The clinical characteristics of adult hemophagocytic lymphohistiocytosis treated with haploidentical donor hematopoietic stem cell transplantation / 中华内科杂志

Objective To analyze the clinical characteristics of adult patients with hemophagocytic lymphohistiocytosis (HLH) receiving haploidentical donor hematopoietic stem cell transplantation (HID HSCT).Method We retrospectively reviewed 20 adult patients with HLH from August 2009 to August 2014.The clinical features and outcome were analyzed.Results Conditioning regimens consisted of total body irradiation/etoposide/cyclophosphamide (TBI/VP-16/CTX) and busulfan (Bu)/VP-16/CTX in HLH with anti-thymocyte globulin (ATG) 8 mg/kg.The stem cells were mobilized from donors' peripheral blood.Median time to white blood cell engraftment was 13 (9-27) days.Median time to platelet engraftment was 14 (10-28) days.Mixed chimerism after transplantation developed in 4 patients and no patient presented graft failure.Eight patients developed grade Ⅱ to Ⅲ acute graft-versus-host disease (GVHD),while as chronic GVHD occurred in 9 patients.Among 12 patients with EB virus (EBV) reactivation,2 patients developed post-transplant lymphoproliferative disorder (PTLD),7 were suspected as PTLD and 3 were considered as relapse of primary disease.With a median follow-up of 20 months (range:0.5-108 months) after transplantation,the estimated 2-year overall survival (OS) rate was (60.0 ± 11.0) ％ in all patients.During the follow-up,12 patients survived,8 died including 5 within 100 days after HSCT.Among 5 non-remission patients before HSCT,4 patients died within 100 days after HCT.Conclusions HID HSCT is an effective treatment for adult patients with HLH to achieve remission and long-term survival.High proportion of mixed chimerism has been seen at early stage after transplantation.EBV reactivation and early transplant-related mortality are common.

Practical Evaluation of Engraftment and Mixed Chimerism Using PCR Amplification of a Microsatellite in the Class II Eb Gene in Murine MHC-mismatched, Nonmyeloablative Bone Marrow Transplantation / 대한혈액학회지

BACKGROUND: Although engraftment following murine allogeneic bone marrow transplantation (BMT) is most commonly confirmed by H2 typing using flow cytometry, recipient mice can be seriously injured during peripheral blood (PB) sampling. Therefore, we developed an alternative DNA-based assay that does not require the large volume of PB necessary for flow cytometry. METHODS: A minute volume of PB from the tail vein was used to evaluate the engraftment by PCR amplification of a microsatellite in the class II Eb gene. Dilution experiments were performed to evaluate the sensitivity of this assay for detecting donor cells in mixed cell populations compared with flow cytometry analysis. RESULTS: Early engraftment and mixed chimerism were confirmed, based on the length variation of the microsatellite in the class II Eb gene. The degree of donor chimerism in the donor-recipient cell mixture could be estimated semiquantitatively in a dilution experiment. The sensitivity of this assay by the naked eye approached 10% of the degree of donor chimerism. CONCLUSION: PCR amplification of a microsatellite in the class II Eb gene can be a useful alternative to flow cytometry for evaluating early engraftment and mixed chimerism following murine nonmyeloablative BMT.

IL-2 Pathway Blocking in Combination with Anti-CD154 Synergistically Establishes Mixed Macrochimerism with Limited Dose of Bone Marrow Cells and Prolongs Skin Graft Survival in Mice

To facilitate the establishment of mixed chimerism with limited dose of bone marrow (BM) cells, and to achieve tolerance in skin graft model, combined blocking of costimulatory pathway and IL-2 pathway was used in minimally myeloablative model using busulfan. BM cells (2.5 x 10(7)) of BALB/c were injected into C57BL/6 mice at day 0 with full thickness skin graft after single dose injection of busulfan (25 mg/kg) on day-1. Recipients were grouped and injected the anti-CD154, CTLA4-Ig, anti-IL-2R at days 0, 2, 4, and 6 according to protocol. Mixed macrochimerism were induced in groups treated with anti-CD154+anti-CTLA4-Ig, anti-CD154+anti-IL-2R, and anti-CD154+anti-CTLA4 Ig+anti-IL-2R. Three groups having chimerism enjoyed prolonged graft survival more than 6 months. Superantigen deletion study revealed deletion of alloreactive T cells in combined blockade treated groups. In graft versus host disease model using CFSE staining, CD4+ T cell and CD8+ T cell proliferation were reduced in groups treated with CTLA4-Ig or anti-IL-2R or both in combination with anti-CD154. However, anti-IL-2R was not so strong as CTLA4-Ig in terms of inhibition of T cell proliferation. In conclusion, IL-2 pathway blocking combined with anti-CD154 can establish macrochimerism with limited dose of BM transplantation and induce specific tolerance to allograft.

IL-2 Pathway Blocking in Combination with Anti-CD154 Synergistically Establishes Mixed Macrochimerism with Limited Dose of Bone Marrow Cells and Prolongs Skin Graft Survival in Mice

To facilitate the establishment of mixed chimerism with limited dose of bone marrow (BM) cells, and to achieve tolerance in skin graft model, combined blocking of costimulatory pathway and IL-2 pathway was used in minimally myeloablative model using busulfan. BM cells (2.5 x 10(7)) of BALB/c were injected into C57BL/6 mice at day 0 with full thickness skin graft after single dose injection of busulfan (25 mg/kg) on day-1. Recipients were grouped and injected the anti-CD154, CTLA4-Ig, anti-IL-2R at days 0, 2, 4, and 6 according to protocol. Mixed macrochimerism were induced in groups treated with anti-CD154+anti-CTLA4-Ig, anti-CD154+anti-IL-2R, and anti-CD154+anti-CTLA4 Ig+anti-IL-2R. Three groups having chimerism enjoyed prolonged graft survival more than 6 months. Superantigen deletion study revealed deletion of alloreactive T cells in combined blockade treated groups. In graft versus host disease model using CFSE staining, CD4+ T cell and CD8+ T cell proliferation were reduced in groups treated with CTLA4-Ig or anti-IL-2R or both in combination with anti-CD154. However, anti-IL-2R was not so strong as CTLA4-Ig in terms of inhibition of T cell proliferation. In conclusion, IL-2 pathway blocking combined with anti-CD154 can establish macrochimerism with limited dose of BM transplantation and induce specific tolerance to allograft.

Chimerism Analysis after Allogeneic Stem Cell Transplantation in Children by Genetic Polymorphism of Hypervariable Regions and Fluorescent in situ Hybridization for X-Chromosome / 대한소아혈액종양학회지

PURPOSE: In order to monitor the clinical outcome of pediatric allogeneic stem cell transplantation (SCT), serial evaluations of chimerism were performed to compare the risk of relapse or graft rejection between patients with complete chimerism (CC) and mixed chimerism (MC). METHODS: Between January, 1996 and April, 2004, 64 cases who underwent SCTs were prospectively enrolled. Serial genotyping of VNTR (variable number of tandem repeats) /STR (short tandem repeats) loci and/or X-chromosome-specific FISH (fluorescent in situ hybridization) were performed at regular intervals. RESULTS: The "informative loci" were found in all 64 patient/donor pairs. CC was persistently detected in 44 cases (68.7%), while MC was detected at least once in 20 (31.3%). In cases with malignancy (n=40), relapse was more frequently encountered in MC group (7/8) than in CC group (7/32) (P < .001), as was death (75% vs. 28%, P < .05). The Kaplan-Meier 5-year overall survival was higher in CC than in MC (69.1% vs. 16.6%; P < .05). In cases with non-malignancy, MC group showed higher rate of graft rejection than CC group (7/12 vs. 1/12, P < .01). Survival was not different between the two groups. The chimerism status was not influenced by sex, donor type, source of stem cells, and inclusion of radiation in conditioning. CONCLUSION: Detection and sequential assessment of MC might be an important tool to predict relapse of disease in malignant diseases as well as to portend graft rejection in non-malignant illnesses. Earlier intervention to circumvent those life-threatening complications should be pursued based on the chimerism analyses.

Clinical Significance of Mixed Chimerism after Hematopoietic Stem Cell Transplantation / 대한진단검사의학회지

BACKGROUND: Chimerism analysis used to be one of the most valuable methods for monitoring patients after allogeneic hematopoietic stem cell transplantation (SCT). The relationship between the mixed chimerism status and the risk of relapse has been controversial. We analysed the clinical significance of mixed chimerism for the prediction of relapse after SCT. METHODS: Between October 2000 and January 2002, 16 patients with haematologic malignancies treated with SCT were included in this study. The median follow-up periods were 11.5 months (range 5-32 months) after SCT. For chimerism analysis, STR (D13S317, D5S818, D7S820) and VNTR (D1S80, D17S30) loci were amplified by PCR. Patients who exhibited complete donor hematopoiesis at all times during the follow-up period were defined as CCG (complete chimerism group) and those who showed mixed chimerism at least once at any time were definded as the MCG (mixed chimerism group). Relapse was considered based on clinical, hematologic and cytogenetic findings. RESULTS: MCG was 63% (10/16). Relapse was observed in 80% (8/10) of MCG and none of CCG (P>0.05). Among 8 relapsed patients, two patients showed MC 1 month prior to relapse and 4 patients changed to MC from CC at relapse status. The remaining 1 patient continued to show CC. CONCLUSIONS: Mixed chimerism seems to be associated with a high risk of relapse. For early detection of relapse, chimerism analysis may need to be performed at shorter time intervals than once a month.

A Case of DNA Chimerism Analysis as a Marker of Donor Lymphocyte Infusion / 대한혈액학회지

Donor lymphocyte infusion (DLI) has some benefit effects as graft-versus-leukemia effect, reducing the relapse of leukemia and inducing of a complete remission. But it has also a graft-versus-host-disease (GVHD) effect. So it is required a proper marker test when DLI is performing. The DNA chimerism analysis can be a marker test in DLI. Variable number of tandem repeats (VNTR) are highly polymorphic DNA markers in the human genomic DNA and used as primers of DNA chimerism analysis. A 43-year-old male who had been diagnosed acute myelogenous leukemia was transplanted with allogeneic peripheral blood stem cells. The initial chimerism analysis showed complete chimerism but it changed to mixed chimerism after 7 months of transplantation. We predicted the relapse of leukemia and performed DLI. The patient could obtain the complete chimerism after DLI. We report a case of chimerism analysis which was useful to predict the relapse of leukemia and perform the DLI.

ADVANCES IN RESEARCH OF TRANSPLANTATION TROLERACE / 解放军医学杂志

Recent advances in transplantation tolerance were comprehensively reviewed. The contents included the mechanisms and methods of induction and maintenance of transplantation tolerance. Some experimental protocols were introduced including mixed chimerism of allogeneic bone marrow, blockade of co-stimulation signal and transgene technology for transplantation tolerance induction. The problems and the future of clinical transplantation tolerance were objectively evaluated here.

Effects of Mixed Chimerism and Immune Modulation on GVHD, Disease Recurrence and Survival after HLA-identical Marrow Transplantation for Hematologic Malignancies

BACKGROUND: The success of allogeneic bone marrow transplantation(allo-BMT) is affected by underlying disease relapse. Although mixed chimerism(MC) is not necessarily a poor prognostic factor, several groups have suggested that MC is associated with an increased risk of disease relapse. There is evidence that patients with MC benefit from additional immunotherapy if the treatment is started in minimal residual disease status(mixed chimerism status), not in frank hematological relapse. The purposes of this study are to evaluate 1) the risk for relapse or graft rejection in correlation to persistent MC status after allo-BMT, and 2) the possibility of preventing relapse by immune modulation treatments (withdrawal or rapid taper-off of post-transplant immuno-suppression, additional interferon treatment, or the administration of donor lymphocytes) in hematologic malignancies. PATIENTS AND METHODS: Of 337 allogeneic donor-recipient pairs between March 1996 and August 1998, 12 patients who showed persistent or progressive MC and who received immune modulation treatments were evaluated. Twelve patients, median age 31 years(range 9 to 39 years), received an allo-BMT for: acute myelogenous leukemia(AML, n = 5), chronic myelogenous leukemia(CML, n = 4), acute lymphocytic leukemia(ALL, n = 3). Serial polymerase chain reaction(PCR) analysis of YNZ 22-, 33.6-minisatellites or Y chromosome-specific PCR analysis at short term intervals(pre- and post-transplant 1, 3, 6, 9, ... months) was performed. Once MC was detected, immune modulation treatments on the basis of increasing MC in an early phase of recurrence of underlying disease were started. RESULTS: Nine of 12 patients converted to complete chimerism(CC) (AML 5/5, CML 3/4, ALL 1/3). Four of 9 CC patients developed graft-versus-host disease(GVHD) grade < or = 2 during immune modulation. All were treated successfully with steroids. Three patients who were not converted to CC showed relapse of underlying diseases or graft failure. CONCLUSION: The results demonstrate that, in patients with hematologic malignancies after allo-BMT, persistent MC is associated with relapse of underlying diseases or graft failure. Furthermore, when patients receive early immune modulation treatment, MC can be changed to complete donor pattern chimerism and ultimately prevent relapse.

Detection of Mixed Chimerism after Sex-Mismatched Bone Marrow Transplantation Using Fluorescence in situ Hybridization: Engraftment and Follow-up Evaluation / 대한혈액학회지

BACKGROUND: Interphase fluorescence in situ hybridization (FISH) analysis following sex-mismatched bone marrow transplantation (BMT) is a sensitive and quantitative method to better assess the engraftment state and mixed chimerism. METHODS: Twelve patients (allogeneic 11 cases, unrelated 1 case) who underwent sex-mismatched BMT at Chonnam University Hospital from April 1996 through October 1998 were anlaysed chimerism employing FISH. Interphase FISH studies on peripheral blood cytospin slides were performed by using chromosome X alpha-satellite probe in early post-transplant periods at intervals of 3 days and in follow-up periods 6~15 months after BMT. RESULTS: In 11 engrafted patients, the mean percentage of host cells was 3.0% at the period of engraftment (15~25 days). Follow-up interphase FISH studies for ten patients with hematologic remission states after engraftment showed mixed chimerism with variable degree (mean, 4.7%: ranges, 0.5~19.0%). Although three of these patients showed host cells above 5.0% (13.2, 8.0 and 7.0%) transiently, they maintained hematologic remission states. One patient who showed 8.5% (344 days) and 14.5% (596 days) host cells proved to be an engraftement failure. CONCLUSION: Engrafted patients who obtained hematological remission showed variable mixed chimerism by FISH. When the host cells were low rate or increased transiently, mixed chimerism was not related to relapse. However, consecutive increasing host cells suggested engraftment failure or relapse. More sensitive long term follow-up FISH studies will help to evaluate and monitor engraftment status and degree of chimerism.