[Haploidentical hematopoietic stem cell transplantation in the treatment of acquired aplastic anemia in children: a single center retrospective analysis].

Objective: To investigate the safety and efficacy of haploidentical hematopoietic stem cell transplantation with different intensity conditioning regimen in the treatment of childhood aplastic anemia (AA) . Methods: Thirty-seven AA patients who underwent haploidentical transplantation in BaYi Children's Hospital Affiliated to PLA Army General Hospital from January 2013 to January 2017 were enrolled. According to the dosage of conditioning regimen, 34 patients excluding 3 other conditioning regimens were divided into high-dosage group (regimen 2, 22 cases) and low-dosage group (regimen 3, 12 cases). The data of Engraftment, graft-vs-host disease (GVHD), hematopoietic reconstitution, relapse, infection, overall survival (OS) were analyzed. The comparison between the two groups was tested by χ(2) test. Results: A total of 35 of 37 patients achieved primary engraftment; 2 cases died of regimen-related toxicity and severe infection before the infusing of the grafts. The activation rate of CMV and EBV was 60% (21/35) . Post-transplant lymphocyte disease (PTLD) of lung occurred in one case. The cumulative incidences of acute GVHD grade Ⅰ-Ⅳ and chronic GVHD were 29% (10/35) and 34% (12/35) respectively and the incidence of extensive chronic GVHD was 6% (2/35) . The median follow-up time was 18.8 (2.9-44.1) months, the OS was 92% (34/37) .All survived patients were no longer dependent on blood transfusion and none of them had recurrence. Comparing the rates of overall survival(86%(19/22) vs.100%(12/12)) and rates of chronic GVHD(40%(8/20) vs. 17%(2/12)) in regimen 2 and regimen 3 group, there were no significant difference (χ(2)=1.742, 1.841, all P>0.05) . Significant difference was found at the incidence of Ⅰ-Ⅳ acute GVHD (10% (2/20) vs. 50% (6/12) ,χ(2)=6.200, P=0.013). Conclusions: Haploidentical hematopoietic stem cell transplantation is effective and safe. It is suitable for patients who are not eligible for matched donor transplantation. Application of reduced dose preconditioning in haploid transplantation is worth exploring.

Autologous bone marrow mixed with HLA-haploidentical allogeneic marrow transplantation for treatment of patients with malignant blood diseases.

We have previously demonstrated that syngeneic marrow mixed with H-2 haploidentical marrow transplantation could provide not only protection against graft-versus-host disease (GVHD) but also anti-leukemic (GVL) effects in mice. In the present studies, we report clinical observations using autologous marrow mixed with HLA-haploidentical allogeneic marrow transplantation for treatment of patients with malignant blood diseases. Sixteen cases, including 12 with acute leukemia and four with advanced malignant lymphoma, were treated by autologous marrow, which was purged in vitro by hyperthemia (42.5 degrees C for 70 min) following incubation for 5 days with interleukin 2 (IL-2) in liquid culture and mixed with HLA haploidentical marrow cells from their sibling or parent. Acute GVHD was not observed in any patient after transplantation. Hematological rescue in the clinical setting was demonstrated in all cases but one who died early from hepatic veno-occlusive disease (VOD). Five cases who were transplanted at the time of CR2 or CR3 and in advanced phase of lymphoma, relapsed 4 to 7 months after transplantation. The relapse rate was 31.3%. None of eight patients who received allogeneic BMT within 2 h after ABMT relapsed with median follow-up of 12 months and two of them died from procedure-related complications. Seven cases are still alive and disease-free with a median follow-up of 12 months. Mixed chimerism was found in 3/6 cases, who had different sex donors, by analysis of sex chromosomes. These results show that mixed transplantation is a safe, effective and new approach to treating patients with malignant tumors. In order to detect the effects of GVL, studies are now in progress in our clinic.

[In vitro separation of bone marrow nuclear cells with methylcellulose and its application to autologous bone marrow transplantation in patients with leukemia].

We studied bone marrow separated with 0.05% methylcellulose and cryopreservatized with liquid nitrogen. The separation time was 43 minutes. The collection rates of nuclear cells and CFU-GM were 79.0 +/- 5.10% and 93.0 +/- 3.10% in normal marrow samples and 83.4 +/- 15.45% and 91.0 +/- 8.32% in those of acute leukemia patients. The rate of residual erythocytes was 12.5% and the bone marrow volume could be reduced by 55.2%. Application of this technique to autologous bone marrow transplantation in four patients with acute leukemia reconstituted all of their hemopoietic functions and no toxic side effect was found after transplantation.

Prolonged disease-free survival after ablative chemoradiotherapy and autologous bone marrow transplantation in adult malignant lymphoma.

From June 1983 to December 1991, 21 adult patients with intermediate or high-grade malignant lymphoma (ML) were treated by ablative chemoradiotherapy, including vincristine, cytosine arabinoside, BCNU and cyclophosphamide plus total lymphoid or body irradiation with boost irradiation over bulky and original tumor areas (Hd-VCCA+TL(B) I) together with autologous bone marrow transplantation (ABMT). Five patients were in advanced stage, 2 in drug-resistant relapse, 6 in drug sensitive relapse, 6 in first complete remission (CR1) and 2 in CR2. One with marrow involvement at ABMT. The 8-year disease-free survival after ABMT in patients with Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL) in 89% and 63%, respectively, with a median follow-up up to 34 months. This study demonstrated that our Hd-VCCA+TL(B) I regimen and ABMT performed early in CR or drug-sensitive relapse of adult poor prognosis lymphoma, may potentially cure more than 70% of them. The toxicity of the present treatment is tolerable. The results confirm the value of ABMT in the treatment of adult ML, and suggest that it is necessary to purge the residual tumor cells in the bone marrow at ABMT in patients with marrow infiltration, or lymphoblastic lymphoma.

Polymerase chain reaction: a method for monitoring tumor cell purge by long-term culture in BCR/ABL positive acute lymphoblastic leukemia.

We report the successful purging of leukemia cells bearing the Philadelphia chromosome and BCR/ABL transcripts by long-term marrow culture (LTC), and subsequent grafting of the purged marrow in a case of refractory acute lymphoblastic leukemia. The efficiency of the purge was evaluated by polymerase chain reaction (PCR) for BCR/ABL transcripts. In two LTCs initiated in the blastic stage, we demonstrated the selective effect of three culture media (serum dependent, serum-free (SF) supplemented or not with IL3 and GM-CSF) on the proliferative potential of normal hematopoietic (CFU-GM/BFU-E) and leukemic progenitors (CFU-ALL). BCR/ABL positive cells disappeared after 3 to 4 weeks of culture. The addition of IL3 and GM-CSF to the SF medium enhanced the growth of CFU-GM/BFU-E and shortened the purging period. We therefore carried out a LTC in the presence of IL3 and GM-CSF with marrow harvested in morphological remission. BCR/ABL positivity was detected at the outset, although no leukemia cells could be identified. The BCR/ABL was no longer found by PCR in the 7 and 14 day LTCs. The patient, consolidated by high dose polychemotherapy and total body irradiation, was infused with the 14 day LTC. This study indicates that PCR is a useful and sensitive technique for monitoring tumor cell reduction after LTC prior to autografting.

[Autologous bone marrow transplantation for the treatment of malignant lymphoma in adults].

Seventeen adult patients with malignant lymphoma, including Hodgkin's disease(HD) during relapse after first-line chemotherapy (6 cases) or in advanced stage (2 cases) and non-Hodgkin's lymphoma (NHL) of high grades after staging (9 cases) were treated with high-dose chemoradiotherapy (Hd-VCCA+TLI) and autologous bone marrow transplantation(ABMT). 16 cases (94.1%) obtained complete remission (CR) after ABMT. The current long-term disease-free probability is 86% for HD group and 62% for NHL group. One case with marrow involvement proved by marrow harvesting is in prolonged unmaintained CR for more than 3 years after ABMT with marrow purging in vitro by hyperthermia (42 C x 60 min). 4 cases with advanced disease relapsed died within two years. 2 cases with advanced lymphoblastic lymphoma relapsed and died of acute lymphoblastic leukemia. These results confirmed the value of ABMT in the treatment of adult malignant lymphoma and suggest that it is necessary to purge the residual tumor cells in the bone marrow before ABMT in the patients with marrow involvement or lymphoblastic lymphoma.

Serum-free liquid marrow culture in patients with acute lymphoblastic leukaemia: a potential application to purge marrow for autologous transplantation.

We have previously established a serum-free (SF) culture medium, which supports normal haemopoietic progenitor cell growth for at least 4 weeks as does conventional serum dependent (SD) medium. In the present study, we investigated the efficacy of such a defined SF liquid medium which sustained in vitro residual normal haemopoietic proliferation of marrow derived from ALL patients and which was detrimental for the leukaemic population. Evidence for a potential selective effect of SF culture was obtained by a leukaemic progenitor cell assay (ALL-CFU) and the detection of the bcr/abl translocation by polymerase chain reaction (PCR). In 13 experiments including 12 patients, morphological blast cells and ALL-CFU were dramatically reduced within 3 weeks of incubation in both SF and SD cultures. Likewise, in 5/5 experiments in SD and 2/5 experiments in SF conditions, leukaemic cells expressing the bcr/abl fusion gene disappeared within 3-4 weeks. In contrast, the absolute numbers of supernatant cells harvested weekly from SF and SD cultures were similar. No difference in CFU-GM production was detected for the two culture systems. Erythropoiesis in SF medium exhibited a slower decline than that found in SD. These results indicate that liquid marrow culture may selectively deplete leukaemic lymphoblastic cells and enable repopulation by residual normal haemopoietic cells. This technique may be useful to purge leukaemic cells for clinical autologous bone marrow transplantation in patients with ALL.

Studies on sensitivity of human GM-CFU and L-CFU to hyperthermic killing in vitro.

The thermal sensitivity of normal myeloid and leukaemic cells was compared using morphology, cytochemistry and cultures of granulocyte-macrophage and leukaemic progenitor cells (GM-CFU and L-CFU). We have clearly demonstrated that blast cells from eight cases of acute nonlymphoblastic leukaemia (ANLL) showed greater morphological deterioration and loss of cytoplasmic enzymes with continuous heating at temperatures of 40-43 degrees C than normal marrow mononuclear cells obtained from ten controls. Survival of L-CFU also decreased exponentially with rising temperature whereas GM-CFU were not markedly affected, even at a temperature of 43 degrees C for 30 min. These results suggest that human L-CFU are more sensitive to hyperthermic killing than normal human GM-CFU and that hyperthermia might selectively purge residual leukaemic cells in vitro. Hyperthermia may have a role in clinical autologous bone marrow transplantation (ABMT) for acute leukaemia.

In vitro studies on the pathogenesis of aplastic anemia in Chinese patients.

We studied the hemopoietic progenitor cell assays for granulocyte-macrophage colony-forming units (CFU-GM) and fibroblast colony-forming units (CFU-F) and the effects of patient marrow cells or serum on the growth of normal CFU-GM in 30 Chinese patients with aplastic anemia. We found the pathogenetic mechanisms to be complex. Defects of hemopoietic stem cells, the supporting microenvironment, and abnormal immune regulation alone or in combination were present. We found no CFU-GM or CFU-F growth in cases of chloramphenicol-induced aplastic anemia, suggesting damage not only to hemopoietic progenitors but also to marrow stromal cells. Our data demonstrate that aplastic anemia is a heterogeneous disease with multiple mechanisms leading to clinical cytopenia. The use of these techniques allows better delineation of the pathogenesis of aplastic anemia in individual patients.

Chromosomal analysis of bone marrow stromal fibroblasts in allogeneic HLA compatible sibling bone marrow transplantations.

Karyotyping was performed on bone marrow stromal fibroblasts and marrow haemopoietic cells on six patients who received bone marrow transplants from siblings of the opposite sex. Three patients with severe aplastic anaemia received unmanipulated donor bone marrow cells. Three other patients with leukaemia and conditioned with high dose chemotherapy (+ total body irradiation in two patients) received T-cell depleted marrow mononuclear cells. Marrow chromosomal analysis was performed on samples obtained between 6 weeks and 1 yr post-transplant. All marrow fibroblasts studied were of recipient origin, whereas all haemopoietic cells were of donor origin. These results demonstrate that in allogeneic sibling marrow transplantation, recipient bone marrow stromal cells regenerate and repopulate the bone marrow and this is not influenced by the conditioning regimen used, the type and dose of marrow cells given and the pre-existing disease.

Studies of hemopoietic stromal fibroblastic colonies in patients undergoing bone marrow transplantation.

We report here the results of serial bone marrow fibroblast cultures from recipients of histocompatible allogeneic bone marrow transplants (37 patients with acute leukemia, and seven with severe aplastic anemia). The mean value of marrow CFU-F growth for recipients at day 21 after transplant was significantly lower than that for normals and for patients before transplant. There were no obvious differences in the morphologic, ultrastructural, and cytochemical characteristics of the CFU-F between the normals and the patients who received transplants. The number of stromal CFU-F cells in S phase was increased during the early period after transplantation, as demonstrated by a significant reduction of CFU-F growth after short exposure to hydroxyurea. However, the return to normal was rapid, within six weeks. The presence of graft-vs-host disease (greater than or equal to grade II) and the choice of immunosuppressive drug, i.e., methotrexate or cyclosporine, did not affect the CFU-F recovery. The findings in the present study show that the marrow stromal compartment is compromised after marrow transplantation, but its regeneration is rapid. No in vitro CFU-F growth was obtained from peripheral blood of donors and patients, either before or after bone marrow transplant.