非清髓性造血干细胞移植后并发移植物抗宿主病的相关因素分析

目的 探讨和分析非清髓性造血干细胞移植(NST)后并发移植物抗宿主病(GVHD)的相关因素.方法 选择34例血液病患者,其中重型再生障碍性贫血(SAA)15例,重型β-地中海贫血(TM)1例,肿瘤性血液病18例;进行无关供者脐带血造血干细胞移植(UCBT)11例,同胞供者骨髓联合外周血干细胞移植7例,外周血造血干细胞移植(PBSCT)16例.移植前采用以抗胸腺细胞球蛋白(ATG)、抗淋巴细胞球蛋白(ALG)或者氟达拉滨强效免疫抑制为基础的非清髓性预处理方案.GVHD的预防采用短程的甲氨蝶呤(MTX)联合环孢素A(CsA).观察非清髓性造血干细胞移植后的临床特点以及急、慢性移植物抗宿主病的发生情况;分析发生慢性移植物抗宿主病(cGVHD)的相关因素.结果 NST的植入率为91.2%.移植后7例肿瘤性血液病患者形成了供、受者造血细胞混合嵌合体(MC),给予供者淋巴细胞输注(DLI)2～9次后,例由MC转变为供者造血细胞完全嵌合体(FDC).随访12(3～96)个月,共发生Ⅰ～Ⅱ度急性移植物抗宿主病(aGVHD)5例,GVHD 15例.经统计学分析,发现年龄大的肿瘤性血液病患者经以ATG为基础的NST后,再给予DLI,其cGVHD的发生率高,且合并感染,对治疗的反应差;而以氟达拉滨为基础的NST患者发生cGVHD后治疗反应较好.移植100 d前后患者分别死亡3例和5例,其中3例死于广泛性cGVHD.结论 患者的年龄大、有合并症、以ATG为基础的预处理方案、肿瘤性血液病是NST后患者并发cGVHD的危险因素.     

非清髓性外周血干细胞移植后白血病复发的防治

目的探讨非清髓性造血干细胞移植后白血病复发的防治。方法7例合并有其它系统疾病的白血病患者接受非清髓性外周血干细胞移植,移植后采用环孢素A及短程甲氨蝶呤预防移植物抗宿主病(GVHD),造血细胞植入后,动态检测骨髓单个核细胞嵌合体的变化,根据嵌合体状态,采用供者淋巴细胞输注(DLI),并配合环孢素A用量的调整来防治白血病复发。结果7例患者移植后造血功能得到恢复,3例移植前合并的疾病有所加重,经处理缓解,移植早期相关并发症少而轻。移植后有3例嵌合体由混合嵌合体(MC)转为完全供者型嵌合体(CC),再转为MC,伴Ph染色体阳性,DLI5~6次后Ph染色体转为阴性,嵌合体状态又转为CC,其中2例发生广泛皮肤慢性GVHD;3例的嵌合体持续为CC,无复发,其中2例接受了1次DLI,此2例在环孢素A减量过程中发生GVHD;1例的嵌合体持续为MC,虽经3次DLI,但无效,患者死于白血病复发。结论非清髓性造血干细胞移植后应动态监测嵌合体状态,采用供者淋巴细胞输注,并配合环孢素A用量的调整,对白血病的复发有一定的效果。     

供者淋巴细胞输注在恶性血液病非清髓性骨髓移植中的应用

目的探讨供者淋巴细胞输注(DLI)治疗非清髓性异基因造血干细胞移植后血液病复发的疗效。方法5例恶性血液病患者接受非清髓性造血干细胞移植,在形成混合性嵌合体和血液学部分缓解(例1～4)或进步(例5)后,进行DLI。移植后的4～5周进行第1次DLI,首次输注T淋巴细胞数量为(0.5～1.0)×105/kg,以后每隔3～4周逐渐增加输注的淋巴细胞数量,至(0.5～2.0)×108/kg,平均行DLI4.6次(3～8次)。结果例1～4分别经过7、3、2、3次DLI后,性染色体及DNA指纹图由混合性嵌合体转变为完全性嵌合体;例2、3经过DLI后消除了微小残留病,除例5仍然为混合性嵌合体和进步状态外,4例均达血液学完全缓解。例1、2和例3、4分别出现Ⅰ/Ⅱ度急性移植物抗宿主病和广泛/局限型慢性移植物抗宿主病,例2、4出现骨髓抑制。结论DLI可使异基因造血干细胞移植后短暂的混合性嵌合体向完全性嵌合体转变,并可清除微小残留病。     

非清髓性无关供者脐血移植与同胞供者骨髓移植治疗再生障碍性贫血的比较

目的 对非清髓性无关供者脐血移植与同胞供者骨髓移植治疗重型再生障碍性贫血(SAA)的临床效果进行评价和比较.方法 回顾性分析15例SAA患者进行非清髓性造血干细胞移植的临床资料,根据造血干细胞(HSC)来源的不同,将患者分为骨髓移植组(BMT组;6例)和脐血移植组(UCBT组;9例).对两组患者术后的外周血象、骨髓象、细胞嵌合体状态、移植物抗宿主病(GVHD)以及存活率等长期随访结果进行了统计学分析.结果 BMT组和UCBT组造血干细胞植入率分别为100%和66.7%,两组比较,差异有统计学意义(P＜0.05).UCBT组移植后大多数形成了供、受者型细胞混合嵌合体,BMT组大多数形成了供者型完全嵌合体.BMT组血象恢复正常的中位时间为25 d、UCBT组为120 d,BMT组骨髓象恢复正常的中位时间为25 d,UCBT组为150 d.BMT组慢性GVHD的表现以肝功能异常为主,而UCBT组则以皮疹为主.UCBT组术后早期感染率为33.3%,BMT组为16.7%.结论 非清髓性无关供者脐血移植和同胞供者骨髓移植均可成功治疗SAA;但与BMT比较,UCBT的造血功能恢复较慢、血型转变少而延迟、早期感染率较高、而慢性GVHD的程度却较轻.     

伊马替尼联合供者淋巴细胞输注治疗造血干细胞移植后慢性粒细胞白血病复发

目的 探讨伊马替尼联合供者淋巴细胞输注(DLI)治疗异基因造血干细胞移植后慢性粒细胞白血病(CML)复发的效果.方法 3例CML(慢性期)患者,在接受预处理后,例1接受其胞妹外周血造血干细胞移植,例2接受其胞兄的骨髓移植,例3接受其胞弟的骨髓与外周血造血干细胞联合移植.例1移植后采用环孢素A(CsA)和霉酚酸酯(MMF)预防移植物抗宿主病(GVHD),例2采用CsA、短程甲氨蝶呤(MTX)、抗胸腺细胞球蛋白及抗CD25单克隆抗体预防GVHD,例3应用CsA、MTX和MMF预防GVHD.采用细胞遗传学及荧光原位杂交技术动态监测治疗效果.移植后发生血液学复发时,给予伊马替尼口服,并行DLI.结果 例1移植后30 d行DLI,输注CD3+T淋巴细胞0.5×107 /kg,移植后50 d和70 d,再次行DLI,分别输注CD3+ T淋巴细胞1.0 × 107 /kg和2.0×107 /kg,短串联重复序列(STR)检测提示为完全供者嵌合(DC).移植后120 d,疾病进展,给予伊马替尼400 mg/d,同时输注供者CD3+ T淋巴细胞2.5 × 107/kg.移植后180 d,STR检查提示仍为DC.患者最终于移植后17个月因髓外复发死亡.例2的染色体核型于移植后35 d转变为46,XY,XY为100%,BCR-ABL融合基因阴性.移植后100 d,原发病复发.停用免疫抑制剂,输入供者CD3+ T淋巴细胞3.9×107 /kg,同时口服伊马替尼500 mg/d.DLI联合伊马替尼治疗后30 d,患者的染色体核型为46,XY,XY为100%,BCR-ABL融合基因阴性,患者至今无病存活53个月.例3移植后5 d造血功能获得重建,移植后60 d,染色体核型为46,XY.移植后120 d,确诊CML复发,遂给予伊马替尼400 mg/d,并行DLI,共输注供者CD3+ T淋巴细胞8×107 /kg,1个月后,患者的染色体核型再次转为46,XY,患者至今无病存活50个月.结论 伊马替尼联合DLI对造血干细胞移植后CML复发具有一定的治疗效果.     

Tju103和CTLA4-Ig联合诱导主要组织相容复合物半相合小鼠骨髓移植耐受

目的观察吲哚亚甲基异烟腙(Tju103)和细胞毒性T淋巴细胞相关性抗原4免疫球蛋白(CTLA4-Ig)联合应用，对主要组织相容复合物(MHC)半相合小鼠骨髓移植的植入以及移植后移植物抗宿主病(GVHD)、移植物抗白血病(GVL)和抗感染的影响；探索一条既能降低GVHD能保留GVL和抗感染能力的移植途径。方法体外以受者(正常CB6F1鼠，H-2^bd)抗原为特异性免疫耐受诱导原，MHC半相合的供者(C57BL／6鼠，H-2^b)T淋巴细胞经和Tju103、CTLA4-Ig共育后，与供者骨髓细胞混合输入经预处理的受者体内。观察Tju103和CTLA4-Ig联合作用对移植后造血重建、GVHD、GVL和抗感染的影响。结果单纯照射组(A组)：全部(10只)白血病小鼠于照射后11d内死于造血功能衰竭，大部分(8只)死于照射后4～7d。环磷酰胺(CTX)治疗组(B组)：全部(10只)小鼠于接种白血病细胞后16～23d(移植后11～18d)死于白血病，但CTX治疗延长了白血病小鼠存活期。单纯移植组(C组)：全部(10只)小鼠于移植后21d内死亡，均死于GVHD。CsA预防组(D组)：4只小鼠于移植后8～22d内死亡，其中1只死于白血病，2只死于感染，1只死于GVHD；6只存活超过30d。Tju103处理组(E组)：4只小鼠于移植后9～26d内死亡，其中1只死于白血病，1只死于感染，2只死于GVHD；6只存活超过30d。CTLA4-Ig处理组(F组)：3只小鼠于移植后14～23d内死亡，其中1只死于感染，2只死于GVHD；7只存活超过30d。Tju103 CTLA4-Ig处理组(G组)：仅1只小鼠于移植后第19d死于GVHD；9只存活超过30d。结论CsA、Tju103或CTLA4-Ig单独处理均可以明显延长小鼠移植后生存期，降低GVHD的发生和程度；CTLA4-Ig有抗感染和GVL作用，而Tju103和CsA则没有此作用；CTLA4-Ig和Tju103两者联合处理供者T淋巴细胞对延长受者生存期和降低GVHD作用更明显。     

异基因造血干细胞移植后嵌合体的检测及临床意义

异基因造血干细胞移植后供者细胞是否成功植入，需要有可靠的证据。对移植后供者细胞植入证据的检测不仅对判断移植是否成功，而且对移植后免疫抑制剂的应用、供者淋巴细胞输注(DLI)、移植物抗宿主病(GVHD)的预防以及探讨移植后供、受者混合嵌合体形成、对疗效影响的评估皆有重要意义。     

ABO血型主要不合异基因造血干细胞移植后纯红细胞再生障碍的临床分析

目的探讨ABO血型主要不合者异基因造血干细胞移植（allo-HSCT）后并发纯红细胞再生障碍（PRCA）的危险因素、临床转归以及PRCA的治疗和预防。方法42例行allo-HSCT，其中供、受者AN）血型主要不合者33例，主次双向不合者9例，27例受者血型为O型。预处理后，13例行骨髓移植，25例行外周血干细胞移植，4例行脐血移植。6例移植前行供者型血浆置换。移植后采用环孢素A（CsA）及短程甲氨蝶呤（MTX）联用预防移植物抗宿主病（GVHD）。结果42例均获得供者细胞植入，11例移植后并发PRCA（26．2％），11例的血型均为O型，其供者9例为A型，2例为B型；移植前行供者型血浆置换的O型受者，移植后均未发生PRCA。并发PRCA的11例中，8例经红细胞输注后自然缓解，2例行供者型血浆置换，其凝集素滴度下降后缓解，1例予利妥昔单抗治疗后缓解。单因素分析表明，O型受者、A型供者以及A型供给O型者与PRCA的发生相关，多因素分析表明，A型供给0型者是发生PRCA的独立危险因素（RR为10．999，95％可信区间为1．975-61．258，P〈0．05）。结论A型供给O型者与PRCA的发生密切相关；移植前行供者型血浆置换可预防PRCA的发生；供者型血浆置换和利妥昔单抗可有效治疗PRCA。     

减低强度预处理造血干细胞移植联合低剂量环孢素A治疗恶性血液病

目的 探讨以减低强度的氟达拉滨、白消安(Bu)和环磷酰胺(CTX)为预处理方案的异基因外周血造血干细胞移植(HSCT)联合低剂量环孢素A(CsA)的疗效及并发症发生情况.方法 恶性血液病患者11例,接受同胞间HSCT,供、受者问HLA配型,HLA全相合10例.5个抗原相合1例.预处理包括移植前第9～4天给予氟达拉滨30～35 mg·m-2·d-1,移植前第4、3天给予白消安3.2 mg·kg-1·d1,移植前第2、1天给予CTX 60mg·kg1·d-1.移植后联合使用CsA和短程甲氨蝶呤(MTX)预防移植物抗宿主病(GVHD),供者细胞植入后,降低CsA用量.结果 移植后早期11例造血功能均获得重建,骨髓细胞为完全供者型.随访3～17个月,9例并发急性GVHD,主要侵犯肝脏和皮肤;9例并发慢性GVHD,均侵犯口腔和肝脏,其中1例为广泛性慢性GVHD,其余为局限性慢性GVHD.增加CsA用量或者加用甲泼尼龙后,急、慢性GVHD均能得到控制,仅1例需加用霉酚酸脂.11例中,2例的原发病复发,其中1例病情得到控制,1例失访.结论 HSCT时采用氟达拉滨、白消安和环磷酰胺(CTX)预处理方案,并将白消安的用量减为常用剂量的一半,移植后采用低剂量CsA,细胞的植入率高;急、慢性GVHD的发生率较高,但对糖皮质激素和CsA的治疗反应良好.     

氟达拉滨联合阿糖胞苷化疗治疗异基因造血干细胞移植后急性白血病复发

目的 观察氟达拉滨联合阿糖胞苷(FA方案)大剂量化疗对异基因造血干细胞移植(allo-HSCT)后急性白血病复发的治疗效果.方法 急性白血病患者10例,7例接受亲缘供者造血干细胞移植,3例接受非亲缘供者造血干细胞移植,所有患者移植后均获得造血重建,为完全供者型.10例于首次移植后38～213 d(中位数为126 d)急性白血病复发,其中完全供者型复发1例,完全受者型复发4例,混合嵌合体型复发5例.原发病复发后均采用FA方案化疗,氟达拉滨用量为25 mg/m2,阿糖胞苷用量为1.5 g/m2,用5 d,其中6例于化疗结束后第2天还接受原供者外周造血干细胞(PBSC)移植,未使用预防移植物抗宿主病(GVHD)的药物.结果 除1例早期(8 d)死亡外,其余9例再次获得造血重建,经外周血DNA序列分析证实为完全供者型.获得造血重建的9例,4例由于复发、感染、多脏器功能衰竭等原因死亡,5例无病存活至随访结束,存活时间分别为585、442、405、213和243 d.治疗后,3例未发生GVHD,其余7例发生急性或慢性GVHD;7例并发肺部真菌感染,3例并发出血性膀胱炎,4例并发巨细胞病毒血症.10例的6个月实际无病存活率为60%,2年预期无病存活率为53%.结论 对于allo-HSCT后的急性白血病复发,采用FA方案化疗,如条件允许联合原供者造血干细胞输注,可能是目前可采取的一种有效治疗方案.     

Chimerism does not influence graft-versus-myeloma and graft-versus-host disease in reduced intensity setting

In this study we serially evaluated the chimerism status in 20 multiple myeloma patients allotransplanted with a reduced intensity regimen. All patients engrafted, with total 75% overall responses and 35% of CRs. After a median follow-up of 35 months, seven patients (35%) died, three of them due to disease progression. Four patients died before day +100, with a TRM of 20%. Nine patients (45%) developed aGVHD and six (40%) had cGVHD. Twenty-five percent of patients achieved full donor chimerism (FDC) before day +100, 42% before day +200 and 75% 24 months after graft. In our series, level of chimerism did not correlate with either the quality of response or aGVHD. No significant differences were found between bone marrow and peripheral blood samples. Analogously, even if donor DNA percentage often resulted higher in the PMN fraction than in the mononuclear one, these differences were not significant after statistical analysis. On the other hand, cGVHD was associated with increased rates of FDC, with 6/6 cases showing a full donor pattern in concomitance of the cGVHD versus 5/9 cases presenting a FDC in the group of patients without cGVHD (p=0.057). The Kaplan-Meier estimates of OS and PFS at 2 years were 59% and 58%, respectively; chimerism pattern did not impact in the predicting clinical outcome. In summary, our study shows that a stable engraftment and high frequency of donor chimerism are achievable after a reduced intensity conditioning regimen. Moreover, even as result of a single center experience, we suggest that chimerism, graft-versus-myeloma and GVHD would represent distinct entities that require larger immunological studies for further clarification.     

Non-myeloablative allogeneic stem cell transplantation for metastatic renal cell carcinoma

Between 1999 and 2004, 11 patients with metastatic renal cell carcinoma (RCC) underwent non-myeloablative stem cell transplantation (NST) with conditioning using fludarabine-based regimens in two institutions of Korea. Among 11 patients, only one patient showed partial response (response rate: 9%), three showed stable disease, and six progressive disease. Three patients developed acute graft-versus-host disease (GVHD), and among them, one developed grade III acute GVHD which caused early death at day 60 after transplantation, and this patient showed partial response at day 30. Six patients developed chronic GVHD, three limited, and three extensive GVHD, respectively. Survival after one yr was 18% in transplanted patients. Median overall survival for entire cohort was 4.3 months. Eight patients died from progressive disease and three (27%) from treatment-related mortality. Only one patient survived 51.2 months after NST with slowly progressive disease. This patient received donor lymphocyte infusion three times after NST and achieved complete donor chimerism. NST does not lead to durable response and prolonged overall survival in the majority of patients with RCC in our series.     

WT1-specific CTL cells of recipient origin may exist in the peripheral blood of patients achieving full donor chimerism soon after nonmyeloablative transplantation

This study was performed to assay whether leukemia-associated antigen (LAA)-specific CTLs of recipient origin existed in the blood of patients who achieved full donor chimerism (FDC) soon after nonmyeloablative transplantation (NST). In 15 patients who received haplo-identical NST, WT1(+) CD8(+) CTLs were detected with WT1/HLA-A*0201 pentamer, and the donor-recipient chimerism levels were analyzed by three methods. Results showed that WT1(+) CD8(+) CTLs could be detected in patients with HLA-A*0201 expressing only in recipient, and cells of recipient origin existed in the blood of patients who achieved FDC, which suggested that LAA-specific CTLs of recipient origin may exist in patients achieving FDC soon after NST.     

Evaluation of mixed chimerism in bone marrow transplantation program in Croatia

In this study we monitored mixed chimerism in 36 patients with various hematologic disorders. All of them underwent a classic conditioning regimen, 31 patients for related bone marrow transplantation (BMT) and 5 patients for unrelated BMT. DNA was isolated from peripheral blood, and samples were polymerase chain reaction (PCR) amplified for 5 short tandem repeat (STR) loci (TH01, VWA31, FES/FPS, F13A01, and SE33) and for one variable number of tandem repeats locus (D1S80). Samples were run on a 6% polyacrylamide gel in an automated ALFexpress sequencer. In all 36 donor-recipient pairs we found differences for at least two STR loci. In most cases the difference was observed for SE33 and D1S80 loci. Mixed chimerism (MC) was detected in 18 patients: 4 with unrelated BMT and 14 with related sibling donors. In 11 patients MC was detected in the early period after BMT, but was soon followed by full donor chimerism (FDC) in peripheral blood. In 5 cases patients MC appearing after FDC was established, and was predictive for the relapse. One patient showed alternating MC and FDC, but at the end showed only recipient cells and graft rejection. In conclusion, the PCR-STR analysis is a highly informative, fast, and simple screening method for monitoring chimerism in a BMT program.     

A second prophylactic MHC-mismatched bone marrow transplantation protects against rat acute myeloid leukemia (BNML) without lethal graft-versus-host disease

BACKGROUND: We have employed a rat model for human acute myeloid leukemia, a promyelocytic leukemia in the BN rat strain (BNML), to develop new protocols for immunotherapy in combination with allogeneic bone marrow transplantation (alloBMT). The status of mixed chimerism in allotransplanted rats provided an opportunity for immunotherapy using alloreactive donor cells. In addition to T or natural killer (NK) cells, we introduced a second infusion of bone marrow cells as prophylactic donor lymphocyte infusions (DLI) to test whether an effective graft-versus-leukemia (GVL) response could be obtained without clinical graft-versus-host disease (GVHD). METHODS: BN rats were sublethally irradiated and transplanted with T-cell depleted bone marrow cells from either fully major histocompatibility complex (MHC)-mismatched (PVG) donor rats or MHC-matched (PVG.1N) as controls. Seven days after transplantation, rats were given 500 leukemic cells to mimic minimal residual disease. Additional cellular therapy was given at day +7. The efficiency of DLI was monitored by chimerism analysis in peripheral blood. RESULTS: Rats receiving infusions of NK cells succumbed to leukemia. T-DLI induced complete donor T-cell chimerism and lethal GVHD. A second alloBMT protected against leukemia. This effect was dependent on an MHC incompatibility between the donor and host and also on the presence of alloreactive T cells in the second bone marrow inoculum, resulting in an increased, mixed donor T-cell chimerism. CONCLUSION: A second prophylactic transplantation influenced the degree of T-cell chimerism to balance favorably between GVL and GVHD. If applicable to humans, repeated alloBMT may provide a novel approach to leukemia therapy.     

Molecular monitoring of T-cell chimerism early after allogeneic stem cell transplantation may predict the occurrence of acute GVHD grades II-IV

Mixed chimerism (MC) within CD4+ and CD8+ T cell days 7 and 10 after allogeneic stem cell transplantation (SCT) was compared with the occurrence of acute graft-vs.-host disease (GVHD) in 34 patients after SCT. Acute GVHD was diagnosed in 22 patients within the first 3 months after SCT, 15 of these developed acute GVHD grades II-IV. The difference in the clearance rate of host T cell between the two days were compared. We found a significantly higher risk (p = 0.005) for developing acute GVHD grades II-IV in patients with complete donor CD4+ T-cell chimerism day 7 after SCT together with patients who increased 50% or more in donor CD4+ T cells between days 7 and 10 after SCT. Our data suggest that molecular monitoring of MC early after transplantation may be useful as a diagnostic tool in predicting the occurrence of moderate to severe acute GVHD after SCT.