| Objectives:Thalassemia is one of the most widely disseminated hereditary hemolytic hemoglobinopathy in the world.0-thalassemia is associated with defective synthesis of ?-globin subunits of hemoglobin A,resulting in the excess of ?-globin chain,which form unstable homotatramers that precipitate as inclusion bodies in the red blood cells,leading to ineffective hematopoiesis and hemolysis.WHO has estimated that about 1.5%of the world's population might be carriers of ?-thalassemia.Southern China is one of the most epidemic area of ? thalassemia,especially in Guangdong,Guangxi and Sichuan.Patients suffer from ?-thalassemia major(?-TM)rely on transfusion and iron chelation therapy whole life long.Hemolysis caused by thalassemia and frequent red blood cell transfusion can produce iron overload,which can cause damage to the vital organs including liver,heart and endocrine system,and increase the risk of death.In the developing countries,patients suffer from ?-thalassemia major usually do not live to 20 years old,because of lacking of blood products,costly iron chelation therapy and insufficient medicare.Currently allogeneic hematopoietic stem cell transplantation(allo-HSCT)is the only way to cure ?-thalassemia major.Bone marrow transplantation(BMT)is the most classic choice,the overall thalassemia-free-survival(TFS)is about 80%?90%in the younger and low risk patients who have matched sibling donors.Compare with BMT,peripheral blood stem cell transplantation(PBSCT)is characterized by easy acquisition,faster hematopoietic reconstitution,lower incidence of infection and little harm to the donors.But PBSC contains a lot of T lymphocytes,which attribute to graft versus host disease(GVHD).Cord blood transplantation has the advantage of prompt availability,no risk of donor refusal,reduced risk of both acute and chronic GVHD,possibility to perform transplantation using 1-or 2-antigen HLA-disparate donors,low risk of viral contamination(ie.human cytomegalovirus and Epstein-Barr virus).But cord blood has less hemopoietic stem cells,which might cause delay in neutrophil and platelet recovery,increase risk of rejection,lack of transfer T memory lymphocytes,delay in immune reconstitution,increased risk of infection.Indeed infection is one of the leading cause of transplantation related motality after cord blood transplantation.In this study,we compared the outcome and immunue reconstitution in 0-thalassemia major after co-transplantation of cord blood and bone marrow with co-transplantation of peripheral blood stem cell and bone marrow.Studied the basic rules of immunohematologic reconstitution would help us to improve the outcome of HSCT(especially CBT)for ?-thalassemia major in the future.Methods:1.Analysis of clinic data From April 2005 to January 2015,154 children with ?-thalassemia major underwent allo-HSCT from a human leukocyte antigen(HLA)-identical or 1-antigen mismatched sibling in our hospital.74 patients received co-transplantation of cord blood and bone marrow,80 patients veceived co-transplantation of peripheral blood stem cell and bone marrow.The median age was 4 years old(range 2?16 years).109 patients were male,64 patients were female.All patients were diagnosed by ?-thalassemia gene examination,the genotypes were ?-thalassemia homozygotes or double heterozygotes,with Pesaro ??? degree.The median follow up was 24 months(range 4-118 months).149(97%)patients transplanted from a HLA-identical sibling,5(3%)patients transplanted from a HLA-1-antigen mismatched sibling.103(67%)patients were ABO matched,29(19%)patients were ABO major mismatched,22(14%)patients were ABO minor mismatched.All patients received a short course of hydroxyurea 45?11 days before HSCT.All patients received Bu+Cy+Flu+ATG as conditioning regimen.CB+BM group received CsA±MMF as GVHD prophylaxis,PBSC+BM group received CsA+MTX+MMF as GVHD prophylaxis.Documented the total nucleated cell dose and the CD34+ cell dose transfused to the recipients,and the engraft time of neutrophil and platelet.To evaluate the chimerism status after transplantation,we used a polymerase chain reaction(PCR)technique amplifying short tandem repeats(STRs)or sex chromosome.Documented the incidence of aGVHD,cGVHD,opportunistic infection(ie.cytomegalovirus,bacteria or fungi)and transplantation related motality(TRM).The SPSS 17.0 package was used for analysis of the data.The Kaplan-Meier survival curves and the Log-Rank test were used to evaluated the OS and TFS in both groups.2.Flow cytometry analysis Collected peripheral blood of CB+BM group,and PBSC+BM group before HSCT and 1,3,6,12 months after HSCT.Collected peripheral blood of 20 healthy children as control group.Flow cytometry was used to evaluate the following T lymphocyte subgroups before HSCT and 1,3,6,12 months after HSCT,T cells(CD3+),helper T cells(CD3+CD4+),naive CD4+T cells(CD3+CD4+CD62L+CD45Ra+CD45Ro-),memory CD4+T cells(CD3+CD4+CD45Ro+),cytotoxic T cells(CD3+CD8+),naive CD8+T cells(CD3+CD8+CD45Ra+),memory CD8+T cells(CD3+CD8+ CD45Ro+),regulatory T cells(Treg cells)(CD3+CD4+CD25+Foxp3+).According to absolute number of white blood cell(WBC)obtained from the automatic blood cell analyzer,we could calculate the absolute number of each T lymphocyte subgroup.The SPSS 17.0 package was used for analysis of the data.Mann-Whitney U test was used to compare the data from two groups.3.TCR-V ? repertoire spectra typing Collected peripheral blood of CB+BM group and PBSC+BM group.Peripheral blood mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation,extracted RNA.RT-PCR was used to amplify the 24 subfamilies of TCR-V 0 repertoire.Gene scan was used to analyze the sequences of each subfamilies that could result in a functional TCR were considered to be productive rearrangements.Results:1.153(99.4%)patients demonstrated engraftment except one patient in the CB+BM group.Myeloid engraftment occurred at a median of 15 days(range 10?24 days)and 11 days(range 8?16 days)in the CB+BM and PBSC+BM group,respectively(P=0.009).The median time to achieve platelet engraftment were 25 days(range 11?42 days)and 16 days(range 11?39 days)in the CB+BM and PBSC+BM group,respectively(P=0.000).13 patients showed mixed chimerism,8 were in the CB+BM group,5 were in the PBSC+BM group(P=0.331).Only one patient in the CB+BM group had aGVHD.17 patients in the PBSC+BM group had aGVHD(P=0.001),among them,4 were with ?°aGVHD,6 were with ?°0 aGVHD,7 were with ?°aGVHD.One patient in the CB+BM group and 6 patients in the PBSC+BM group had cGVHD,respectively(P=0.034).HC occurred in 19 patients in the CB+BM group and 30 patients in the PBSC+BM group,respectively(P=0.115).VOD occurred in 8 patients in the CB+BM group and 6 patients in the PBSC+BM group,respectively(P=0.475).CMV reactivation occurred in 21 patients in the CB+BM group and 7 patients in the PBSC+BM group,respectively(P=0.001).Infection occurred in 8 patients in the CB+BM group and 2 patients in the PBSC+BM group,respectively(P=0.037).Invasivee fungal disease(IFD)occurred in 6 patients,all 6 patients were in the CB+BM group(P=0.009).Pneumonia occurred in 18 patients in the CB+BM group and 8 patients in the PBSC+BM group,respectively(P=0.018).The total TRM rate was 6.5%.9 patients in the CB+BM group and 1 patients in the PBSC+BM group died from TRM,respectively(P=0.018).The 2-year accumulative TRM rate was 12.3%in the CB+BM group and 1.3%in the PBSC+BM group,respectively(P=0.006).143 patients were alive,among them 142 patients were thalassemia free.The 3-year estimated OS was 92.9%and TFS was 92%,for all the patients.The 3-year estimated OS for the CB+BM group and BSC+BM group were 86.3%and 98.8%,respectively(P=0.003).The 3-year estimated TFS for the CB+BM group and PBSC+BM group were 86.3%and 97.5%,respectively(P=0.011).2.CD3+,CD4+ and CD8+ T lymphocyte declined in the 1st month after transplantation,then recovered gradually.12 months after HSCT,CD3+T lymphocyte fully recovered as it was before HSCT.CD4+T lymphocyte recovered much more slowlier and didn't reach the level before HSCT.CD8+T lymphocyte grew faster and reached normal level at 3 months after HSCT.In the early period after HSCT,quickly expansion of CD3+CD8+T lymphocyte led to consistent inversion of CD4+/CD8+.At 12 months after HSCT the CD4+/CD8+ratio was 0.78±0.45.The absolute number of T lymphocyte subgroups of the CB+BM group and PBSC+BM group were analyzed by Mann-Whitney U test,before HSCT and 1,3,6,12months after HSCT.The absolute number of MemCD8+T lymphocyte at3 months after HSCT was significantly higher in the PBSC+BM group.The absolute number of Treg T lymphocyte at 12 months after HSCT was significantly higher in the CB+BM group.The absolute number of NavCD4+T lymphocyte at 1 and 12 months after HSCT was significantly lower in the patients with CMV reactivation,and so did the MemCD4+T lymphocyte at 1 month after HSCT.The absolute number of Treg lymphocyte at land 3 months after HSCT was significantly lower in the patients with CMV reactivation too.The absolute number of CD4+T lymphocyte at 1 and 12 months after HSCT was significantly lower in the patients with infections,and so did the MemCD4+T and NavCD4+T lymphocyte at 1 month after HSCT.The absolute number of Treg lymphocyte at 1 and 3 months after HSCT was significantly lower in the patients with infections,too.3.Both CB+BM group and PBSC+BM group recovered all 24 subfamilies of TCR V ? within 1 year after HSCT.CB+BM group showed reducing rate of TCR V ? oligoclone.The patients with CMV reactivation seemed to show more TCR V ? abnormities.Conclusion1.Both CB+BM transplantation and PBSC+BM transplantation were effective ways to cure ?-thalassemia major.2.The hematopietic reconstitution was slower in the CB+BM group,and the incidence of GVHD was lower in the CB+BM group.But the incidence of CMV reactivation and infections were higher in the CB+BM group,which resulting in a higher TRM rate.3.Quick expansion of CD3+ CD8+T lymphocyte led to consistent inversion of CD4+/CD8+ after HSCT.At 12 months after HSCT the CD4+/CD8+ratio was 0.78±0.45.The absolute number of MemCD8+ T lymphocyte at 3 months after HSCT was significantly higher in the PBSC+BM group.The absolute number of Treg lymphocyte at 12 months after HSCT was significantly higher in the CB+BM group.4.NavCD4+,MemCD4+ and Treg cells in the patients with CMV reactivation and infections were significantly lower at different time points after HSCT,which indicated that low CD4+T lymphocytes and slow immune reconstitution were risk factors for opportunistic infections.5 Both CB+BM group and PBSC+BM group recovered all 24 subfamilies of TCR V ? within 1 year after HSCT. |
PDF Full Text Request