Ki-67、p53、bcl-2的表达与恶性淋巴瘤自体造血干细胞移植预后的相关性

 目的　研究bcl-2、p53、Ki-67在恶性淋巴瘤组织中的表达以及与自体造血干细胞移植（AHSCT）预后的相关关系。方法　采用免疫组织化学IHC法检测33例行AHSCT治疗的患者淋巴瘤组织切片中Ki-67、p53、bcl-2的表达。并分析Ki-67、p53、bcl-2的表达与预后的相关性。生存率统计采用Kaplan-Meier生存曲线,Log-Rank检验,多因素分析采用COX风险回归模型。结果　p53 的表达与33例AHSCT患者预后无关,bcl-2阳性表达组和阴性表达组移植后3年无瘤生存率（DFS）分别为35.71 %和88.89 %（P＜0.05）;Ki-67阳性表达组和阴性表达组3年DFS分别为43.75 %和85.71 %（P <0.05）,提示bcl-2和Ki-67的表达与AHSCT的预后相关。COX多因素分析显示,Ki-67和bcl-2是影响淋巴瘤患者AHSCT后无瘤生存的相关因素（P＝0.0437）。结论　bcl-2、Ki-67蛋白阳性表达的淋巴瘤患者,AHSCT后易复发,可作为移植后预后判断的指标之一。Ki-67和bcl-2是影响淋巴瘤患者AHSCT无瘤生存的独立相关因素。     

自体造血干细胞移植治疗恶性淋巴瘤的临床研究进展*

高剂量治疗联合自体造血干细胞移植（high-dose therapy/autologous hematopoietic stem cell transplantation ,HDT/AHSCT ）是目前治疗复发/难治侵袭性淋巴瘤的标准方案。然而,HDT/AHSCT作为淋巴瘤一线治疗的地位尚存在争议,对于其在不同亚型淋巴瘤中的应用仍有很多问题需要解决。本文就HDT/AHSCT治疗恶性淋巴瘤的临床研究进展作系统回顾。      

自体造血干细胞移植治疗恶性淋巴瘤的研究进展

自体造血干细胞移植(AHSCT)是治疗恶性淋巴瘤(ML)的重要方法,其治疗过程较为复杂,疗效也受诸多因素影响.近年来AHSCT在ML的治疗中应用更加广泛,寻找有效的预后因素以进一步区分可能从AHSCT中获益的患者以及制订合理的移植方案是临床医生关注的重要问题.文章就AHSCT治疗ML的研究进展作一综述.     

自体造血干细胞移植治疗复发难治恶性淋巴瘤十例效果观察

目的 探讨自体造血干细胞移植(AHSCT)治疗复发难治恶性淋巴瘤的疗效和安全性.方法 回顾性分析济南军区总医院2011年8月至2015年6月收治的10例接受AHSCT治疗的复发难治恶性淋巴瘤患者的临床资料,其中男性6例,女性4例;中位年龄34岁(20~50岁);复发4例,难治6例;霍奇金淋巴瘤(HL)5例,非霍奇金淋巴瘤(NHL)5例.移植前经过多个疗程的放化疗,予大剂量甲氨蝶呤(CTX)+粒细胞集落刺激因子(G-CSF)动员外周血造血干细胞,采用BEAM(卡莫司汀+依托泊苷+阿糖胞苷+美法仑)、CBV(环磷酰胺+卡莫司汀+依托泊苷)或全身照射(TBI)方案进行预处理.结果 10例AHSCT患者单个核细胞(MNC)中位计数为7.385×108/kg,移植后8例完全缓解,2例复发.中位随访时间为18个月(20~50个月),患者总生存率及无病生存率均为80%(8/10).患者均出现不同程度的恶心、呕吐、腹泻、口腔黏膜炎等不良反应,均可耐受.结论 AHSCT是治疗复发难治恶性淋巴瘤的有效方法,安全性较高.     

自体造血干细胞移植治疗恶性淋巴瘤

目的观察白体造血干细胞移植（AHSCT）治疗恶性淋巴瘤的疗效。方法自1998年1月至2003年2月,用AHSCT治疗恶性淋巴瘤15例。外周血千细胞动员均采用CHOP方案＋VP16＋粒细胞集落刺激因子（G—CSF300μg／d,5～7d）。预处理方案为CBV或改良CHOP方案。结果全部患者均获得缓解,随诊至2003年12月,3年无瘤生存率为86．1％,无移植相关死亡。结论AHSCT联合大剂量化疗对中高度、中晚期恶性淋巴瘤疗效佳,能够提高生存率并优于常规化疗。     

自体造血干细胞移植联合利妥昔单抗治疗CD+20B细胞淋巴瘤

 采用自体造血干细胞移植（AHSCT）治疗中,高危侵袭性淋巴瘤已获得了较好疗效,但仍有部分移植患者因复发而死亡。复发的根源主要为微小残留病变,包括体内残留的肿瘤细胞和移植物中的肿瘤细胞污染。利妥昔单抗可靶向性清除CD+20 B细胞,因此,对CD+20 B细胞淋巴瘤患者,移植前后应用利妥昔单抗可起到体内净化和清除残留病灶作用,AHSCT联合利妥昔单抗有望进一步提高CD+20 B细胞淋巴瘤的疗效。     

自体造血干细胞移植治疗恶性淋巴瘤15例临床分析

目的:评价自体造血干细胞移植(AHSCT)治疗恶性淋巴瘤患者的疗效。方法:采用AHSCT治疗恶性淋巴瘤患者15例,其中霍奇金淋巴瘤患者3例(均为复发病例),非霍奇金淋巴瘤患者12例(Ⅲ、Ⅳ期或复发病例,IPI评分2-4分)。采集外周血造血干细胞前均经化疗及动员剂动员(CHOP方案9例,CHOP+MTX 3例,CEP、大剂量MTX、单用G-CSF各1例)。预处理方案为联合化疗10例(BEAC、CBV方案为主),联合化疗加放射治疗5例(TBI、TLI各1例,提前局部照射3例)。结果:移植后白细胞≥1.0×109/L的中位时间为10(9-13)天,血小板≥50×109/L的中位时间为14(11-17)天。随访时间为1-110.5个月。中位生存时间为43(1-110.5个月)个月,3年总生存率(OS)为66.7%。结论:AHSCT是一种治疗复发难治恶性淋巴瘤的安全有效的方法。     

T淋巴母细胞淋巴瘤自体干细胞移植后的长期随访观察

目的评价自体造血干细胞移植（autologous hematopoietic stem cell transplantation,AHSCT）治疗复发难治T淋巴母细胞淋巴瘤（TLBL）的临床疗效及安全性。方法本文回顾性分析AHSCT治疗后长期随访的TLBL16例,预处理方案主要为BEAM和BEAC。结果 15例患者可评价疗效,1例失访。中位随访37个月（12~132个月）,中位无进展生存时间（PFS）34.5个月。预计中位总生存时间49月。1、3、5年总生存率分别为60%、53%、32%。初治患者一线治疗有效者,接受AHSCT者预计中位总生存时间为108个月,5年总生存率、无进展生存率分别为62%、63%;复发患者挽救治疗后接受AHSCT者,中位总生存时间为22.8个月,5年总生存率、无进展生存率分别为33%、22%。初始治疗未达CR/PR的难治患者,中位生存仅21个月,5年生存率和无进展生存率分别为20%和29%。结论 AHSCT常规化疗治疗TLBL安全、有效,可提高复发难治的T淋巴母细胞淋巴瘤的远期生存,延长初治TLBL患者的无进展生存期,但复发率仍偏高,值得开展大规模临床试验进一步深入研究。     

T淋巴母细胞淋巴瘤自体干细胞移植后的长期随访观察

目的评价自体造血干细胞移植(autologous hematopoietic stem cell transplantation,AHSCT)治疗复发难治T淋巴母细胞淋巴瘤(TLBL)的临床疗效及安全性。方法本文回顾性分析AHSCT治疗后长期随访的TLBL16例,预处理方案主要为BEAM和BEAC。结果 15例患者可评价疗效,1例失访。中位随访37个月(12~132个月),中位无进展生存时间(PFS)34.5个月。预计中位总生存时间49月。1、3、5年总生存率分别为60%、53%、32%。初治患者一线治疗有效者,接受AHSCT者预计中位总生存时间为108个月,5年总生存率、无进展生存率分别为62%、63%;复发患者挽救治疗后接受AHSCT者,中位总生存时间为22.8个月,5年总生存率、无进展生存率分别为33%、22%。初始治疗未达CR/PR的难治患者,中位生存仅21个月,5年生存率和无进展生存率分别为20%和29%。结论 AHSCT常规化疗治疗TLBL安全、有效,可提高复发难治的T淋巴母细胞淋巴瘤的远期生存,延长初治TLBL患者的无进展生存期,但复发率仍偏高,值得开展大规模临床试验进一步深入研究。     

大剂量化疗联合自体造血干细胞移植治疗外周T细胞淋巴瘤的回顾性研究

目的 :探讨大剂量化疗联合自体造血干细胞移植(high-dose therapy/autologous hematopoietic stem cell transplantation,HDT/AHSCT)在外周T细胞淋巴瘤(peripheral T-cell lymphoma,PTCL)治疗中的价值。方法:对50例接受HDT/AHSCT治疗的PTCL患者的病历资料进行回顾性分析,并进行随访。结果 :50例患者均未发生HDT/AHSCT相关死亡。中位随访时间为13个月(1~136个月),2年无进展生存率为59.0%,2年总生存率为65.0%。单因素分析结果显示,移植前达完全缓解(complete remission,CR)患者的2年无进展生存率和总生存率均明显优于非CR患者(2年无进展生存率:72.8%vs 41.9%,P=0.003;2年总生存率:88.2%vs41.9%,P=0.002),一线治疗敏感患者接受HDT/AHSCT的预后优于二线治疗敏感患者(2年无进展生存率:76.8%vs 30.8%,P=0.001;2年总生存率:81.1%vs 46.2%,P=0.015)。此外,移植前的红细胞沉降率是2年无进展生存率和2年总生存率的影响因素(P=0.004,P=0.018),移植前血清乳酸脱氢酶水平是2年无进展生存率的影响因素(P=0.044)。多因素分析结果显示,仅移植前达CR是2年总生存率的独立影响因素[相对风险=4.879(95%可信区间:1.583~15.034),P=0.006];未发现与2年无进展生存率相关的独立影响因素。亚组分析显示,一线治疗达CR的血管免疫母细胞性T细胞淋巴瘤及晚期自然杀伤细胞/T细胞淋巴瘤患者接受HDT/AHSCT可有生存获益。结论 :HDT/AHSCT作为PTCL患者一线巩固治疗或挽救治疗的安全性较高,并且可以部分改善PTCL患者的预后。今后有待开展前瞻性随机对照研究以验证HDT/AHSCT在不同病理亚型及移植前状态下的应用价值。     

Mobilization of peripheral blood progenitor cells after DHAP regimen with or without rituximab: a large multicenter comparative study in patients with malignant lymphoma

DHAP regimen is commonly used in patients with lymphoma. It is routinely used in combination with the monoclonal anti-CD20 antibody rituximab (R-DHAP), particularly for peripheral blood stem cell (PBSC) mobilization. The aim of this study was to assess the impact of rituximab on PBSC mobilization in patients with lymphoma receiving DHAP chemotherapy. We retrospectively reviewed the data of patients treated by DHAP or R-DHAP regimens as PBSC mobilization protocol between July 1998 and June 2005. Sixty-nine patients were included in the study: 21 in the DHAP group and 48 in the R-DHAP group. Both groups were not statistically different in term of clinical and biological presentation of the disease. The first cytapheresis was performed at day 10 in the R-DHAP group versus day 11 in the DHAP group. In contrast, the number of circulating CD34⁺ cells was higher, but not significant, in the R-DHAP group than the DHAP group, namely 9.7×10⁶ CD34⁺ cells/kg and 6.1×10⁶ CD34⁺ cells/kg, respectively. Finally, the complete remission status at time of harvest was the only one factor associated with poor mobilization on multivariate analysis. In conclusion, our results show that rituximab does not impair PBSC collection.     

High-dose rituximab does not negatively affect peripheral blood stem cell mobilization kinetics in patients with intermediate-grade non-Hodgkin's lymphoma

Rituximab, an anti-CD20 human-mouse chimeric monoclonal antibody has been shown to improve response rates when it is combined with standard salvage chemotherapy in patients with relapsed or refractory intermediate-grade B-cell non-Hodgkin's lymphoma. A vast majority of these patients subsequently undergo high-dose therapy followed by stem cell transplantation. However, the impact of rituximab on stem cell mobilization kinetics is not well characterized. The purpose of this study was to study the effect of high-dose rituximab given with chemotherapy on stem cell mobilization in patients with intermediate-grade B-cell non-Hodgkin's lymphoma. Thirty-six patients received ifosfamide, etoposide, and rituximab followed by filgrastim for stem cell mobilization. The chemotherapy regimen was well tolerated. Thirty-four of 36 patients (94%) were able to mobilize at least 2 × 10⁶ CD34+ cells/kg body weight after a median of 2 apheresis procedures. The median CD34+ cell dose collected per kilogram of recipient body weight was 6.5 × 10⁶ (range, 4.65-31.15). All patients who subsequently underwent high-dose chemotherapy and stem cell transplantation experienced sustained engraftment. In conclusion, high-dose rituximab given during stem cell mobilization does not negatively affect stem cell mobilization kinetics.     

Successful mobilization of peripheral blood stem cells with the DHAP regimen (dexamethasone, cytarabine, cisplatinum) plus granulocyte colony-stimulating factor in patients with relapsed Hodgkin's disease

High-dose chemotherapy followed by autologous stem cell transplantation can improve the outcome of relapsed and refractory Hodgkin's disease (HD) patients. The objective of the trial was to determine the mobilizing potential of the DHAP salvage regimen (dexamethasone, cytarabine, cisplatin) for the collection of peripheral blood stem cells (PBSC) in patients with relapsed HD. The target yield of harvesting CD34 + cells was ≥ 2 × 10⁶/kg in order to support the subsequent myeloablative chemotherapy. Most of the 105 patients included were intensively pre-treated with different combination chemotherapy regimens prior to mobilization. The use of DHAP followed by granulocyte colony-stimulating factor (G-CSF; 10 μg/kg) resulted in the successful collection of adequate numbers of PBSC in 97.1% of patients (102 of 105) with a median harvest of CD34 + cells of 13 × 10⁶/kg (range 2.6 - 85.1). More than 2.0 × 10⁶ CD34 + cells/kg were achieved in 65 of 103 (63%) patients after 1 apheresis, the maximum number of aphereses for all patients was 3. It was found that the optimal time of PBSC harvest was at days 13 - 16 after initiating the mobilization regimen.

These results demonstrate that the salvage chemotherapy regimen, such as DHAP combined with G-CSF, can be successfully used to mobilize PBSC in HD patients.     

普乐沙福用于外周血干细胞动员的研究进展

大剂量化疗联合自体外周血干细胞（peripheral blood stem cell,PBSC）移植是淋巴瘤和多发性骨髓瘤（multiple myeloma,MM）的有效治疗手段。PBSC的常规动员方案包括粒细胞集落刺激因子（granulocyte colony-stimulating factor,G-CSF）单用或联合化疗。部分患者使用常规动员方案无法采集到目标剂量的CD34+细胞,无法进行造血干细胞（hematopoietic stem cell,HSC）移植治疗。因此,针对动员不佳患者以及降低具有危险因素患者动员失败的风险,根据个体情况有效调整动员策略十分必要。普乐沙福是一种新型动员剂,联合G-CSF可显著提高CD34+细胞采集量,降低动员失败率的同时缩短采集天数,进而提高自体造血干细胞移植（autologous hematopoietic stem cell transplantation,ASCT）效率,改善患者长期预后。本文对普乐沙福在PBSC动员的研究进展进行综述,旨在探讨普乐沙福适宜的动员人群、干预时机和路径,以优化PBSC的动员策略。      

自体造血干细胞移植联合大剂量放化疗治疗恶性淋巴瘤32例

目的观察自体造血干细胞移植(AHSCT)治疗恶性淋巴瘤的疗效.方法自1991年6月至2000年4月,用AHSCT治疗恶性淋巴瘤32例.其中非霍奇金淋巴瘤(NHL)23例,霍奇金病(HD)9例;行自体骨髓移植(ABMT)12例,自体外周血干细胞移植(APBSCT)20例.外周血干细胞动员方法均采用常规化疗(CE或CHOP)加细胞集落刺激因子(G-CSF或GM-CSF;或G-CSF+GMCSF)10μg*kg-1*d-1.预处理方案为BEAM方案和MEL140mg/m2(或+Vp-16200mg)+单次全身照射(TBI)8Gy.结果全部患者移植后均重建造血,随访至2000年5月30日,中位随访1020d.处于无病生存者24例(75.0%),1,2年无病生存分别为78.1%(25/32)和46.9%(15/32),最长存活8年.8例(25.0%)复发.全组患者无移植相关死亡.结论AHSCT联合大剂量放化疗对预后不良复发或敏感的恶性淋巴瘤疗效佳,优于常规化疗.APBSCT造血恢复比ABMT快.预处理方案中含TBI的放疗组与单用联合化疗组疗效差异无显著性,但含放疗组副作用大.     

PBSC mobilization in lymphoma patients: analysis of risk factors for collection failure and development of a predictive score based on the kinetics of circulating CD34+ cells and WBC after chemotherapy and G‐CSF mobilization

Autologous stem cell transplantation (ASCT) is a potentially curative treatment of lymphoma, but peripheral blood stem cell (PBSC) mobilization fails in some patients. PBSC mobilizing agents have recently been proved to improve the PBSC yield after a prior mobilization failure. Predictive parameters of mobilization failure allowing for a preemptive, more cost‐effective use of such agents during the first mobilization attempt are still poorly defined, particularly during mobilization with chemotherapy + granulocyte colony‐stimulating factor (G‐CSF). We performed a retrospective analysis of a series of lymphoma patients who were candidates for ASCT, to identify factors influencing PBSC mobilization outcome. Premobilization parameters—age, histology, disease status, mobilizing protocol, and previous treatments—as well as white blood cell (WBC) and PBSC kinetics, markers potentially able to predict failure during the ongoing mobilization attempt, were analyzed in 415 consecutive mobilization procedures in 388 patients. We used chemotherapy + G‐CSF in 411 (99%) of mobilization attempts and PBSC collection failed (<2 × 10⁶ CD34+ PBSC/kg) in 13%. Multivariable analysis showed that only a low CD34+ PBSC count and CD34+ PBSC/WBC ratio, together with the use of nonplatinum‐containing chemotherapy, independently predicted mobilization failure. Using these three parameters, we established a scoring system to predict risk of failure during mobilization ranging from 2 to 90%, thus allowing a selective use of a preemptive mobilization policy. Copyright © 2014 John Wiley & Sons, Ltd.     

Flow Cytometric Analysis of Protein Phosphorylation in the Hematopoetic System

Cellular growth and differentiation in blood cells are regulated by the phosphorylation status of growth factor receptors and downstream proteins. Protein kinases and phosphatases balance the homeostasis of protein phosphorylation. Various diseases are associated with alterations in these tightly regulated processes. Aberrations have been proved to be of diagnostic value and might enhance the pathophysiological insight into the origin of the disease. However, quantitation of protein phosphorylation is currently not feasible in a clinical situation.

We developed a flow cytometric methodology which enables for direct investigation of protein phosphorylation in cell populations defined by multi-color flow cytometry. This assay does not only overcome drawbacks of traditional methodologies (e.g. Western blotting) but also allows quantitative analyses even in rare cell populations.

We accurately examined phosphorylation levels in different cell populations of hematological interest and especially analyzed CD34⁺ hematopoetic progenitor cells. CD34⁺ cells in bone marrow and in cord blood contained similar, low levels of phosphotyrosine. Circulating pheripheral blood system cells PBSC in patients exposed to G-CSF for stem cell mobilization exhibited significantly increased levels of phosphotyrosine. In vitro exposure of CD34⁺ progenitors to growth factors (G-CSF, IL-3, SCF) raised the levels of tyrosine phosphorylation in bone marrow and cord blood. Effects were dose and time dependent. Interestingly, in vivo stimulated CD34⁺ PBSC could not be further stimulated in vitro.

In conclusion, we present a new powerful methodology for analysis of protein phosphorylation in hematological specimens. The method does not only allow for accurate detection of phosphorylation levels in vivo, but also enables for quantitative analysis of growth factor receptor stimulation in vitro and in vivo.