| Background and ObjectiveAplastic anemia (AA) is a life-threatening bone marrow failure. It first reported by Ehrich in1888. Chauffard presented the name "aplastic anemia" in1904.The characteristic of this disease is extremely low bone marrow hyperplasia. There are two or three manifestations in the peripheral blood should meet the following criteria: neutrophils<0.5x109/L, platelet<20x109/L, reticulocyte<1%. The treatment of severe aplastic anemia (SAA) to the disease itself is very important, apart from supportive care like blood transfusion supporting and infection control. Now the main treatment for SAA is immunosuppressive therapy (1ST) using the combination of antithymocyte immunoglobulin (ATG) and ciclosporin A (CSA) allogeneic hematopoietic stem cell transplantation (allo-HSCT).1ST is effective therapy for severe aplastic anemia. It is main applying in non-severe aplastic anemia that depending on blood transfusion, SAA or very severe aplastic anemia (VSAA) patients who were older than40years of age and patients who were younger than40years of age without matched sibling donor (MSD).IST usually takes3-6months to response. It also has a small number of patients are enforced delayying to1year. The response rate of single drug in IST is30%～45%, but significantly improved in combination drug therapy. Currently ATG+CSA is the standard treatment. It is associated with response rates of50%to80%, with current5-year survival rates of75%to85%. Allogeneic hematopoietic stem cell transplantation is one of the effective therapy of severe aplastic anemia.Human leukocyte antigen(HLA)-matched allogeneic hematopoietic stem cell transplantation is recommended as the first-line therapy for SAA or VSAA patients younger than40years. The long-term survival rate is70%～90%. Cyclophosphamide (Cy)-based conditioning regimen with antithymocyte globulin has been adopted in the majority of HSCT for severe aplastic anemia patients. It is reported that the engraft rate was84%—95%and the long-term survival rate was70-90%if adopted this conditioning regimen in recent years. However graft rejection rates of HSCT for severe aplastic anemia with cyclophosphamide-based conditioning regimen still ranged from5%～16%. This is one of problems which troubled clinical studies in hematopoietic stem cell transplantation of severe aplastic anemia in recent years. So how to increase the rate of sustained engraftment and decrease the rejection after transplantation for severe aplastic anemia is still worthy to be studied. The reasons of graft failure and rejection for severe aplastic anemia may be include donor and receptor failed in building immune tolerance, serious infection, the influence of acute graft versus host disease and chronic graft versus host disease, the number of stem cells infused and immunosuppressive therapy before transplantation. But the fact that patients with graft failure and rejection could be successful grafted after more intensity conditioning regimen in second transplantation prompts us that more intensive immunosuppression in conditioning regimen could decrease the graft failure for severe aplastic anemia. So could it improve the engraftment and reduce rejection after transplantation in allo-HSCT for severe aplastic anemia if increase the immunosuppression in cyclophosphamide-based conditioning regimen? Fludarabine (Flu) was added in cyclophosphamide-based conditioning regimen to enhance the immunosuppressive for22cases of severe aplastic anemia patients in allo-SCT. The purpose is to observe the impact of adding fludarabine to the Cy-based conditioning regimen on engraftment of hematopoietic stem cells and analyze the long-term survival and transplantation-related complications of patients.Methods The research object:From January2000to June2011, twenty-two patients with severe aplastic anemia received allogeneic bone marrow and peripheral blood stem cell transplantation or simply peripheral blood stem cell transplantation at the Department of Hematology, Zhujiang Hospital, Southern Medical University. Among the22patients, twelve were males, ten were females. The patients with a median age of19.5years (ranging from5to52years old), underwent allo-HSCT from HLA matched (N=14) or unmatched (N=8) related donors. The median time of transplant following diagnosis was five months (ranging from1to125months). Twelve patients received a variety of immune inhibitors therapies, including cyclosporine before transplantation. There were no patients using ATG. Twelve were ABO-compatible and ten were ABO-incompatible (two were major blood-group incompatible, six were minor blood-group incompatible, two were major and minor blood-group incompatible). Male to female were7cases, female to male were8cases, parents to their children were4cases. The severe aplastic anemia patients who were free of significant functional deficits in major organs and without serious fungal and bacterial infection were included in this study.The selection of the donors and HLA matching:We choose the healthy immediate family members of patients with severe aplastic anemia. We use sequenceing-based typing (SBT) testing for HLA matching. The degree of HLA matching consistency was the main selection criteria for the donors. We choose relatives of patients with HLA matching at least5/10as donors. Blood type consistency or not was not exclusion criteria for the donors. But we choose the one with the same blood type as donor when they have the same degree of HLA matching consistency.Conditioning Regimen:All patients accepted that FBCA conditioning regimen. The conditioning regimen composed of fludarabine (30mg/m2/dx5d), busulfex(3mg/kgx2d, between2000and2004patients received drug by oral way, after2004patients received drug by intravenous way), cyclophosphamide (60mg/kg/dx2d) and ATG (2.5mg/kg/d×5d, Rabbit Anti-human Thymocyte Immunoglobulin, Imtix-Sangstat, France).Hematopoietic stem cell mobilized, collected and transfused:The donors were all injected with G-CSF (lO^g/kg body weight/day) subcutaneously twice daily for5-6consecutive days. On day4, bone marrow stem cells were collected. On day5, leukaphereses were performed via a blood cell separator (CS3000plus) using ACD-A as the anticoagulant. The circulating blood volume was10-12L each time. The median infused cell dose of nucleated cells and CD34-positive cells of22patients was7.8(3.1-13.9) X108/kg(weight of recipient) and8.0(4.2～13.5)×106/kg.GVHD prevention:Graft-versus-host disease prevention post-transplantion composed of CSA, short-range methotrexate (+1d15mg/m2,+3d,+6d,+11d10mg/m2). Cyclosporine was administered intravenously at a dose level of3-4mg/kg daily, beginning on day-1, with subsequent adjustment to maintain a trough level between100and300ng/ml. Methylprednisolone (1～2mg/kg·d) or other immunosuppressors were used to treat acute graft-versus-host disease. Some patients with serious acute graft-versus-host disease could infuse mesenchymal stem cells to deal with.The prophylaxis of HC:During the conditioning, we use hydrantion、 alkalined the urine、mesna、diuretics to prophylaxis HC.The prophylaxis of HVOD:Heparin(100U/Kg/day) and prostaglandin E1were begun to use at the first day of conditioning, and with the monitor of liver function and APTT.It would be stop at day30after transplant,if there was no symptom of HVOD.The prevention of infection:Before transplantation, the patient need to visit the oral、ophthalmic、ENT、anorectal、obstetrics and gynecology department for consultation, and clear out the infection that may exist. Before the transplantation, the patients need to take oral antibiotics to clean out the intestinal tract, and take ganciclovir to prophylaxis viral infections. All patients have to admit to the Laminar flow ward. The staff and nursing staff need well disinfection. Use antibiotics to prevent the infection in the agranulocytosis period after transplantation.The chimerism status observation:Engraftment was documented by analysis of short tandem repeats with polymerase chain reaction (STR-PCR) on approximately day+30,+90,+180,+1year and+2year after SCT, respectively.Data analysis:Analyze the engraftment of hematopoietic stem cell, long-term survival and transplantation-related complications in22SAA patients. ResultsAll patients obtained prompt and sustained hematopoietic reconstitution. Three patients died of cytomegalovirus pneumonia (N=1), acute graft-versus-host disease (N=1) and severe pulmonary infection (N=1). The media survival time was24months (ranging from0.5to140.5months).Hematopoietic reconstitution:The median infused cell dose of nucleated cells and CD34-positive cells of22patients was7.8(3.1～13.9)×108/kg(weight of recipient) and8.0(4.2～13.5)×106/kg. All patients obtained prompt and sustained hematopoietic reconstitution. Median time for neutrophil and platelets(PLT) engraftment was fifteen days (range11～22days) and sixteen days (range12-27days) respectively.The sustained engraftment after allo-HSCT:All patients were full donor chimerism evaluated by STR-PCR on day+30,+60,+90,+180,+360,+720post transplantation except one patient died of severe pneumonia on+15, one died of Ⅲ acute graft-versus-host disease on+45and one died of severe cytomegalovirus pneumonia on+150. No patients was mixed chimeras.The transplant-related complications:(1) aGVHD:After transplantation, two patients (9.1%) developed aGVHD. One was grade I. Its specific manifestation was only partial skin itching, without having to glucocorticoid to treat.The other was grade III. Its specific manifestation was severe diarrhea, hematochezia and serious damage of hepatic function, following large dose of methylprednisolone and FK506to therapy, the patient died on day+45.(2) cGVHD:Local cGVHD occurred in three patients with mild clinical symptoms and the CI was15.8%.(3) CMV infection:After transplantion,10patients had a increase in CMV titers, including nine cases of CMV viremia, which turned to negative after the use of ganciclovir for two weeks. One patient developed severe CMV pneumonia and died on5months after transplantation because of treatment is actually ineffective.(4) Bacterial and fungal infection:One patient developed severe pneumonia (bacterial and fungal infection). The patient died on day+15after powerful, broad spectrum antibacterial and antifungal therapy. Fungal and bacterial sepsis occurred in two patients following transplantation. One case was intestinal infection and the onther case was oral infection.They all controlled by positive and effective anti-infection treatment.Survival situation:The follow-up was up to June1st,2013. The median follow-up time was24months (1～141months).19patients survived disease-free. The2years cumulative survival rate of the enrolled22patients was86.4%.The deaths occurred mainly in half a year after transplantation.Conclusions1. More intensity of FBCA conditioning regimen may favour donor stem cell sustained engraftment in allogeneic stem cell transplantation for severe aplastic anemia patients;2. The incidence of treatment-related complications in severe aplastic anemia patients such as acute graft-versus-host disease, chronic graft-versus-host disease, infections, hepatic vein occlusion disease and posttransplant lymphoproliferative disease not increased obviously. |
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