Abstract

BACKGROUND: There is no consistently effective therapy for patients with steroid-refractory acute graft-versus-host disease (GVHD). A variety of alternative approaches have been tested, including antithymocyte globulin, mycophenolate mofetil (MMF), pentostatin, and monoclonal antibodies; however, these treatments have been only modestly successful.
OBJECTIVE: To further evaluate the efficacy of human adipose-derived stem cells (ASCs) as the salvage therapy for steroid-refractory acute GVHD.
DESIGN: A clinical trial.
SETTING: Department of Haematology, Henan Institute of Haematology, Henan Tumor Hospital.
PARTICIPANTS: The clinical trial was performed at the Henan Institute of Haematology from September 2002 to August 2005. Eight patients were treated with ASCs for grades Ⅲ-Ⅳ steroid-resistant acute GVHD. The study was approved by the Ethics Committee at Henan Tumor Hospital and informed consent was obtained from patients and ASCs donors before they enrolled.
METHODS: Eight patients with steroid-refractory grades Ⅲ-Ⅳ acute GVHD received intravenous infusions of ASCs. The ASCs dose was 1.0×106/kg. Seven patients were treated once and one patients twice. Four patients received ASCs from haplo-identical family donors and four from unrelated mismatched donors.
MAIN OUTCOME MEASURES: The efficacy of human ASCs as the salvage therapy for steroid-refractory acute GVHD.
RESULTS: No side effects were noted after the ASCs infusions. Acute GVHD disappeared completely in seven of eight patients and six of these seven patients are still alive after the median follow-up of 30 months (range 11-90 months) after the initiation of ASCs therapy. All four surviving patients were in good clinical condition and in remission of their hematological malignancy. Two patients died-one with no obvious response to ASCs died of multiorgan failure and one of relapse of leukemia.
CONCLUSION: These results suggest that ASCs is a very promising treatment for severe steroid-resistant acute GVHD.

INTRODUCTION

Corticosteroids are the backbone of most treatment regimens for acute graft-versus-host disease (GVHD), but fewer than 50% of patients show a durable response to corticosteroids[1]. Steroid-resistant acute GVHD is associated with high morbidity and mortality and is extremely difficult to manage[2-5].
Ex vivo expanded mesenchymal stem cells (MSCs) have been infused in phase Ⅰ studies to reduce neutropenia after autologous hematopoietic stem cell transplantation (HSCT) and allogeneic HSCT (ASCT) to correct inborn errors of metabolism and osteogenesis imperfecta without adverse effects[6-9]. These stem cells are not immunogenic and escape recognition by alloreactive T cells and natural killer cells[10]. MSC given intravenously have been well tolerated[7]. Furthermore, they are immunosuppressive and inhibit the proliferation of alloreactive T cells[11]. Preliminary reports of transplantation of MSC show a reduction in acute GVHD[12,13]. Here we describe our experience of transplanting human adipose-derived stem cells (ASCs) as the salvage therapy for steroid-refractory acute GVHD in a series of eight patients.

SUBJECTS AND METHODS

Subjects
Between September 2002 and August 2005, eight patients who had received an allogeneic related or unrelated HSCT were treated with ASCs for grades Ⅱ -Ⅳ steroid-resistant acute GVHD (Table 1). This study was approved by the ethics committee at Henan Tumor Hospital. Informed consent was obtained from patients and family members before they enrolled. Some necessary steps were taken to insure the protection of the rights of minors (children).

Methods
Conditioning regimen
Conditioning therapy was modified BUCY in HLA-matched sibling transplantations consisting of cytarabine (2 g/m2 per day) intravenously on days -10 to -9; busulfan (4 mg/kg per day) orally on days -8 to -6; cyclophosphamide (1.8 g/m2 per day) intravenously on days -5 to -4; and Me-CCNU (250 mg/m2) orally once on day -3. In HLA-matched unrelated donor transplantations, patients received an identical regimen to the HLA-matched patients, along with antithymocyte globulin (ATG) (thymoglobuline, 2.5 mg/kg per day; Sang Stat, Lyon, France, now marketed by Genzyme, Cambridge, MA) intravenously for 4 consecutive days (days -5 to -2). In a patient given a cord blood transplant, the preparative regimen consisted of fractionated total body irradiation to a total dose of 10 Gy in five doses in three days (days -8 to -6) with lungs shielded at 8 Gy, fludarabine i.v., total dose of 120 mg/m2 (30 mg/m2/day, days -5 to -2),cyclophosphamide i.v., 120 mg/kg total dose (60 mg/kg/day over 1 hour, days -3 and -2) and ATG(Sang Stat, Lyon, France) 30 mg/kg on day -1.

GVHD prophylaxis and treatment
All patients received cyclosporine A (CsA), mycophenolate mofetil (MMF), and methotrexate (MTX) to prevent GVHD. The dose of MTX was 15 mg/m2 intravenously on day +1, followed by 10 mg/m2 intravenously on days +3, +6, and +11 after transplantation. MMF (7.5 mg/kg twice a day) was begun on day -10 and tapered on day +14 in the HLA-matched patients. But in the mismatched patients, tapering of MMF was delayed until beginning on days +30 to +80, based on the presence or absence of severe GVHD, infectious diseases, and relapse risk. The dosage of CsA was 2.5 mg/kg per day intravenously from day 10 before transplantation until bowel function was normal. Then CsA (3.25 mg/kg) was given orally twice a day with trough levels targeted at 150 to 250 ng/mL during the first 40 days and then tapered, taking about 60 days to be fully discontinued in the high-risk patients. Grading and staging of acute GVHD were done according to the consensus conference on acute GVHD grading[14]. The first-line GVHD treatment consisted of a combination of CsA and corticosteroids at a dose of 2 mg/kg of methylprednisolone. Steroid-refractory GVHD was defined as nonresponse to corticosteroids administered for at least seven consecutive days.

ASCs treatment and evaluation of responses
For GVHD that did not respond or that progressed despite the initial treatment, ASCs was administered intravenously at a dose of 1×106/kg. The administration of CsA continued during the ASCs treatment, and corticosteroids were tapered. Responses were assessed for each involved organ. Complete response (CR) was defined as the complete resolution of GVHD. Partial response (PR) was defined as a decrease in organ stage by at least one stage in patients with acute GVHD. No response (NR) was defined as a progressive worsening of GVHD requiring the introduction of additional GVHD treatment.

ASCs preparation
First polymerase chain reaction (PCR) tests was done to determine whether the donor was infected by human immunodeficiency virus, cytomegalovirus, or hepatitis virus, then the suitable donor gave written informed consent, and ASCs were isolated as previously described[15]. Briefly, subcutaneous abdominal adipose tissue obtained from the donor undergoing plastic surgery (lipectomy) was digested with 0.2% collagenase Ⅱ (Sigma, St. Louis, MO, USA) for 30 minutes under constant shaking. After removal of the floating mature adipocytes and erythrocytes, the lower layer was centrifuged (200 g, 10 minutes). After successive filtrations through 100- and 70-μm sieves, the cells were washed with phosphate buffered saline /2% fetal calf serum (FCS; Gibco Life Technologies, Paisley, UK) for two times and then plated in in polystyrene flasks at a density of 2×106/mL. Selective expansion medium contained 57% D-MEM/F-12 (Gibco), 40% MCDB-201 (Sigma), 2% FCS (Gibco), 1×insulin transferrin selenium (Gibco), 1× insulin transferrin selenium (Gibco), 10-9 mol/L dexamethasone (Sigma), and 10-4 mol/L ascorbic acid 2-phosphate (Sigma), 10 ng/mL epidermal growth factor (Sigma), 10 ng/mL PDGF-BB (Sigma), 100 U/mL penicillin, 1 000 U/ mL streptomycin (Gibco). Once adherent cells were more than 70% confluent, they were detached with 0.125% trypsin and 0.01% EDTA (Sigma) and expanded under the same culture conditions. The culture-expanded cells were assayed in a flow cytometer (FACSort, Becton Dickson, San Jose. CA, USA) and the data analyzed with Cellquest software (Becton Dickinson). As we previously described[14], these cells expressed fetal liver kinase, CD166, CD105, CD44, CD29, and HLA class Ⅰ but not CD34, CD45, CD14 or HLA class Ⅱ. The cultured cells before infusion were tested time and again for bacteria, mycoplasma, and fungi by PCR assays, and the results were negative. In addition, the result of enzyme-linked immunoassay to measure the contamination by endotoxin/LPS was also negative.

RESULTS

ASCs as salvage treatment for steroid-refractory acute GVHD
Patient 1 was a 42-year-old male with acute myeloid leukaemia (AML) in first remission had an ASCT from his HLA-identical sister. On day +41 post transplant, he was admitted with grade-Ⅳ acute GVHD of liver, confirmed by liver biopsy. He was treated with methylprednisolone 2 mg/kg intravenously daily. He failed to respond and 10 days later had pulsed methylprednisolone 1 g daily for 5 days. His liver function tests (LFTs) deteriorated. At this stage, he was treated with ASCs intravenously 1×106 ASCs per kg of the patient's weight on day +72 and the steroids were tapered rapidly over 5 days. Approximately 3 days following ASCs treatment, LFTs started to improve. His ALT, ALP and bilirubin normalised on day +87. He is now alive and well 90 months after ASCs therapy.
Patient 2 was a 22-year old man with AML in second remission received a transplant of HSC from his HLA-identical elder brother. After transplant, his hematopoietic reconstitution was rapid, with absolute neutrophil count > 0.5×109 L-1 on day +13, and platelet count > 20×109 L-1 on day +18. However, the patient began to develop diarrhea and skin rash on days +21 and +29, respectively, while receiving a relatively high dose of methylprednisolone (2 mg/kg intravenously daily). The diagnosis of GVHD (grade Ⅲ) was pathologically confirmed by examining the colonoscopy. Methylprednisolone (500 mg/day), infliximab (10 mg/kg), and daclizumab (1 mg/kg) were ineffective. MMF, tacrolimus, and MTX were also tried. In conclusion, this patient seemed to be unresponsive to all types of immunosuppression. At the same time, he was treated for repeated bacterial, viral, and invasive fungal infections. A dose of 1×106 ASCs per kg of the patient's weight was given intravenously on day +64. A significant improvement of skin and liver disease were observed after 3 days and stool normalized after twelve days. He was discharged from the hospital on day +107. He shows no GVHD after 29 months.
Patient 3 was a 39-year-old male had an ASCT for Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL) in first remission. He received a transplant of HSC from an HLA-identical unrelated male donor. On day +26 post transplant, he developed grade-I acute GVHD of the skin, which was treated with methylprednisolone 2 mg/kg intravenously daily for 5 days followed by oral methylprednisolone, which was gradually tapered. He developed grade-Ⅳ acute GVHD of liver 7 months post transplant on CsA taper. His LFTs at that time were as follows: total bilirubin 150 μmol/L, ALT 428 U/L and alkaline phosphatase (ALP) 597 U/L. He was treated with methylprednisolone 2 mg/kg intravenously daily. His LFTs showed a brief improvement before deteriorating further, total bilirubin 209 μmol/L, ALP 712 U/L, and ALT 596 U/L. On day +242, colonoscopy showed grade Ⅳ acute GVHD. Total bilirubin increased to 310 mmol/L. On day +243, he got a dose of 1×106 ASCs per kg of the patient's weight. A decline in total bilirubin, ALP and ALT was noted 6 days after the ASCs transplantation. At the last follow-up 2 years post ASCs therapy, his LFTs are normal. He continues to be in molecular remission and has had no major infective complications following ASCs treatment.
Patient 4 was a 38-year-old female with ALL in second remission received a transplant of hematopoietic stem cells (HSC) from an HLA-identical unrelated female donor. She developed grade Ⅳ acute GVHD of the skin, gut and liver on day +62 post transplant. She showed no response to all types of immunosuppression as reported [12]. She was treated with ASCs on day +69 and his GVHD improved markedly at all occasions. Colonoscopy showed 8% male epithelium using fluorescent in situ hybridization (FISH). However, on day +116, the patient again had diarrhea and mild abdominal pain. Colonoscopy showed mild-to-moderate acute GVHD. She received a transplant of the same batch of ASCs from the donor (1×106/kg) on day +127. After 1 week, her stools were normal and she started to eat again. Bilirubin and serum alanine aminotransferase (ALT) also returned to normal 2 weeks later. She was discharged from the hospital on day +151. Now she is well 18 months after ASCs therapy and has no minimal residual disease in blood and bone marrow.
Patient 5 was a 41-year-old female with AML in second remission received an ASCT from her HLA-identical sister. On day +27, the patient developed a maculopapular rash of the thorax and back that progressed despite treatment with methylprednisolone (2 mg/kg intravenously daily). On day +42, the patient developed severe diarrhea (> 1 500 mL/day) and abdominal pain requiring morphine. Then she stopped eating and was treated with total parenteral nutrition (TPN). Colonoscopy showed IV acute GVHD. Infliximab (10 mg/kg) and daclizumab (1 mg/kg) were ineffective. A dose of 1×106 ASCs per kg of the patient's weight were given on day +76. A complete resolution of diarrhea and significant improvement of skin rash were observed. Briefly, Skin rash disappeared in ten days and stool normalized after two weeks. The steroids were rapidly tapered. However, thirteen months after the HSCT, she died in hospital because of relapse of leukemia.
Patient 6 was a 49-year old femal with ALL in second remission received a transplant of HSC from an HLA-identical unrelated male donor. On day +12, he developed a skin rash and on day +14, diarrhea. The diagnosis of GVHD (grade Ⅲ) was pathologically confirmed by examining the skin biopsy and colonoscopy specimens. Methylprednisolone (2 mg/kg intravenously daily for 10 days) was given, but there were no responses. Methylprednisolone (500 mg/day), infliximab (10 mg/kg), and daclizumab (1 mg/kg) were also ineffective. Infusion of 1×106 ASCs per kg of the patient's weight was given intravenously on day +56 had no effect on diarrhea. On day +70, he got 1×106 ASCs per kg of the patient's weight. There was just a transient improvement of skin rash. At this stage, she developed systemic fungal infections and hemorrhagic cystitis. He died 16 days later. Autopsy showed multiorgan failure.
Patient 7 was a 15-year-old boy with Ph-positive ALL in first remission. He received a cord blood transplant from an HLA-identical unrelated donor. He was discharged from the hospital on day +72. On day +89, the patient was readmitted because of deterioration of general condition, gastrointestinal discomfort with persistent diarrhea, and significant weight loss. During the first week of hospitalization, he had watery diarrhea, with a stool volume ranging from 600 to 900 mL/day. Stool culture was negative for bacterial and fungal infections, and the patient was started on methylprednisolone 2 mg/kg/d for 2 weeks while CsA was switched from oral to intravenous administration. At the same time, he stopped eating and was treated with TPN. The patient did not to respond to the initial treatment. On day +106, the patient developed severe diarrhea (> 1 500 mL/day) and abdominal pain requiring morphine. Colonoscopy showed moderate-to-severe acute GVHD. Methylprednisolone (500 mg/day), infliximab (10 mg/kg), and daclizumab (1 mg/kg) were ineffective. Colonoscopy on day +146 showed IV GVHD. A dose of 1×106 ASCs per kg of the patient's weight were given on day +147. Over the ensuing 2 weeks, a significant improvement of diarrhea was observed. Briefly, Diarrhea decreased from 10-16 times daily (> 1 200 mL/day) to 1-2 times daily (< 150 mL/day). On day +164, his stools normalized and TPN was discontinued. His general condition improved, as demonstrated by significant weight gain (from 39 kg before ASCs administration to 46 kg afterward). Histopathologic improvement was documented by endoscopy on day +168: erosions and apoptotic bodies of villi detected at the time of the first biopsy completely disappeared, but a modest decrease of crypts was still present. He was discharged from the hospital on day +187. Now he is well 341 days after ASCs therapy and has no minimal residual disease in blood and bone marrow.
Patient 8 was a 12-year-old girl with AML in first remission had an ASCT from an HLA-identical unrelated female donor. On day +13, she developed grade-Ⅰ acute GVHD of the skin, which was treated with methylprednisolone 2 mg/kg i.v. daily for 6 days followed by oral prednisolone, which was gradually tapered. She developed grade- Ⅲ acute GVHD of liver on day +62 post ASCT on CsA taper. Her LFTs at that time were as follows: total bilirubin 143 μmol/L and serum ALT 598 U/L. She was treated with methylprednisolone 2 mg/kg intravenously daily. Her LFTs just showed a brief improvement. Tacrolimus and cyclophosphamide were ineffective. Methylprednisolone (500 mg/day), infliximab (10 mg/kg), and daclizumab (1 mg/kg) were also tried. On day +109, liver biopsy showed grade Ⅳ acute GVHD. On day +110, she got a dose of 1×106 ASCs per kg of the patient's weight. She tolerated ASCs without major side effects and her LFTs gradually improved over the next two weeks, normalizing in 3 weeks post treatment. At the last follow-up 2 years post ASCs therapy, her LFTs are normal. She continues to be in molecular remission and has had no major infective complications following ASCs treatment.

Patient outcomes
The patients' outcome are summarized in Table 1.The overall proven response rate was 7/8. Seven patients showed prompt and complete responses, one developed systemic fungal infections. One patient responded twice in liver and gut. Two patients died-one with no obvious response to ASCs died of multiorgan failure and one of relapse of leukemia.

DISCUSSION

Effective treatment for GVHD, which is a significant cause of morbidity and mortality after HSCT, is needed to improve short-term and long-term results of ASCT. The combination of CsA and corticosteroids is currently considered the standard first-line treatment[5]. Patients who fail on standard therapy have poor prognosis. However, currently, there is no standard second line therapy for refractory GVHD. Several candidate drugs have been already tested [16-20]. In addition to age, which is a significant risk factor for GVHD, elderly patients are likely to have several comorbidities, and thereby be more likely to suffer from the side effects of corticosteroids. Therefore, strategies aimed at minimizing the duration of corticosteroids treatment are obviously needed.
MSC may be derived from adult bone marrow, adipose, and several fetal tissues[11,15,21-24]. They escape the immune system in vitro, and this may make them candidates for cellular therapy in an allogeneic setting[25,26]. They also have immunomodulatory effects, inhibit T-cell proliferation in mixed lymphocyte cultures[27-29], prolong skin allograft survival[27,28], and may be therapeutic for the control of GVHD[26,30,31]. Recently, we used ASCs successfully in a 38-year-woman with grade-IV refractory acute GVHD of the gut and liver following ASCT for ALL[12]. Le Blanc et al[13] used bone marrow derived-MSC successfully in a 9-year-old boy with grade-IV refractory acute GVHD of the gut and liver following ASCT for ALL. Ringden et al[32] used ASCs in eight patients (two AML, Three ALL, two solid tumors and one myeloma) with steroid refractory acute GVHD (mainly liver and gut) following ASCT, and six of eight patients showed dramatic response with resolution of both liver and gut GVHD within two weeks. In these three case reports mentioned above, several effects have been noted. Firstly, no side-effects were noticeable after the infusion of MSC; secondly, MSC dramatically affected tissue repair of severe acute GVHD of the gut, and it could be proven that the donor-derived epithelial cells in the colon detected by fluorescence in situ hybridization; thirdly, skin rash disappeared soon after MSC infusion; fourthly, MSC reduced corticosteroid requirements, thereby reducing the risk complications due to iatrogenic immunosuppression. Furthermore, MSC administration did not appear to carry an increased risk of infection in patients that are already immunocompromised. Looking at the data in published studies[18, 33-35], it seems that the results of MSC treatment are at least comparable to those achieved with other drugs in terms of overall survival. Thus, in ASCT, MSCs may be used for hematopoiesis enhancement, as GVHD prophylaxis, and for the treatment of severe acute GVHD.
Our results imply that ASCs can be efficiently used in some cases with steroid-refractory GVHD. Briefly, seven of eight patients responded to ASCs administration (87.5%) with seven achieving complete responses, while the incidence of infectious complications was relatively low in our patients (1 event in eight patients), and this was better than some pharmacological agents like mycophenolate mofetil and antithymocyte globulin[36-38]. The lack of a comparison group precludes any definite conclusions regarding the incidence of infectious complications in patients receiving ASCs vs other GVHD treatments. However, in our study, ASCs administration did not appear to carry an increased risk of infection in patients that are already immunocompromised. Regular monitoring of CMV by PCR and antimicrobial prophylaxis during ASCs treatment should be recommended.
In conclusion, in view of the dismal outcome in patients with acute steroid-refractory GVHD, ASCs can be used safely and has encouraging efficacy, although it should be emphasized that our results may have been influenced by the small number of patients studied, and therefore should be considered suggestive rather than definitive. We wanted to report the positive effects using ASCs for treatment of life-threatening steroid-refractory acute GVHD at an early stage, because of the very poor outcome in such patients. We felt it was urgent to alert physicians treating such patients of this possibility.

ACKNOWLEDGEMENT

We want to thank the staff at the Center of Excellence in Tissue Engineering, Institute of Basic Medical Science &School of Basic Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College.

REFERENCES

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成人脂肪源间充质干细胞治疗顽固急性移植物抗宿主病*☆

房佰俊，李 宁，宋永平，张龚莉，林全德，魏旭东
房佰俊☆，男，1975年生，河南省南阳市人，汉族，2004年协和医科大学毕业，博士，副主任医师，主要从事干细胞与组织工程实验与临床研究。
通讯作者：宋永平，博士，河南省肿瘤医院、河南省血液病研究所，河南省郑州市 450008
河南省杰出青年基金(0612000900)*
摘要
背景：目前为止，医学界仍然没有一种有效的策略治疗对糖皮质激素耐药的、严重的急性移植物抗宿主病；很多药物包括抗胸腺细胞球蛋白、霉酚酸酯、喷司他丁及单克隆抗体等均已经临床尝试，但疗效均不尽人意。
目的：进一步评价成人脂肪源间充质干细胞作为挽救方案用于治疗对糖皮质激素耐药的急性移植物抗宿主病。
设计：临床实验。
单位：河南省肿瘤医院，河南省血液病研究所血液科。
对象：实验于2002-09/2005-08在河南省血液病研究所完成，经河南省肿瘤医院医学伦理委员会批准。共有8例出现Ⅲ~Ⅳ度对糖皮质激素耐药的急性移植物抗宿主病的患者入选本实验，患者及脂肪源间充质干细胞供者对治疗及实验均知情同意。
方法：8例对糖皮质激素耐药的Ⅲ~Ⅳ度急性移植物抗宿主病患者患者均经静脉输入成人脂肪源干细胞（剂量为1.0×106/kg），其中1例患者接受两次成人脂肪源干细胞输注，其余7例患者均接受一次成人脂肪源干细胞治疗。在这8例患者中，4例患者接受的成人脂肪源干细胞来源于HLA配型完全相合的家庭供者，其余4例患者接受的成人脂肪源间充质干细胞来源于无血缘关系的无关供者。
主要观察指标：成人脂肪源间充质干细胞用于治疗对糖皮质激素耐药的急性移植物抗宿主病的疗效。
结果：所有接受成人脂肪源间充质干细胞治疗的8例患者均无明显副作用出现；在接受成人脂肪源干细胞治疗的8例患者中，除1例患者（后死于多器官功能衰竭）对治疗无反应外，另7例患者的病情（对糖皮质激素耐药的急性移植物抗宿主病）很快得以缓解；在对成人脂肪源干细胞治疗反应良好的7例患者中，有6例患者存活11~90个月,中位时间30个月，其中4例患者目前仍处于血液学完全缓解状态且生存状况良好，另1人在接受成人脂肪源干细胞治疗后13月后死于白血病复发。
结论：成人脂肪源间充质干细胞非常有希望用于治疗对糖皮质激素耐药的急性移植物抗宿主病。
关键词：脂肪源间充质干细胞;抗宿主病;治疗
中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2008)03-00587-06
房佰俊,李宁,宋永平,张龚莉,林全德,魏旭东.成人脂肪源间充质干细胞治疗顽固急性移植物抗宿主病[J].中国组织工程研究与临床康复，2008，12(3):587-592
[www.zglckf.com/zglckf/ejournal/upfiles/08-3/3k-587(ps).pdf]
(Edited by Asok Mukhopadhyay/Ji H/Wang L)