非清髓性异基因造血干细胞移植治疗恶性血液病26例

　　作者：苏丽萍,许莲蓉,姜波,叶芳,朱秋娟　　作者单位：山西医科大学第二医院血液科，太原 030001

　　【摘要】为研究非清髓性异基因造血干细胞移植(non-myeloablative allogeneic stem cell transplantation，allo-NST)治疗恶性血液病的疗效及相关技术，选择26例恶性血液病患者作为研究对象(急性白血病10例，慢性髓性白血病14例，多发性骨髓瘤2例)，其中14例采用FAC(fludarabin+ATG+Cy)预处理，12例采用MAC(melfalan/maleran+ATG+Cy)预处理;用G-CSF 600 μg/d或G-CSF 300 μg/d+GM-CSF 300μg/d进行外周血干细胞动员，于第5天开始用Cobe Spectra血细胞分离机连续采集2-3次;用环孢菌素A联合短程甲氨蝶呤预防GVHD;移植后第4周开始供体淋巴细胞输注，首剂1×107/kg，之后依据临床反应及嵌合体形成情况，每4周1次，剂量逐级递增;微卫星短串联重复序列(STR)分析、Bcr/Abl融合基因、Ph染色体、HLA位点分析、性染色体及ABO血型等为植活检测指标。结果显示： 植入率84.62%，aGVHD发生率11.54%，cGVHD发生率23.07%;感染和出血等毒副反应发生率低、反应轻。结论：非清髓性异基因造血干细胞移植治疗恶性血液病疗效确切，毒副作用小，但相关技术，如适应症的选择、预处理方案、移植过程中的免疫治疗等需要进一步深入研究。

　　【关键词】 造血干细胞移植;非清髓性异基因造血干细胞移植; 恶性血液病

　　Nonmyeloablative Allogeneic Hematopoietic Stem cell Transplantation in 26 Cases of Hematological Malignancies　SU Li-Ping，XU Lian-Rong，JIANG Bo，YE Fang，ZHU Qiu-Juan，

　　LU Yu-Jin，CUI Yue-E，ZHU Lei，ZHANG Li，MA Xiang-Lan　Department of Hematology，The Second Teaching Hospital of Shanxi Medical University，Taiyuan 030001，China

　　Abstract The purpose of this study was to investigate the efficacy of non-myeloablative allogeneic stem cell transplantation (allo-NST) and its related technologies in hematological malignancies. 26 patients with hematological malignancies (acute leukemia 10，chronic myeloid leukemia 14，multiple myeloma 2) received allo-NST following conditioning regimens with fludarabin/cyclophosphamide/ATG in 14 cases or busulfan or melphalan/ cyclophosphamide/ATG in 12 cases prior to infusion of 2 or 3 collections of G-CSF(600 μg/d) or G-CSF(300 μg/d) plus GM-CSF(300 μg/d) mobilized blood stem cell on the fifth day. A combination of cyclosporine A (CsA) and methotrexate(MTX) was administered for GVHD prophylaxis. Patients were eligible for donor lymphocyte infusion (DLI) (or donor stem cell infusion (DSI) )given in graded increments according to the chimeric formation and clinical feature. Generally，the dose of the first infusion was 1×l07/kg in 4th week post-transplantation. The engraftment analyses included the detection of microsatellite short tandem repeats (STRs)，bcr/abl fusion gene，Philadelphia chromosome，HLA-locus analysis，sex chromosome and ABO blood type or blood subtype. The results showed that out of 26 patients，22 (84.62%) were engrafted，18/22 were full donor chimerism (FDC) up to now. Acute GVHD occurred in 3/26 (11.54%)，while chronic GVHD was diagnosed in 6 out of 26 (23.07%) patients. The incidence and degree of infection and hemorrhage were low and slight. It is concluded that NST is a safe and effective therapy for hematological malignancies，whereas related technologies such as adaptation selected，conditioning regimen and transplantation immunotherapy should be studied further.

　　Key words hematopoietic stem cell transplantation; nonmyeloablative allogeneic hematopotetic stem cell transplantation; hematological malignancy

　　As conventional allogeneic hematopoietic stem cell transplantation (HSCT) for patients with hematological malignancies both eradicates the patients’ underlying diseases and suppresses their immune responses，they do not reject the subsequent allografts. The allografts serve to “rescue” patients from the marrow lethal effects of the conditioning regimens. Consequently，the conditioning therapy intensity would be limited only by toxicities to non-marrow organs，such as gut，lung，kidney，heart，and liver. These toxicities have restric-ted HSCT to younger，medically fit patients，with treatment administered in specialized hospital wards. Almost no allogeneic HSCT have been carried out in patients>60 years old，and in only a few patients older than 50 years. Since median ages at diagnoses of patients with acute myelocytic leukemias (AML)，chronic myelocytic leukemias(CML)，chronic lymphocytic leukemias(CLL)，non-Hodgkin lymphomas (NHL)，and multiple myelomas (MM) range from 65-70 years，so that conventional allogeneic HSCT benefit only a minority of patients with candidate disease.

　　Nonmyeloablative allogeneic stem cell transplantation (NST) was first described and applied in 1997 by Giralt and colleages[1-3]. Because of their low toxicity，nonmyeloablative allogeneic stem cell transplantation shows a promise to reduce the morbidity and mortality associated with conventional high-dose chemoradiotherapy and allows allogeneic transplants in elderly or medically infirm patients who are not eligible for conventional myeloablative HSCT. So it has been increasingly explored as a safe alternative to conventional high-dose transplantation regimens.

　　We analyzed NST with its adaptation，conditioning regimen，engraftment，immunotherapy and complications，such as GVHD，in details in 26 patients with hematological malignancies who underwent NST from September 1998 to September 2002.

　　Materials and Methods

　　Patient characteristics

　　During the period from September 1998 to September 2002，26 patients were diagnosed as hematological malignancies and treated with NST in our department. Of these patients，male and female were 15 and 11，respectively. Ages ranged from 19 to 58 years (median，40). Ten patients had acute leukemia (3 of B-cell acute lymphoblastic leukemia; 7 of acute myelocytic leukemia，i.e. 3 of M2a，l of M4a，3 of M5a，respectively). Fourteen patients had chronic myelocytic leukemia (11 in chronic phase，2 in accelerated phase，1 in blast phase). Two patients suffered from multiple myeloma.

　　Patient status before transplantation

　　The patients with acute leukemia were all in the first complete remission but one patient with B-cell acute lymphocytic leukemia and one M2a in the second complete remission. Those with chronic myelocytic leukemia were all in the first chronic phase but 1 case was in blast phase and 2 cases were in the second chronic phase. All 2 patients with multiple myeloma showed partial remission.

　　Laboratory parameters

　　Serum cytomegalovirus(CMV)-IgM antibody was determined by enzyme linked immunosorbent assay (ELISA)，serum CMV-DNA was detected by polymerase chain reaction (PCR) in some patients and the results were negative. HBsAg，HBeAg and HBcAb tests were positive in l patient，HBeAb and HBcAb tests were positive in 2 patients. Human leukocyte antigen (HLA)-identification was carried out by serological examination for class I and PCR-SSP for class Ⅱ.

　　Donor

　　The donors were 13 males and 13 females，age ranged from 22 to 52 years (median 38). All donors were sibling but 1 was the patient’s father. Out of these donors，1 was positive in HBsAg，HBeAb and HBcAb. 17 donors were HLA-identical matched，4 were single locus mismatched，4 were two loci mismatched，1 was three loci-mismatched. 11 were the same ABO blood type and 15 were different (7 major incompatible，8 minor incompatible ).

　　Methods of transplantation

　　1 patient was treated by bone marrow transplantation，7 received peripheral blood stem cell transplantation combining with bone marrow transplantation，18 underwent peripheral blood stem cell transplantation.

　　Mobilization，separation and collection of peripheral blood stem cell

　　(1) G-CSF 300 μg，s.c. twice daily; (2) G-CSF 300 μg plus GM-CSF 300 μg，s.c. once a day. Collection started from the fifth day of mobilization with blood cell separator (leukapheresis) known as Cobe Spectra for 2-3 times.

　　Number of MNC and CD34+ cells infused

　　The number of mononuclear cells (MNC) was (4.15-16.28)×108/kg of recipent weight (median，6.87±2.93×108/kg). The number of CD34+ cells determined by flow cytometry was (3.62-19.32)×106/kg (median，5.05±4.3×106/kg).

　　Conditioning regimen

　　(1) 14 patients received fludarabin 50 mg/d for 5 consecutive days (day 9 to 5 pre-transplantation)，anti-T lymphocyte globulin (ATG) 10 mg/(kg•d) for 4 consecutive days (day 7 to 4 pre-transplantation)，cyclophosphamide 30 mg/(kg•d) on day 4 to 3 pre-transplantation[3]; (2) 12 patients underwent melphalan/busulfan 2 mg/(kg•d) on day 8 to 5 pre-transplantation，ATG 10 mg/(kg•d) on days 7 to 4 pre-transplantation (24 patients received the products of Wuhan Institute of biological preparations，2 were treated with IMTIX-SANGSTAT made in France)，cyclophosphamide 30 mg/(kg•d) on day 4 to 3 pre-transplantation.

　　Donor lymphocyte infusion (DLI)[4-5]

　　24 patients were eligible for DLI given once every 4 weeks in graded incresments according to the chimeric formation and clinical reaction. Generally，the dose of the first infusion was 1×107/kg in 4th week post-transplantation. 2 patients underwent donor stem cell infusion (DSI，donor's peripheral blood stem cell infusion) also once every 4 weeks in graded incresments with first infusion of 1×107/kg. All patients need to be infused regularly for 2-3 times.

　　Efficacy assay

　　(1) Short tandem repeats (STRs) [6] assay was on day 10 and 28 post-transplant，followed once a month; (2) HLA-locus analysis; (3) bcr/abl fusion gene and Philadephia chromosome for CML patients bimonthly or trimonthly; (4) Sex chromosome; (5) ABO blood type.

　　Follow-up

　　Patients were regularly followed-up after transplantation to assess disease response and remission status. To November 2002，the follow-up time was 1 month to 30 months (median，21 months). Up to now，13 patients are still in disease-free survival.

　　Results

　　Side effects of hematopoietic system and reconstitution of hematopoietic function

　　The clinical data are listed in Table. The absolute neutrophil counts (ANC) of 26 patients post-transplantation decreased to 0.5，0.2 and 0.1×109/L，respectively. The incidences of ANC approaching to zero were 69.23%，38.46%，57.69%，11.53% respectively. The median time was 6.39，7.20，7.27 and 7.67 days respectively. The incidence of platelet (Plt) constantly dropping to 20×109/L was 57.69% for 0-16 days. The ANC and Plt of 25 patients restored to 0.5 and 20×109/L from 15.5 and 20.57 days post-transplant respectively. Only a patient with CML-CP can not be restored and died of pulmonary infection.

　　Engraftment

　　Twenty-two patients (84.62%) were engrafted，including CML-CP1 (n=10)，CML-CP2 (n=2)，ALL-CR1 (n=2)，M2a (n=2)，M4a-CR1 (n=l)，M5a-CR1 (n=3)，MM (n=2)，who had donor-DNA from 20% to 100% detected by STRs on 1 month post-transplantation，with 18 patients being full donor chimerism (FDC) up to now. Of these patients，15 cases were HLA-matched (88.24%)，4 cases were single-locus mismatched (100%)，3 cases were two-loci-mismatched (75%). 4 patients failed to be engrafted，including ALL-CR1 (n=l) who was HLA-matched，CML-BC (n=l) who was also HLA-matched，M2a-CR2 (n=l) whose stem cells were donated by his father with three-loci mismatched，CML-CP1 (n=l) who was two-locus mismatched.

　　GVHD

　　Acute GVHD (aGVHD) occurred in 3 patients (1154%)，including 1 patient diagnosed as CML-CP1 and performed by MAC preparative regimen，1 M2a-CR1 patient given FAC conditioning regimen and 1 MM patient received FAC conditioning regimen. The first case of aGVHD grade IV in liver occurred on day 30 after transplantation and reoccurred on day 90 after a first DLI，then was controlled by high-dose methylprednisolone and developed chronic GVHD (cGVHD) grade Ⅱ on mucosa and skin. The second case of aGVHD grade Ⅱ on mucosa and skin occurred on day 60 after transplant. The last case of extensive exfoliative dermatitis (aGVHD grade IV) occurred on day 55

　　Note:-represents pre-transplantation，+ represents post-transplantation.Table . Hematopoietic side-effects and reconsititution of 26 cases of hematological malignanies after-transplantation(略)

　　after transplantation because of stopping oral CsA for 2 weeks. He responded promptly to high-dose methyl-prednisolone，but died of progressive dyspnea after 24 hours. All three patients were HLA-matched，whose donors were females，in particular，the last two were 50 and 46 years old respectively. cGVHD occurred mainly on skin and mucosa in 6 patients (23.07%) composed of 2 transformed from aGVHD，CML-CP1 (n=1)，CML-CP2 (n=l)，M4a-CR1 (n=l) and B-ALL-CR1 (n=l)，out of which aGVHD of 1 patient occurred in liver and another in bulbar conjunctiva. Out of the last 4 patients，3 cases received FAC conditioning regimen and 1 MAC，3 cases were HLA-matched and 1 was single-locus mismatched，whose donors were 2 males and 2 females.

　　Infectious complications

　　Staphylococcus aureus infection occurred by catheter in 1 patient，pulmonary staphylococcus aureus infection occurred in 2，all the strains of staphylococcus aureus are sensitive to antibiotics and were controlled by them. One patient suffered from frequent micturition，urgency of urination and urodynia，with severe hyperemia，edema，bladder contracture observed by cystoscopy and CMV negative in serum and urine. Fortunately，these symptoms were relieved by intravenous kenai and γ-globulin. Another patient died of asphyxia on 9 months post-transplantation because of slight interstitial pneumonia evidenced of thoracic CT with CMV negative in serum，urine，sputum but no sensitivity to kenai，ganciclovir and dasansu.

　　Transfusion

　　Platelets less than 20×109/L did not occur in 11 patients who need no platelet transfusion.

　　Complications of DLI

　　One patient was rescued from acute dyshematopoiesis by cytokines after first DLI，and the other had aGVHD in liver after first DLI.

　　Discussion

　　Nonmyeloablative allogeneic hematopoietic stem cell transplantation (NST) is a novel method in an effort to cure hematological malignancies by reduced-intensity chemoradiations，which have been designed to immunosuppress recipients’ immune responses and to permit allogineic hematopoietic stem cells engraftment but li-mited systemic toxicity，and by using immunosuppressants the post-transplants that form chimerism attain double immune tolerance between hosts and grafts and to induce graft versus tumor (GVT) effects to eradicate tumor cells in the hosts or genetically abnormal hematopoietic cells. Most scholars hold that GVT is the main mechanism of radically curing hematological malignancies following the evaluation on the effects of hematopoietic stem cell transplantation for several decades. Therefore，NST is a brave and more rational reform than myeloablative transplantation on conditioning regimens that decrease hematopoisis toxicity but increase immunosuppressive effect. Furthermore，NST enhances immunotherapy during the whole transplant to induce immune tolerance between donors and recipients and enhance the GVT effects.

　　Giralt et al[1] firstly started the clinical study of NST in 1997 that consisted of various diseases，such as ALL，AML，CML，CLL，MM，lymphoma and MDS，and the subjects were elder，infirm and not eligible for conventional myeloablative HSCT. The results showed the higher engraftment and lighter side effects than that in myeloablative HSCT. In our study，12 patients with CML (86.70%)，2 patients with MM (100%) and 8 patients of AL (80%) were engrafted，suggesting that NST is also suitable for AL in stable phase as well as CML and AML in slow progression. Furthermore，NST is a safe and effective therapy for hematological malignancies especially for those in stable phase and advanced in years or in poor performance status.

　　Our study showed that (1) the engraftment of those in unstable status was lower. 20 patients in stable phase，2 with CML-CP2 were all significantly engrafted (P=0.014); (2) the engraftment of those two-loci mismatched was as low as 75%. However，single locus mismatched had few effects on the engraftment that was 100% of engraftment as compared with HLA-matched patients (P=l.000，0.489，respectively); (3) the ages of donors and recipients had no apparent effect on the engraftment (P=1.000); (4) there was no significant difference in engraftment between the FAC groups and the MAC groups (P=0.306).

　　There was no remarkable variation in the incidence of GVHD between two conditioning groups (P=0.391)，demonstrating that NST may reduce the difference on the occurrence of GVHD between different groups because of relative low toxicity of conditioning regimen. Three patients with aGVHD were HLA-matched whose donors were all females，which may correlate with aGVHD (P=0.063). In addition，all these 3 patients were full donor chimerism for STRs on days 28 after transplantation，which may be one of the main reasons for the occurrence of aGVHD. The main influenced factors for GVHD in our study were not definite but the results at least showed that there was no significant association between GVHD and the ages of donors and recipients，sexes，performance status，the degree of HLA-matching.

　　The mixed transplantation of peripheral blood stem cells combined with bone marrow stem cells was used in 7 patients with insufficient PBSC，with 500-750 ml bone marrow aspirate being collected in each of 6 donors as well as 1 000 ml of aspirate in another. Six patients were engrafted (85.67%)，with a 56-year-old patient with CML for 7 years and developing into accelerated phase for 3 years and becoming full donor chimerism on day 28 post-transplant.

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