New strategies for allogeneic bone marrow transplantation and organ allografts

In humans, the success rate of bone marrow transplantation (BMT) across major histocompatibility complex (MHC) barriers is not high due to: (1) graft-versus-host reaction (GvHR); (2) graft rejection, and (3) incomplete T cell recovery. In mice, GvHR can be prevented if T cell- depleted bone marrow cells (BMCs; <2% T cells) are used. Graft rejection can be prevented by either bone grafts (to recruit donor-derived stromal cells) or the injection of donor BMCs via the portal vein (p.v; to induce donor-specific tolerance). T cell functions are recovered by BMT plus bone grafts if the thymic functions of recipients are not completely lost. After the complete loss of thymic functions (due to aging), BMT plus embryonal thymus grafts should be carried out.Recently, we have found that persistent donor-specific tolerance can be induced if allogeneic hemopoietic stem cells are injected via the p.v. Based on these findings, we have established new strategies for organ allografts. Without irradiation, donor BMCs should be injected from the p.v. injection on day 0 plus i.v. injection on day 5, and an immunosuppressant (CsA or FK506) should be used on days 2 and 5. Without using immunosuppressants, sublethal irradiation (7 Gy) followed by skin allografts plus allogeneic BMC injection via the p.v. should be carried out. This leads to a 100% acceptance of skin and pancreas allografts for more than 300 days. The recipient mice show mixed allogeneic chimerism, and spleen cells from the recipients show tolerance to both donor-type and host-type MHC determinants in the assays for mixed lymphocyte reaction and generation of cytotoxic T lymphocytes. We have confirmed that these strategies are applicable to other animals such as pigs and rats. We therefore believe that they will become viable and valuable strategies for human organ allografts.     

T-cell depletion versus methotrexate as GvHD-prophylaxis in allogeneic bone marrow transplantation for leukaemia

Graft-versus-host disease (GvHD) prophylaxis using methotrexate (23 patients) and T-cell depletion of the graft (40 patients) was compared in 63 allogeneic bone marrow transplantations (BMT) for leukaemia. T-cell depletion significantly reduced (p = 0.001) the incidence of GvHD from 68% to 11% and the GvHD-associated mortality from 79% to 5%. Actuarial disease-free survival for low-risk patients (57% with T-cell depletion and 47% with MTX) was not significantly improved, due to graft failure and possibly due to a higher leukaemic relapse rate after T-cell depletion. Prevention of graft failure after T cell-depleted BMT is essential and could also reduce the risk of leukaemic relapse by improved engraftment.     

Second marrow transplants in patients with leukemia who relapse after allogeneic marrow transplantation

Twenty-six patients with recurrent leukemia following allogeneic marrow transplantation received a second marrow transplant between 1.5 and 78 months (median 26) after the initial transplant. Preparative regimens for second transplant included multi-agent chemotherapy with total body irradiation, 2.0-10.0 Gy (five patients), dimethylbusulfan alone (one patient), and dimethylbusulfan or busulfan plus cyclophosphamide (20 patients). One patient died before engraftment of infection and 18 died after engraftment from veno-occlusive disease (4), infection (2), idiopathic pneumonia (3), cytomegalovirus pneumonia (3), leukemia (5) and encephalopathy (1). Seven patients (27%) survive 12-38 months (median 26); five (19%) are disease-free and two have recurrent leukemia. Two of the five disease-free survivors have chronic graft-versus-host disease. All of the surviving patients received dimethylbusulfan or busulfan plus cyclophosphamide and six of the seven surviving patients were among 11 patients transplanted more than 2 years after the first transplant whereas only one was among the 15 transplanted in less than 2 years. Those who have second marrow transplants one or more years after their initial transplant are more likely to benefit, while those who are less than 1 year from initial transplant appear to benefit the least.     

Prospective comparative trial of autologous versus allogeneic bone marrow transplantation in patients with non-Hodgkin's lymphoma

A prospective comparative trial of allogeneic versus autologous bone marrow transplant (BMT) was conducted. Sixty-six consecutive patients (median age, 41; range, 15 to 60; female:male ratio = 21:45) entered this clinical trial. Priority for allogeneic BMT was given to patients who were 55 or younger and had a major histocompatibility complex- matched or 1-antigen-disparate sibling donor. Autologous BMT was offered to all other patients whose age was 60 or younger. Patients who had no sibling donor and who had BM involvement at the time of evaluation were not eligible. Thirty-one patients received an allograft, and 35 patients received an autograft. Thirteen patients received a BM graft purged with 4-hydroperoxycyclophosphamide because of previous BM involvement. Patients who had previous radiation to the thoracic and/or abdominal areas of more than 20 Gy received a preparative regimen consisting of cyclophosphamide (1,800 mg/m2/d for 4 days), VP-16 (200 mg/m2 every 12 hours for 8 doses), and 1,3-bis(2- chloroethyl)-1-nitrosourea (600 mg/m2 as 1 dose). Other patients received cyclophosphamide 1,800 mg/m2/d for 4 days followed by total body irradiation of 12 Gy administered as a single daily fraction over 4 days. With a median follow-up of 14 months, the progression-free survival (PFS) for autograft and allograft recipients was 24% +/- 8% (+/- SE) and 47% +/- 9%, respectively, (P = .21). However, the probability of disease progression was significantly higher in the autologous group (69% +/- 9%) than in the allogeneic group (20% +/- 10%; P = .001). When other confounding prognostic factors were adjusted in the multivariate analysis, chemosensitive disease and allograft were found to have a significant favorable influence on probability of disease progression (P = .03 and .003), but only chemosensitive disease had a significant influence on the PFS (P < .002). Our results suggest the existence of graft-versus-lymphoma effect and also support the rationale of using immunotherapy after autologous BMT. Allogeneic BMT should be preferable to autologous BMT in younger patients with lymphoma.     

Mycobacterium tuberculosis infection in allogeneic bone marrow transplantation patients.

Bone marrow transplant (BMT) recipients are prone to bacterial, viral and fungal infections. Mycobacterium tuberculosis infection can occur in these patients, but the incidence is lower than that of other infections. This report describes four patients with Mycobacterium tuberculosis infection identified from 641 adult patients who received a BMT over a 12-year period (prevalence 0.6%). The pre-transplant diagnosis was AML in two patients and CML in the other two. Pre-transplant conditioning consisted of BU/CY in three patients and CY/TBI in one. Graft-versus-host disease (GVHD) prophylaxis was MTX/CsA in three patients and T cell depletion of the graft in one patient. Sites of infection were lung (two), spine (one) and central nervous system (one). Onset of infection ranged from 120 days to 20 months post BMT. Two patients had co-existing CMV infection. One patient had graft failure. The two patients who received anti-tuberculous (TB) therapy recovered from the infection. Although the incidence of tuberculosis in BMT patients is not as high as in patients with solid organ transplants, late diagnosis due to the slow growth of the bacterium can lead to delay in instituting anti-TB therapy. A high index of suspicion should be maintained, particularly in endemic areas.     

Lymphotropic herpesviruses in allogeneic bone marrow transplantation

Human herpesvirus-6 (HHV-6), human herpesvirus-7 (HHV-7), Epstein-Barr virus (EBV), and human cytomegalovirus (CMV) DNA were repeatedly assayed in peripheral blood leukocytes from 37 allogeneic bone marrow transplant (BMT) patients by polymerase chain reaction. Before BMT, HHV- 6 DNA was detected in 8 (22%) patients. HHV-7, EBV, and CMV DNA were detected in 21 (57%), 10 (27%), and 1 (3%) patient, respectively. After BMT, HHV-6 DNA was detected in 26 (70%), HHV-7 in 21 (57%), EBV in 28 (76%), and CMV in 21 (57%) patients. Thirty-two (87%) patients were positive with more than one virus. HHV-6, HHV-7, and EBV DNA were found earlier than CMV DNA in most patients after BMT. The proportions of HHV- 6-positive samples during the first 3 months after BMT were higher in the patients with either delayed granulocyte engraftment (P = .04, Fisher's exact test) or delayed platelet engraftment (P = .001, Fisher's exact test). The HHV-6 DNA in samples from the patients with delayed engraftment was confirmed to be variant B. The detection of any lymphotropic herpesvirus was not related to the development of acute graft-versus-host disease (aGVHD). High-dose acyclovir (ACV) prophylaxis significantly (P < .01) reduced the proportion of HHV-6- positive samples and tended to lower HHV-6 DNA levels (P = .06). Our data indicate that HHV-6 variant B can inhibit marrow engraftment and that high-dose ACV may be beneficial to engraftment after BMT by preventing HHV-6 reactivation. No relation between the proportions of HHV-7-, EBV-, and CMV-positive samples in the first 3 months and engraftment or aGVHD was found.     

The impact of early CD4+ lymphocyte recovery on the outcome of patients who undergo allogeneic bone marrow or peripheral blood stem cell transplantation

Background

Different factors influence the clinical outcome of allogeneic transplants, the foremost being good immune recovery.

Materials and methods

The purpose of this study was to evaluate the influence of different factors, such as stem cell source, type of donor, conditioning regimen and acute graft-versus-host disease, on early lymphocyte recovery after transplantation. We then analyzed the impact of early CD4+ cell count on overall survival, transplant-related mortality and disease-related mortality.

Results

Univariate analysis with Spearman’s rho showed a significant correlation between early CD4+ cell recovery and overall survival, transplant-related mortality, stem cell source and type of donor. In multivariate analysis CD4+ cell count was significantly associated with (i) stem cell source, being higher in patients whose haematopoietic progenitor cells were obtained by apheresis than in those whose source of grafted cells was bone marrow, and (ii) type of donor, being higher in patients transplanted from sibling donors than in those whose graft was from an alternative donor. The ROC curve of CD4+ cell count indicated that a cut-off of 115 CD4+ cells/mL could differentiate groups with different outcomes. At 2 years follow-up, patients achieving this CD4+ cell count had significantly lower cumulative transplant-related mortality compared to patients who did not have this count (10%±4% versus 40%±8%, p=0.0026). At the 5-year follow-up, the overall survival rates were 77.5%±0.6% and 36%±7% (p=0.000) in patients with a CD4+ cell count ≥115/mL and in patients with CD4+ cell count ≤ 115/mL, respectively.

Conclusion

Early CD4+ cell recovery after allogeneic transplantation has a relevant impact on overall survival and transplant-related mortality and is influenced by two factors: stem cell source and type of donor.     

骨髓移植术后急性肠道移植物抗宿主病内镜及病理学诊断研究

目的评价内镜和病理学诊断在急性肠道移植物抗宿主病(GVHD)中的作用。方法回顾性总结和分析2001—2005年北京大学人民医院血液病学研究所临床已确诊的23例急性肠道GVHD患者的内镜及病理学资料。结果内镜下表现可分为:黏膜大致正常、血管纹理模糊或消失、弥漫性黏膜充血、水肿和脆性增加,重者出现糜烂、溃疡,甚至黏膜脱落或出血;病理学表现提示:隐窝上皮细胞凋亡、缺失,隐窝结构破坏,上皮和黏膜固有层不同程度的淋巴细胞浸润。结论内镜和病理学检查可以用于急性肠道GVHD的诊断,尤其是内镜在急性肠道GVHD的早期诊断方面具有重要作用,同时结合病理学检查最终确立诊断。     

Differences between lymphocyte subsets, after allogeneic bone marrow transplantation, in patients who received tacrolimus and patients who received cyclosporin A

The subsets of peripheral blood lymphocytes after allogeneic bone marrow transplantation were compared in 20 patients who received tacrolimus and 34 patients who received cyclosporin A (CsA) prophylactically. The phenotypes of CD3, CD4, CD8, and D8/CD57 were analyzed by flow cytometry. The percentage of CD3+ cells in the tacrolimus group (58.8% +/- 21.6%) was significantly lower than in the CsA group (77.2% +/- 12.8%) (P = .0239). The percentage of CD8+CD57+ cells in the patients receiving tacrolimus and developing acute graft-versus-host disease (GVHD) (grade I, 20.1% +/- 10.6%; grade II-IV, 13.2% +/- 6.3%) was significantly higher than in the patients receiving CsA and developing acute GVHD (grade I, 10.7% +/- 5.2%; grade II-IV, 7.7% +/- 4.0%) (grade I, P = .0036; grade II-IV, P = .0255). The absolute number of CD8+CD57+ cells in the patients with grade II-IV acute GVHD was also significantly higher in the tacrolimus group compared with the CsA group. There was no difference in the incidence of acute GVHD in the 2 groups. Recovery from acute GVHD in the tacrolimus group (16.6 +/- 13.6 days) was more rapid than in the CsA group (30.8 +/- 24.8 days) (P = .0124). These results suggest that, compared with CsA, tacrolimus administered prophylactically induces more CD8+CD57+ lymphocytes when acute GVHD occurs and accelerates the recovery from acute GVHD more rapidly.     

Conditioning regimens for allogeneic bone marrow transplantation.

Conditioning regimens for transplantation are important in determining transplant outcome. This review focuses on transplantation in aplastic anemia and leukemia using marrow from HLA-identical siblings. Results of conditioning with newer regimens such as busulfan plus cyclophosphamide and etoposide plus total body irradiation are reviewed and compared to results achieved with cyclophosphamide and total body irradiation. The potential for improved results using recent innovations such as dose adjustment of busulfan, agents which may decrease transplant-related toxicity, and directed radiation are discussed.     

Risk-adapted pre-emptive therapy for cytomegalovirus disease in patients undergoing allogeneic bone marrow transplantation

We prospectively evaluated a risk-adapted pre-emptive treatment with ganciclovir for CMV diseases in patients undergoing allogeneic bone marrow transplantation (BMT). High-level CMV antigenemia (10 or more positive cells on two slides) or CMV antigenemia at any level in patients with grade II-IV acute graft-versus-host disease (aGVHD) were chosen as risk factors. We also retrospectively evaluated virus reactivation in plasma using quantitative real-time polymerase chain reaction (PCR). Fifty patients were evaluable. None of the 27 patients with or without grade I aGVHD developed high-level CMV antigenemia or CMV disease. Among the 23 patients with grade II-IV aGVHD, 12 patients (52%) developed CMV antigenemia and were treated pre-emptively, of whom two developed CMV gastroenteritis or retinitis in spite of therapy. Six of the remaining 11 patients developed CMV gastroenteritis before CMV antigenemia was detectable. All of the eight patients with CMV diseases were successfully treated with ganciclovir and no deaths directly related to CMV disease occurred. In four of the seven evaluable patients with CMV gastroenteritis, real-time PCR was able to detect virus reactivation earlier than CMV antigenemia. Although our risk-adapted pre-emptive therapy effectively reduced CMV-related mortality, further refinements of this approach, particularly in the prevention of CMV gastroenteritis, may be achieved by incorporating real-time PCR.     

Constitution and telomere dynamics of bone marrow stromal cells in patients undergoing allogeneic bone marrow transplantation

We evaluated the genotypic origin of mesenchymal stem cells (MSC) following sex-mismatched allogeneic bone marrow transplantation (BMT), and investigated the telomere dynamics in MSC in normal individuals and patients after BMT. The study population consisted of 11 patients with hematologic disorders who showed complete chimerism after BMT. Telomere length was measured in MSC using Southern blotting analysis in eight patients and 18 healthy subjects as a control group. Following culture, MSC were identified by the expression of SH2 and SH4, and lack of CD14, CD34, and CD45. All MSC showed the recipient genotype, based on the results of fluorescent in situ hybridization analysis using X-chromosome satellite probes or microsatellite DNA polymorphism analysis. The mean telomere length in MSC from normal controls was 7.2+/-0.53 kb (range, 6.12-7.78), and progressive telomere shortening was seen with age. There was no significant difference in MSC telomere length between the BMT group and age-matched controls. This study confirmed that the MSC isolated from the recipients of allogeneic BMT did not have the donor genotype, despite complete chimerism. Moreover, MSC were demonstrated to show progressive loss of telomere length with age, but the telomeres in MSC were not affected by BMT.     

Cyclosporine as prophylaxis for graft versus host disease in adults undergoing allogeneic bone marrow transplantation

Fifteen adult patients undergoing allogeneic bone marrow transplantation (BMT) received cyclosporine (CSP) as prophylaxis of graft versus host disease (GVHD). In our patients eleven were hematologic malignancies, and four were severe aplastic anemia. Twelve patients were HLA-matched, and three were one locus mismatched. Three patients received CSP only, twelve received CSP and short term methotrexate. Seven patients had acute GVHD, but GVHD over Grade II were seen in only 3 patients who were transplanted from HLA-one locus mismatched donor. 7 patients had chronic GVHD. CSP were given intravenously at 3-5 mg/kg, starting 1 day before BMT. From about day 30, CSP was given orally. CSP concentrations when patients were given orally were lower in patients who had chronic GVHD. Although hypertension and water retention were seen in 8 patients, and renal dysfunction was seen in 3 patients, the side effects of CSP were mild and transient. There were no correlations between serum concentrations and the side effects of CSP. Three patients had the disturbance of hematopoiesis. Ten of fifteen patients are alive at median follow-up of 18.5 months (8-41 months) after BMT.     

Lung function in allogeneic bone marrow transplantation recipients

In order to investigate the incidence of pulmonary function complications following bone marrow transplantation (BMT), 17 patients with leukaemia and 8 with aplastic anaemia were sequentially assessed over a one year period. Before BMT, all the patients were free of respiratory symptoms and had both normal chest X-ray and routine lung function tests. However, 5 patients disclosed airway hyperreactivity. Aplastic anaemia patients had significantly lower haemoglobin-adjusted diffusing capacity for carbon monoxide (DLCO) than those with leukaemia, a finding significantly related to the lower haemoglobin values shown in the former individuals. Following BMT there were transient mild to moderate reductions in DLCO and static lung volumes; moreover, patients with leukaemia had lower DLCO than those with aplastic anaemia. Fourteen of the 25 patients had ventilatory defects, including 10 individuals with bronchial hyperresponsiveness. Post-BMT lung function changes were transiently accompanied by mild to moderate symptoms of respiratory disease in most of the patients.     

Factors affecting survival in allogeneic bone marrow transplantation

From 1979 to 1988, 82 allogeneic and 2 syngeneic bone marrow transplants (BMT) were performed in 78 patients (age range 13-49 years) with the following diagnoses: acute myelogenous leukemia (AML) (21 patients); acute lymphoblastic leukemia (ALL) (15 patients); chronic myelocytic leukemia in chronic, accelerated, or blastic phase (CML-CP, AP or BC) (25 patients); myelodysplastic syndrome (MDS) (1 patient); multiple myeloma (MM) (1 patient); Hodgkin's disease (HD) (1 patient); diffuse poorly differentiated lymphoma (DPDL) (1 patient); aplastic anemia (AA) (13 patients). Univariant analyses were carried out to determine factors of importance in predicting outcome. AML patients receiving transplants in remission had 12/19 (63%) survivors. Only one of seven ALL patients receiving transplants in remission survives free of disease, and none of eight patients receiving transplants in relapse survived. Six ALL patients relapsed. In CML, 6 of 16 (40%) patients receiving transplants in CP survive; two of nine patients (22%) in AP or BC survive. Of the 13 aplastic anemias, 8 (62%) survive. Graft-vs.-host disease (GVHD) was evaluated in 75 patients, 24 of 33 (73%) who developed GVHD died, compared to 24 of 44 (55%) who did not develop GVHD. Of the 30 patients given the combination of methotrexate (MTX) plus cyclosporine (CSP), only 23% developed GVHD, compared to 58% of those not given the combination. Interstitial pneumonia (IP) occurred in 16 patients and was fatal in 15. The introduction of daily acyclovir and weekly intravenous gamma globulin in 1985 was associated with little reduction in the frequency of IP (from 20% to 18%). However, survival increased from 21% to 47%.(ABSTRACT TRUNCATED AT 250 WORDS)