Clinical Study On Chimerism Of Blood Cell Subsets Following Allogeneic Peripheral Blood Stem Cell Transplantation

Chimerism testing following allogeneic hematopoietic stem cell transplantation (allo-HSCT) can be used to monitor donor cell engraftment, diagnose graft failure or disease relapse and guide immunotherapy. Because of abundant polymorphism, PCR amplification of short tandem repeats (STR) in DNA sequence became the most sensitive approach for chimerism testing. However, the qualitative testing of STR-PCR cannot obtain the exact ratio of donor cell to recipient cell in mixed chimerism. Since cells from donor increase gradually during engraftment and decrease gradually during rejection, evaluating engraftment status, predicting impending relapse and adjusting immunotherapy depend on accurate quantitative testing of chimerism.Whole blood and bone marrow are mixture of different cells, the chimerism values of whole blood and bone marrow reflects the mixture results of those cells. As different cell engrafts and subsides at different tempo during engraftment and relapse, lineage specific chimerism should be analyzed.By constructing quantitative technique of STR-PCR and combining flow cytometry sorting technique, We observed the engraftment process of blood cell subsets and analyzed the relationship between lineage specific chimerism and graft failure, disease relapse and graft versus host disease (GVHD), in order to find the stable engraftment marker and molecular omen of graft rejection or disease relapse, therefore guide immunotherapy and improve overall survival.Part 1. Construction of quantitative method of short tandem repeats bymultiple PCR amplification[Objective]To build quantitative method of STR-PCR on the basis of qualitative method. [Methods]For standard curve construction, blood samples from donor and recipient were mixed with respect to the concentration of their white blood cells at ratio of 95:5, 90:10, 80:20, 70:30, 60:40 和 50:50. Multiple PCR amplification of STR labeled with fluorescence was performed after extracting genome DNA from these mixed samples. 9 STR loci and gender locus Amelogenin were amplified in one PCR tube at the same time. Since PCR product content was correlate to the STR peak area in quantitative detection, after electrophoresis chimerism values were calculated from the observed peak areas of the informative loci. [Results]It was 20 μ l PCR reaction system, which included 12μl reaction mix (8. 4 μl PCR reaction mix, 0. 5 μl Taq DNA polymerase, 4. 4 μl primer set) and 8μl (2. 5ng) DNA template. After an initial denaturation of 11 min at 95℃, 28 cycles were performed at 94℃ for 1 min, 58℃ for 1 min and 72℃ for 1 min, and additional incubation step at 60℃ for 45℃ was added after the cycles. The detection limit was 5%, i.e. 5% minor cells in mixed sample could be detected.The amount of DNA template in PCR reaction was the key factor affecting the quantitative analysis. Too much or too little DNA template would cause stutter peak, split peak or imbalance amplification of alleles, which might interfere the accuracy of the quantitative analysis. [Conclusion]Quantitative method of STR-PCR was successfully built by optimizing the procedure of qualitative method, which enabled accurate analysis of chimerism following transplantation.[Objective]1. To observe the engraftment kinetics of blood cells following allogeneic peripheral blood stem cell transplantation (allo-PBSCT);2. To observe the rejection kinetics of blood cells during graft rejection and disease relapse;3. To find the stable engraftment marker of allogeneic stem cell and molecular omen of graft rejection and disease relapse;4. To analyze the relationship of GVHD and chimerism;5. To analyze the relationship of blood group shifting and chimerism. [Methods]28 cases of allo-PBSCT in our hospital were observed, their median age was 38(15-54). Their diagnosis were AML (n=7), ALL (n=2), CML (n=13), MM (n=4), MDS (n=l) and refractory and relapse ovarian cancer (n=l). 13 patients had progressive diseases and 15 were in stable status. 22 patients received graft from HLA-identical sibling donors, 5 from HLA-identical unrelated donors and 1 form HLA hyploidentical mother. Myeloablative conditioning was given to 12 patients and non-myeloablative conditioning to 16 patients. 15 recipients and their donors were ABO blood group mismatched.FACS sorted granulocyte, T lymphocyte, B lymphocyte and NK cell from peripheral blood of all patients were analyzed in 7 days intervals for 1 month starting from the day of PBSCT, and 1 month interval for 6 months, and then 3 months interval to 1 year. The purity of sorted cells was above 96%. Short tandem repeats polymorphisms were analyzed on these cell subsets. Count of blood cells, bone marrow smear, bone marrow chromosomes, blood group and XY dual color FISH were analyzed serially. Clinical manifestation and GVHD were also observed. [Results]On day 7 posttransplant, NK cell chimerism was the highest in 88. 5% patients, which meant NK cell was the first cell engrafted. In myeloablative transplant, granulocyte and T cell began to engraft later than NK cell, while B lymphocyte was the latest. In non-myeloablativetransplant, T lymphocyte engrafted earlier, granulocyte and B lymphocyte were the latest. Granulocyte, B lymphocyte and NK cell reached complete chimerism (CC) quickly on median day 14 posttransplant, while T lymphocyte was the latest one to reach CC in both myeloablative and nonmyeloablative transplant on median day 21 and day 28 posttransplant respectively.7 patients suffered from myeloid malignancy had molecular omen of graft rejection or disease relapse. When graft rejection or disease relapse occurred, all 7 recipients had progressive mixed chimerism. 3/7 patients had T cell donor chimerism shifting from CC to MC only, while other cells remained CC. 2/7 patient' s T cell chimerism remained the lowest compared with other cells. T cell became decreasing MC in 5 cases after discontinuance of immunosuppressant and donor lymphocyte infusion (DLI), 3 out of them achieved CC again.8 cases developed acute GVHD in 19 stable CC patients, 7/8 had GVHD after CC was achieved by all cell subsets. While none of the 7 patients developed GVHD before they had graft rejection or relapse. The rate of acute GVHD of CC group was higher than MC group ( x~2=4. 25, P<0. 05) .ABO mismatched and matched groups were not different in their chimerism of blood cell subsets at the same time (P>0.5). There was no difference between the two groups in blood cell count recovery. The time of blood group shifting to donor type correlated to granulocyte, B cell and T cell chimerism. All recipients' blood group changed to donor type after CC was achieved except 2 cases with hemolysis.It took longer time for T lymphocyte reached CC than peripheral blood and bone marrow (P<0.01) . The changing of T lymphocyte chimerism was earlier than PB and BM in graft rejection and disease relapse. [conclusions]1. The intensity of conditioning regimen affected the engraftment kinetics of different cells. NK cell was the first one to engraft, T lymphocyte was the last one to reach CC.2. Donor T lymphocyte was the first one to subside in graft rejection and disease relapse in myeloid malignancy. Patients responding to immunotherapy could achieve CC in T lymphocyte fraction again.3. GVHD usually occurred after CC was achieved in all cell lineages.4. ABO mismatch did not affect the engraftment of granulocyte, T lymphocyte, B lymphocyte and NK cells, did not affect recovery of blood granulocyte and platelet count. Blood group changed to donor type usually after CC was reached.5. CC of T lymphocyte could be a marker of stable engraftment of donor cells. Progressive MC of T cell was the omen of graft rejection and disease relapse. Chimerism analysis of T lymphocyte was superior to PB and BM.6. Individualized immunotherapy should adopt according to the chimerism of different patients. For patients reached CC in T cell fraction in one month after transplant, GVHD prevention was the most important; therefore the patients might be given full dose and longer period of immunosuppressant in 3 months posttransplant. For patients did not, prevention of graft rejection and disease relapse were more important, thus the immunosuppressant might be tapered 1 month after transplant, in order to promote the engraftment of donor cell and avoid disease recurrence.