Contrasting The Clinical Outcome And Immunological Reconstitution From HLA Haploidentical And HLA-matched Peripheral Blood Hematopoietic Stem Cell Transplantation Without In Vitro T-cell Depletion

Objective:1) To explore the clinical application of human leukocyte antigen (HLA) haploidentical allogeneic peripheral blood stem cell transplantation (allo-PBSCT) for malignant hematological diseases using a new SIAGP protocol.2) To contrast clinical outcome of transplantation from HLA haploidentical and HLA-matched allo-PBSCT without in vitro T-cell depletion for malignant hematological diseases at the same time.3) To contrast the difference of immune function by detecting T,B,NK lymphocyte subsets and relationship with main complications from HLA haploidentical and HLA-matched allo-PBSCT. Method:1) 52 patients with malignant hematological diseases, age from 11 to 47 years (median 38 years), were transplanted with stem cells from an HLA-haploidentical family donor with 1-3 mismatched loci of HLA antigens.9 patients had 1 locus mismatched donors,43 patients had 2-3 loci mismatched donors. Patients were classified as having standard-risk (36 patients), high-risk (16 patients) based on the status of their leukemia. The patients have received Ara-C+Bu+Cy or Bu/Cy myeloablative conditioning regiment. All patients were given a combination of cyclosporine (CsA), a short course of methotrexate (MTX) and MMF.2-3 loci HLA-mismatched patients were given antihuman thymocyte globuline (ATG) and CD25 mono-colony antibody on days+1 and +4. CsA intravenously was started on day-1 and continued until patients were able to tolerate oral medication, it was tapered on days+180 and about 12 month to be fully discontinued if no evidence of cGVHD. On day+1, MTX (15mg/m2) was administered intravenously and then 10 mg/m2 was given on days+3,+6, and+11 after transplantation. MMF (1g/d twice a day) was begun on day-1 and tapered half dose on day+40,+90 to+100 day to be fully discontinued. aGVHD was treated with 1 to 2 mg/kg per day of methyllprednisolone (MP), cGVHD was treated with prednisolone and azathioprine. Donors received granulocyte colony-stimulating factor (G-CSF) at 7～10μg·kg-1·d-1 for 5 consecutive days and test donors blood routine test per day. On the 5th and 6th days, peripheral blood stem cells were collected with COBE Cell Separator without in vitro T-cell depletion, then to count MNC and CD34+ cells.2) 111 patients with malignant hematological diseases underwent PBSCT without in vitro T-cell depletion at the same time, including 51 patients with HLA-haploidentical and 60 patients with HLA-matched. All patients have received Ara-C+Bu+Cy or Bu/Cy myeloablative conditioning regimen. A two-agent based graft-versus-host disease (GVHD) prophylaxis was used as cyclosporine A and a short course of methotrexate in HLA-haploidentical and HLA-matched patients. MMF was added for 1 locus mismatched patients. MMF, antithymocyte globulin and CD25 mono-colonal antibody were added for 2-3 loci mismatched patients. The grafts were granulocyte colony-stimulating factor-mobilized peripheral blood stem cells without in vitro T-cell depletion.3) 67 patients undergoing HLA-matched (n=33) or HLA haploidentical (n=34) allo-PBSCT performed during the same time period were compared in our hospital.The indirect immunofluorescence assay was employed to detect T,B,NK lymphocyte subsets before transplantation and on months 1,3,6,12 and 18 after transplantation, and lymphocyte subsets of 100 healthy people as normal control. The main process were prepared mononuclearcell suspension and detected T lymphocyte subsets(CD3+,CD4+,CD8+), B lymphocyte subsets (CD19+), NK lymphocyte subsets(CD16+,CD56+). The comparison of immunological reconstitution and relationship with main complications were taken statistical analysis. Result:1) 52 patients achieved sustained, full donor-type engraftment, the median of MNC cells were13.9 (8.6～31.0)×108/kg×108/kg, the median of CD34+ cells were 10.1 (4.16～21.00)×106/kg×106/kg. The median time to reach an absolute neutrophil count above 0.5×109/l was 14 days (range,11-24 days) and engrafted to platelet count exceeding 20×109/l with a median time of 16 days (range,9-26 days).23 patients occurred HC, they were all late-onset HC, gradeⅠin 5 cases, gradeⅡin 13 case, gradeⅢin 3 case, gradeⅣin 1 case.26 patients developed aGVHD, the gradeⅠaGVHD occurred in 20 and gradeⅡin 6.35 patients developed cGVHD with limited in 30 and extensive in 5, the 4-year cumulative incidences of cGVHD were 70.4%,66.7% in standard-risk,62.5% in high-risk. No patient died of GVHD. Patients had been leukemia free with a median follow-up of 15 months (range,4 to 69 momths),7 patients relapsed, including 5 standard-risk patients and 2 high-risk patients, the cumulative incidence was 17.6%. The 3-year probabilities of leukemia-free survival (LFS) for patients were 71.8%, 76.4% in standard-risk patients and 60.2% in high-risk patients.2) 111 patients achieved sustained, full donor-type engraftment, the median of MNC and CD34+ cells were 14.5 (8.6～31.0)×108/kg and 10.9 (4.16～21.00)×106/kg in 51 HLA-haploidentical patients, the median of MNC and CD34+ cells were 10.59 (7～19.9)×108/kg and 5.9 (2.86～10.2)×106/kg in 60 HLA-matched patients,. The median time to reach an absolute neutrophil count above 0.5×109/L was 14 days and engrafted to platelet count exceeding 20×109/L with a median time of 15 days in 51 HLA-haploidentical patients, the median time were 12 days and 13 days in 60 HLA-matched patients. In 51 HLA-haploidentical patients,25 patients developed aGVHD, gradeⅠaGVHD occurred in 20 cases and gradeⅡin 5 cases, the cumulative incidences of aGVHD were 49%,48.6% in standard-risk,50% in high-risk.33 patients developed cGVHD with limited in 20 and extensive in 3, the 4-year cumulative incidences of cGVHD were 70.4%,71.4% in standard-risk,66.2% in high-risk. The 3-year probabilities of LFS were 74.5%, standard and high risk patients were respectively 77.3% and 68.2%.7 patients relapsed,the the cumulative incidences of relapse were 18%,17.7% in standard-risk,18.3% in high-risk; In 60 HLA-matched patients,14 patients developed aGVHD, gradeⅠaGVHD occurred in 10 cases, gradeⅡin 2 cases and gradeⅢin 2 cases, the cumulative incidences of aGVHD were 23.3%, 24.4% in standard-risk,20% in high-risk.37 patients developed cGVHD with limited in 32 and extensive in 5, the 4-year cumulative incidences of cGVHD were 58.1%,57.8% in standard-risk,60% in high-risk. The 3-year LFS were 72.1%, standard and high risk patients were respectively 77.6% and 52.7%.10 patients relapsed,the the cumulative incidences of relapse were 20.2%,18.3% in standard-risk,27.5% in high-risk. The incidence of aGVHD in HLA-haploidentical cohort was higher than HLA-matched cohort, it has statistical difference (P<0.05). The incidence of cGVHD, incidence of relapse and LFS were no statistical difference (P>0.05) between HLA-haploidentical and HLA-matched cohorts.3) Compared the total 67 patients with normal control, the CD3+%,CD4+%,CD4+/CD8+ at 1 month, the CD4+% CD4+/CD8+ at 3 month and CD4+% at 6 month after PBSCT were lower, the CD8+% at 3 month and 6 month were higher;In HLA haploidentical group, the CD3+% at 1 month decreased obviously and recovered at 3 month,the CD4+% decreased obviously at 3 month and recovered at 6 month,the CD8+% increased aftre transplantation and recovered at 18 month, the CD4+/CD8+ decreaed significantly at 3 month and recovered at 12 month; Compared HLA haploidentical with HLA-matched, the CD3+% at 1 month was lower, the CD4+% was lower and last to 6 month, the CD4+/CD8+ was still lower at 6 month.it all has statistical difference; Compared aGVHD cohort and non-aGVHD cohort in HLA haploidentical,the CD3+% and CD4+% at 3 month were higher than aGVHD cohort, it has statistical difference; The CD3+% was lower than normal control in HLA haploidentical and HLA-matched cohort at 1 month. The CD4+% was lower than normal contral in HLA haploidentical and HLA-matched cohort at 1,3,6 month. The CD8+% was higher than normal contral in HLA haploidentical and HLA-matched cohort at 3,6 month. The CD4+/CD8+ was lower than normal control in HLA haploidentical and HLA-matched cohort at 3,6 month;The immune function of severe infection patients and non-infection patients in HLA haploidentical were no statistical difference. The CD3+% and CD8+% in non-infection patients of HLA-matched were lower than non-infection patients in HLA haploidentical at 3 month. The CD4+% in non-infection patients of HLA-matched were higher than non-infection patients in HLA haploidentical at 6 month. The CD19+% and CD56+% in non-infection patients of HLA-matched were higher than non-infection patients in HLA haploidentical at 12 month;The immune function of patients with chronic graft-versus-host disease (cGVHD) between HLA haploidentical and HLA-matched allo-PBSCT were no statistical difference, the immune function of patients without cGVHD in two groups were no statistical difference; The immune function of relapsed 8 patients and non-relapsed patients were no statistical difference. Conclusion:1) HLA-haploidentical PBSCT is feasible and safe for malignant hematological diseases to use myeloablative conditioning regiment combination of intensive immunosuppressants without in vitro T cell depletion.2) HLA-haploidentical with peripheral blood as a stem cell source can be performed safely on donors and can be taken many times, no risk of anesthesia, no significant risk of graft rejection, no increased incidence of severe aGVHD and extensive cGVHD.3) The clinical outcome of cGVHD,incidence of relapse and LFS were similar between HLA-haploidentical and HLA-matched cohorts, the incidence of aGVHD in HLA-haploidentical cohort was higher than HLA-matched cohort.4) The severe aGVHD and the extensive cGVHD were similar in HLA-haploidentical and HLA-matched cohorts.5) Compared with normal control,the immune function of patients recovered at 12 month. The immune function has no statistical difference HLA haploidentical and HLA-matched allo-PBSCT.6) The immunologic injury in HLA haploidentical was more severe than HLA-matched allo-PBSCT within 6 month after transplantation, the immunologic reconstitution was delay in HLA haploidentical cohort. 7) The change of T lymphocyte subsets were most significance in T,B,NK lymphocyte subsets after HLA haploidentical and HLA-matched allo-PBSCT.