The Primary Clinical Study Of HLA Haploidentical Stem Cell Transplantation For Patients With Non-malignant Hematologic Diseases

[Background and Objective]Nonmalignant hematologic diseases is a heterogeneous group of diseases, including aplastic anemia (AA), thalassemia, anemia, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, sickle cell anemia disease,et al. Theoretically speaking, hematopoietic stem cell transplantation (HSCT) can treat all congenital hematopoietic system disorders, including the above mentioned nonmalignant hematologic diseases. In clinical work, allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a very important treatment for severe aplastic anemia (SAA), heavy β thalassemia and Fanconi anemia. The human leucocyte antigen(HLA) identical donor is the most suitable donor for allo-HSCT, but the proportion of full-matched donor is only about25%in slibing,and the probability to find a unrelated donor which is matched is1/50000to1/100000or less. HLA identical allogeneic hematopoietic stem cell transplantation is limited by the problem of donor limits, but more than90%of patients who need transplants have the right haploidentical donor. In recent years, HLA haploidentical hematopoietic stem cell transplantation has been used in treatment of hematologic malignancies, and it has achieved encouraging results. But can HLA haploidentical hematopoietic stem cell transplantation be applied to nonmalignant hematologic diseases? How is its efficacy? Do they have severe graft-versus-host disease? Few reports talk about it. In this study, a retrospective analysis of13patients with nonmalignant hematologic diseases using the FBCA conditioning regimen to HLA haploidentical hematopoietic stem cell transplantation, and we evaluate the clinical efficacy and safety.[Methods]1. The selection of the patientsAll patients in the study were chozen from the whole patients treated in our department from2001to2013.13patients were selected, including9cases of severe aplastic anemia(4males and5females, median age18years old (8-21years old));3cases of severe β-thalassemia(1male and2females, median age was3years old (3-3.5years old));1case of pure red cell aplasia (female,6years old). Severe aplastic anemia and pure red cell aplasia were diagnosed by bone marrow pathology, severe P-thalassemia were diagnosed by gene analysis.2. The selection of the donors and HLA matchingWe choose the healthy immediate family members of patients with nonmalignant hematologic disease. The degree of HLA matching consistency was the main selection criteria for the donors. Before2005, we use serological testing for HLA matching.And after2005, we use PCR reaction (sequence specific primer, PCR-SSP) method for HLA matching.3. The mobilization and collection of the donor’s hematopoietic stem cell We use glycosylated G-CSF to mobilize peripheral blood stem cells, a dose of10ug/kg, continuous application for five days.And in the fifth day we use the CS3000blood cell separator to collect the peripheral blood stem cells, some donors need to collect bone marrow in the6th day,collection bone marrow for400ml volume.4. The conditioning regimeThe conditioning regime consisted of Fludarabine (30mg/m2/d×5d), Busulfan (3.2m g/kg/d×4d),Cyclophosphamide (60m g/kg/d×2d),Rabbit anti-human lymphocyte globulin (2.5mg/kg/d×5d).5. The prophylaxis of graft-versus-host diseaseFor preventing GVHD, Cyclosporin A and short term MTX were used. CsA was use in the day before infusion of stem cells for3mg/kg/d. MTX was used at1,3,6and11days after the transplantation, and the dose was15mg/m2,10mg/m2,10mg/m2, mg/m2.6. The prophylaxis of HVODHeparin(100U/Kg/day) and prostaglandin El were 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.7. The prophylaxis of HCDuring the conditioning, we use hydrantion、alkalined the urine、mesna、 diuretics to prophylaxis HC.8. The prevention of infectionBefore 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 patient 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.9. Hematopoietic reconstitution and chimerism monitoringAfter transplantation, monitor the CBC of the patients to make sure the hematopoietic reconstitution.If the patient and donor are in the different gender, use fluorescence in situ hybridization to detect changes.If the patient and donor are in the same gender,use polymerase chain reaction with short tandem repeat(STR-PCR) method to analysis of D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317,D7S820,AM etc.10. Statistical MethodsUse SPSS13.0statistical software for statistical analysis.Use Kaplan-Meier method to draw out the survival curves.[Results]All13patients were successfully grafted, and they all have long and stable complete-donor-chimerism. The median follow-up time was13months, in addition to1case of aplastic anemia patient and1case of pure red aplastic anemia which died for infection,11patients are disease-free survival.All of the11surviving patients achieved a hematologic CR.1. HLA matching and blood type of the donor and recipientThree HLA loci mismatched in4cases, two HLA loci mismatched in8cases and one HLA locus mismatched in1case.5cases of ABO-incompatible between donors and recipients,including two cases of major blood group incompatibility, three cases of minor blood group incompatibility.2. EngraftmentThe median infused doses of monuclear cell (MNC) was8.7×108/kg (3.076-15.8×108/kg).The median infused doses of CD34+cell was5.02×106/kg(2.48-11.85×106/kg).All patients achieved hematopoietic reconstitution successfully.The median time to reach an absolute neutrophil count above0.5×109/L was+11day (+9d～+14d),and platelet up to20×109/L need a median time of+13day (+9d～+27d).All the laboratory test results showed absolute stable donor chimerism.3. Transplant-related complicationsGVHD:The incidence of grade1-2acute graft-versus-host graft-versus-host disease (aGVHD) was30.8%, and that of grades3-4was7.7%. The cumulative incidence of total chronic GVHD (cGVHD) was23%.Infectioin:After the transplantion,8patients had a increase in CMV titers, including seven cases of CMV viremia, which turned to negative after the use of ganciclovir for two weeks.And one case of CMV pneumonia with invasive fungal infections, which death in+44days. Fungal infections occurred in5patients, except one case of pulmonary fungal infections and one case of pulmonary fungal infections with CMV pneumonia who were dead, other patients were all treated.4cases of bacterial infection, including one case of positive blood cultures (Proteus mirabilis), one case of urinary tract infections, one case of oral infections, one case of mixed infection of the lungs, and all patients were treated.Three HLA loci mismatched in4cases, two HLA loci mismatched in8cases and one HLA locus mismatched in1case.4. Protopathy changesExcept two cases of severe aplastic anemia patients who died for infection, seven cases of severe aplastic anemia patients、one case of pure red aplastic anemia patients three cases of severe β-thalassemia patients with primary disease have been cured.All11patients can live without transfusion of any blood products.And severe aplastic anemia patients live without severe infections and bleeding after transplantion.5.Survival situationThe current median follow-up time was13months (2-145), except1case of aplastic anemia patient and1case of pure red aplastic anemia which died for infection(CMV pneumonia, pulmonary fungal infection),11patients had disease-free survival.[Conclusion]HLA haploidentical stem cells transplantation is an effective and safe therapy for nonmalignant hematologic disease.