem cell transplantation with high numbers of peripheral blood stem cells followed by granulocyte colony-stimulating factor administration. Bone Marrow Transplant 2000;25(2):228-9.
25. Spitzer TR. Engraftment syndrome following hematopoietic stem cell transplantation. Bone Marrow Transplant 2001;27(9):893-8.
26. Oyama Y, Cohen B, Traynor A, et al. Engraftment syndrome:a common cause for rash and fever following autologous hematopoietic stem cell transplantation for multiple sclerosis. Bone Marrow Transplant 2002;29(1):81-5.
27. Kishi Y, Kami M, Miyakoshi S, et al. Early immune reaction after reduced-intensity cord-blood transplantation for adult patients. Transplantation 2005;80(1):34-40.
28. Lee YH, Lim YJ, Kim JY, et al. Pre-engraftment syndrome in hematopoietic stem cell transplantation. J Korean Med Sci 2008;23(1):98-103.
29. Radford J, Rest E, Weisdorf D. Cytokine-stimulated engraftment syndrome after high-dose chemotherapy with bone marrow and G-CSF-primed peripheral stem cell rescue. Blood 1994,84(10 Suppl.1):490a
30. Patel KJ, Rice RD, Hawke R, Abboud M, Heller G, Scaradavou A,et al. Pre-engraftment syndrome after double-unit cord blood transplantation:a distinct syndrome not associated with acute graft-versus-host disease. Biol Blood Marrow Transplant 2010;16(3):435-40.
31. Kanda J, Kaynar L, Kanda Y, Prasad VK, Parikh SH, Lan L, et al. Pre-engraftment syndrome after myeloablative dual umbilical cord blood transplantation:risk factors and response to treatment. Bone Marrow Transplant 2013;48(7):926-31.
32. Park M, Lee SH, Lee YH, Yoo KH, Sung KW, Koo HH,et al. Korean Cord Blood Transplantation Working Party. Pre-engraftment syndrome after unrelated cord blood transplantation:a predictor of engraftment and acute graft-versus-host disease. Biol Blood Marrow Transplant 2013;19(4):640-6.
33. Hong KT, Kang HJ, Kim NH, Kim MS, Lee JW, Kim H, Park KD, Shin HY, Ahn HS. Peri-engraftment syndrome in allogeneic hematopoietic SCT. Bone Marrow Transplant 2013;48(4):523-8.
34. Spitzer TR. Engraftment syndrome following hematopoietic stem cell transplantation. Bone Marrow Transplant 2001;27(9);893-8.
35. Wang X, Liu H, Li L, Geng L, Ding K, Liu X, et al. Pre-engraftment syndrome after unrelated donor umbilical cord blood transplantation in patients with hematologic malignancies. Eur J Haematol 2012; 88(1):39-45.
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1. Giralt S, Bishop MR. Principles and overview of allogeneic hematopoietic stem cell transplantation. Cancer Treat Res 2009;144:1-21.
2. Norkin M, Hsu JW, Wingard JR. Quality of life, social challenges, and psychosocial support for long-term survivors after allogeneic hematopoietic stem-cell transplantation. Semin Hematol 2012;49(1):104-9.
3. Appelbaum FR. Pursuing the goal of a donor for everyone in need. N Engl J Med 2012;367(16):1555-1556.
4. Delaney C, Gutman JA, Appelbaum FR. Cord blood transplantation for haematological malignancies:conditioning regimens, double cord transplant and infectious complications. Br J Haematol 2009;147(2):207-16.
5. Moscardo F, Sanz GF, Sanz MA. Unrelated-donor cord blood transplantation for adult hematological malignancies. Leuk Lymphoma 2004;45(1):11-8.
6. Sanz GF, Saavedra S, Planelles D. Unrelated donor cord blood transplantation in adults with Hematologic malignancies. Blood 2001; 98:2332-8.
7. Kim DH, Sohn SK, Kim JG, Suh JS, Lee KS, Lee KB. Clinical impact of hyperacute graft-versus-host disease on Results of allogeneic stem cell transplantation. Bone Marrow Transplant 2004; 33:1025-30.
8. Lee CK, Gingrich RD, Hohl RJ, Ajram KA. Engraftment syndrome in autologous bone marrow and peripheral stem cell transplantation. Bone Marrow Transplant 1995;16(1):175-82.
9. Ravoet C, Feremans W, Husson B, et al. Clinical evidence for an engraftment syndrome associated with early and steep neutrophil recovery after autologous blood stem cell transplantation. Bone Marrow Transplant 1996;18(5):943-7.
10. Kawano C, Muroi K, Kuribara R, et al. Engraftment syndrome after autologous peripheral blood stem cell transplantation with high numbers of peripheral blood stem cells followed by granulocyte colony-stimulating factor administration. Bone Marrow Transplant 2000;25(2):228-9.
11. Spitzer TR. Engraftment syndrome following hematopoietic stem cell transplantation. Bone Marrow Transplant 2001;27(9):893-8.
12. Oyama Y, Cohen B, Traynor A, et al. Engraftment syndrome:a common cause for rash and fever following autologous hematopoietic stem cell transplantation for multiple sclerosis. Bone Marrow Transplant 2002;29(1):81-5.
13. Kishi Y, Kami M, Miyakoshi S, et al. Early immune reaction after reduced-intensity cord-blood transplantation for adult patients. Transplantation 2005;80(1):34-40.
14. Lee YH, Lim YJ, Kim JY, et al. Pre-engraftment syndrome in hematopoietic stem cell transplantation. J Korean Med Sci 2008;23(1):98-103.
15. Naoyuki Takahashi, Mori S-I, Soeda A, Wakeda T, Ohsaki Y, Shiwa M, et al. Profiling of immune-related microRNA expression in human Cord blood and adult peripheral blood cells upon proinflammatory stimulation. European Journal of Haematology 2011;8:1600-1609.
16. Matsuno N, Yamamoto H, Watanabe N, Uchida N, Ota H, Nishida A, et al. Rapid T-cell chimerism switch and memoryT-cell expansion are associated with pre-engraftment immune reaction early after cord blood transplantation. Br J Haematol 2013;160(2):255-8.
17. Harkonen PL, Vaananen HK. Monocyte-macrophage system as a target for estrogen and selective estrogen receptor modulators. Ann N Y Acad Sci 2006;1089:218-27.
18. Dubourdeau M, Pipy B, Rousseau D. Roles of PPAR and p21WAF1/CIP1 in monocyte/macrophage differentiation:are circulating monocytes able to proliferate?. Med Sci (Paris) 2010;26(5):481-6.
19. Van den Bossche J, Malissen B, Mantovani A, et al. Regulation and function of the E-cadherin/catenin complex in cells of the monocyte-macrophage lineage and DCs. Blood 2012; 119(7):1623-33.
20. Castagna A, Polati R, Bossi AM, Girelli D. Monocyte/macrophage proteomics: recent findings and biomedical applications. Expert Rev Proteomics 2012;9(2):201-15.
21. Park M, Lee SH, Lee YH, Yoo KH, Sung KW, Koo HH,et al. Korean Cord Blood Transplantation Working Party. Pre-engraftment syndrome after unrelated cord blood transplantation:a predictor of engraftment and acute graft-versus-host disease. Biol Blood Marrow Transplant 2013;19(4):640-6.
22. Kanda J, Kaynar L, Kanda Y, Prasad VK, Parikh SH, Lan L, et al. Pre-engraftment syndrome after myeloablative dual umbilical cord blood transplantation:risk factors and response to treatment. Bone Marrow Transplant 2013;48(7):926-31.
23. Patel KJ, Rice RD, Hawke R, Abboud M, Heller G, Scaradavou A,et al. Pre-engraftment syndrome after double-unit cord blood transplantation:a distinct syndrome not associated with acute graft-versus-host disease. Biol Blood Marrow Transplant 2010;16(3):435-40.
24. Hong KT, Kang HJ, Kim NH, Kim MS, Lee JW, Kim H, Park KD, Shin HY, Ahn HS. Peri-engraftment syndrome in allogeneic hematopoietic SCT. Bone Marrow Transplant 2013;48(4):523-8.
25. Spitzer TR. Engraftment syndrome following hematopoietic stem cell transplantation. Bone Marrow Transplant 2001;27(9):893-8.
26. Wang X, Liu H, Li L, Geng L, Ding K, Liu X, et al. Pre-engraftment syndrome after unrelated donor umbilical cord blood transplantation in patients with hematologic malignancies. Eur J Haematol 2012;88(1):39-45.
2. Baxter-Lowe LA, Hurley CK. Advancement and clinical implications of HLA typing in allogeneic hematopoietic stem cell transplantation. Cancer Treat Res 2009;144:71-94.
3. Lv M, Huang XJ. Allogeneic hematopoietic stem cell transplantation in China: where we are and where to go. J Hematol Oncol 2012; 18:5-10.
4. Weisdorf D, Forman S. Allogeneic hematopoietic stem cell transplantation for adult acute lymphoblastic leukemia. Cancer Treat Res 2009; 144:441-54.
5. Terwey TH, Kim TD, Arnold R. Allogeneic hematopoietic stem cell transplantation for adult acute lymphocytic leukemia. Curr Hematol Malig Rep 2009;4(3):139-47.
6. Kebriaei P, Poon LM. The role of allogeneic hematopoietic stem cell transplantation in the therapy of patients with acute lymphoblastic leukemia. Curr Hematol Malig Rep 2012;7(2):144-52.
7. Phillips GL. Allogeneic hematopoietic stem cell transplantation (HSCT) for high-risk acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS):how can we improve outcomes in the near future? Leuk Res 2012;36(12):1490-5,
8. Norkin M, Hsu JW, Wingard JR. Quality of life, social challenges, and psychosocial support for long-term survivors after allogeneic hematopoietic stem-cell transplantation. Semin Hematol 2012;49(1):104-9.
9. Gluckman E, Broxmeyer HA, Auerbach AD, et al. Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med 1989;321(17):1174-1178.
10. Appelbaum FR. Pursuing the goal of a donor for everyone in need. N Engl J Med 2012;367(16):1555-1556.
11. Delaney C, Gutman JA, Appelbaum FR. Cord blood transplantation for haematological malignancies:conditioning regimens, double cord transplant and infectious complications. Br J Haematol 2009;147(2):207-16.
12. Moscardo F, Sanz GF, Sanz MA. Unrelated-donor cord blood transplantation for adult hematological malignancies. Leuk Lymphoma 2004;45(1):11-8.
13. Mogul MJ. Unrelated cord blood transplantation vs matched unrelated donor bone marrow transplantation:the risks and benefits of each choice. Bone Marrow Transplant 2000; 25 Suppl 2:S58-60.
14. Wadlow RC, Porter DL. Umbilical cord blood transplantation:where do we stand? Biol Blood Marrow Transplant 2002;8(12):637-47.
15. Anasetti C, Aversa F, Brunstein CG. Back to the future:mismatched unrelated donor, haploidentical related donor, or unrelated umbilical cord blood transplantation? Biol Blood Marrow Transplant 2012;18(1 Suppl):S161-5.
16. Atsuta Y, Morishima Y, Suzuki R, et al. Comparison of unrelated cord blood transplantation and HLA-mismatched unrelated bone marrow transplantation for adults with leukemia. Biol Blood Marrow Transplant 2012;18(5):780-7.
17. Atsuta Y, Suzuki R, Nagamura-Inoue T, et al. Disease-specific analyses of unrelated cord blood transplantation compared with unrelated bone marrow transplantation in adult patients with acute leukemia. Blood 2009; 113(8):1631-8.
18. Takahashi S, Iseki T, Ooi J, et al. Single-institute comparative analysis of unrelated bone marrow transplantation and cord blood transplantation for adult patients with hematologic malignancies. Blood 2004; 104(12):3813-20.
19. Ooi J, Iseki T, Takahashi S, et al. A clinical comparison of unrelated cord blood transplantation and unrelated bone marrow transplantation for adult patients with acute leukaemia in complete remission. Br J Haematol 2002;118(1):140-3.
20. Sanz GF, Saavedra S, Planelles D. Unrelated donor cord blood transplantation in adults with Hematologic malignancies. Blood 2001; 98:2332-8.
21. Kim DH, Sohn SK, Kim JG, Suh JS, Lee KS, Lee KB. Clinical impact of hyperacute graft-versus-host disease on Results of allogeneic stem cell transplantation. Bone Marrow Transplant 2004; 33:1025-30.
22. Lee CK, Gingrich RD, Hohl RJ, Ajram KA. Engraftment syndrome in autologous bone marrow and peripheral stem cell transplantation. Bone Marrow Transplant 1995;16(1):175-82.
23. Ravoet C, Feremans W, Husson B, et al. Clinical evidence for an engraftment syndrome associated with early and steep neutrophil recovery after autologous blood stem cell transplantation. Bone Marrow Transplant 1996;18(5):943-7.
24. Kawano C, Muroi K, Kuribara R, et al. Engraftment syndrome after autologous peripheral blood stem cell transplantation with high numbers of peripheral blood stem cells followed by granulocyte colony-stimulating factor administration. Bone Marrow Transplant 2000;25(2):228-9.
25. Spitzer TR. Engraftment syndrome following hematopoietic stem cell transplantation. Bone Marrow Transplant 2001;27(9):893-8.
26. Oyama Y, Cohen B, Traynor A, et al. Engraftment syndrome:a common cause for rash and fever following autologous hematopoietic stem cell transplantation for multiple sclerosis. Bone Marrow Transplant 2002;29(1):81-5.
27. Kishi Y, Kami M, Miyakoshi S, et al. Early immune reaction after reduced-intensity cord-blood transplantation for adult patients. Transplantation 2005;80(1):34-40.
28. Lee YH, Lim YJ, Kim JY, et al. Pre-engraftment syndrome in hematopoietic stem cell transplantation. J Korean Med Sci 2008;23(1):98-103.
29. Radford J, Rest E, Weisdorf D. Cytokine-stimulated engraftment syndrome after high-dose chemotherapy with bone marrow and G-CSF-primed peripheral stem cell rescue. Blood 1994,84(10 Suppl.1):490a
30. Patel KJ, Rice RD, Hawke R, Abboud M, Heller G, Scaradavou A,et al. Pre-engraftment syndrome after double-unit cord blood transplantation:a distinct syndrome not associated with acute graft-versus-host disease. Biol Blood Marrow Transplant 2010;16(3):435-40.
31. Kanda J, Kaynar L, Kanda Y, Prasad VK, Parikh SH, Lan L, et al. Pre-engraftment syndrome after myeloablative dual umbilical cord blood transplantation:risk factors and response to treatment. Bone Marrow Transplant 2013;48(7):926-31.
32. Park M, Lee SH, Lee YH, Yoo KH, Sung KW, Koo HH,et al. Korean Cord Blood Transplantation Working Party. Pre-engraftment syndrome after unrelated cord blood transplantation:a predictor of engraftment and acute graft-versus-host disease. Biol Blood Marrow Transplant 2013;19(4):640-6.
33. Hong KT, Kang HJ, Kim NH, Kim MS, Lee JW, Kim H, Park KD, Shin HY, Ahn HS. Peri-engraftment syndrome in allogeneic hematopoietic SCT. Bone Marrow Transplant 2013;48(4):523-8.
34. Spitzer TR. Engraftment syndrome following hematopoietic stem cell transplantation. Bone Marrow Transplant 2001;27(9);893-8.
35. Wang X, Liu H, Li L, Geng L, Ding K, Liu X, et al. Pre-engraftment syndrome after unrelated donor umbilical cord blood transplantation in patients with hematologic malignancies. Eur J Haematol 2012; 88(1):39-45.
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11. Spitzer TR. Engraftment syndrome following hematopoietic stem cell transplantation. Bone Marrow Transplant 2001;27(9):893-8.
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14. Lee YH, Lim YJ, Kim JY, et al. Pre-engraftment syndrome in hematopoietic stem cell transplantation. J Korean Med Sci 2008;23(1):98-103.
15. Naoyuki Takahashi, Mori S-I, Soeda A, Wakeda T, Ohsaki Y, Shiwa M, et al. Profiling of immune-related microRNA expression in human Cord blood and adult peripheral blood cells upon proinflammatory stimulation. European Journal of Haematology 2011;8:1600-1609.
16. Matsuno N, Yamamoto H, Watanabe N, Uchida N, Ota H, Nishida A, et al. Rapid T-cell chimerism switch and memoryT-cell expansion are associated with pre-engraftment immune reaction early after cord blood transplantation. Br J Haematol 2013;160(2):255-8.
17. Harkonen PL, Vaananen HK. Monocyte-macrophage system as a target for estrogen and selective estrogen receptor modulators. Ann N Y Acad Sci 2006;1089:218-27.
18. Dubourdeau M, Pipy B, Rousseau D. Roles of PPAR and p21WAF1/CIP1 in monocyte/macrophage differentiation:are circulating monocytes able to proliferate?. Med Sci (Paris) 2010;26(5):481-6.
19. Van den Bossche J, Malissen B, Mantovani A, et al. Regulation and function of the E-cadherin/catenin complex in cells of the monocyte-macrophage lineage and DCs. Blood 2012; 119(7):1623-33.
20. Castagna A, Polati R, Bossi AM, Girelli D. Monocyte/macrophage proteomics: recent findings and biomedical applications. Expert Rev Proteomics 2012;9(2):201-15.
21. Park M, Lee SH, Lee YH, Yoo KH, Sung KW, Koo HH,et al. Korean Cord Blood Transplantation Working Party. Pre-engraftment syndrome after unrelated cord blood transplantation:a predictor of engraftment and acute graft-versus-host disease. Biol Blood Marrow Transplant 2013;19(4):640-6.
22. Kanda J, Kaynar L, Kanda Y, Prasad VK, Parikh SH, Lan L, et al. Pre-engraftment syndrome after myeloablative dual umbilical cord blood transplantation:risk factors and response to treatment. Bone Marrow Transplant 2013;48(7):926-31.
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24. Hong KT, Kang HJ, Kim NH, Kim MS, Lee JW, Kim H, Park KD, Shin HY, Ahn HS. Peri-engraftment syndrome in allogeneic hematopoietic SCT. Bone Marrow Transplant 2013;48(4):523-8.
25. Spitzer TR. Engraftment syndrome following hematopoietic stem cell transplantation. Bone Marrow Transplant 2001;27(9):893-8.
26. Wang X, Liu H, Li L, Geng L, Ding K, Liu X, et al. Pre-engraftment syndrome after unrelated donor umbilical cord blood transplantation in patients with hematologic malignancies. Eur J Haematol 2012;88(1):39-45.