Summary:Within a prospective study we analyzed hematopoietic chimerism in serial peripheral blood samples taken from 55 patients with acute leukemias (ALL 21, AML 20, MDS 14) with a median age of 13.5 years at very short time intervals following allogeneic bone marrow transplantation (allo-BMT). The investigation was performed to determine the implications of mixed hematopoietic chimerism (MC) with regard to the clinical outcome in patients with acute leukemias after allo-BMT. Analysis of chimerism was performed by PCR of variable number of tandem repeat (VNTR) sequences with a maximum sensitivity of 0.8%. Thirteen male patients transplanted with the marrow of a female donor were also studied by amplification of a Y-chromosomespecific alphoid repeat (0.1-0.01% sensitivity). VNTR analysis in 55 patients revealed complete chimerism (CC) in 36 cases, MC in 18 follow-ups and autologous recovery in one patient. Quantitative analysis of MC identified 10/18 patients with increasing autologous patterns in whom 9/10 subsequently relapsed. The patient with autologous recovery relapsed as well. Eight of 18 patients with MC showed decreasing amounts of autologous DNA and became CC upon further follow-up. In contrast, only 7/36 patients with CC in the prior analysis of chimerism status relapsed. However, in 4/7 patients the interval between last CC confirmation and relapse was more than 4 months. In 2/7 patients autologous DNA was not detectable in peripheral blood but in bone marrow aspirates. One of these seven patients developed a fulminant relapse within 3 weeks. The probability of relapse-free survival for patients with CC is 0.67 (n = 36), for patients with decreasing MC 1.0 (n = 8) and for patients with increasing MC 0.1 (n = 10). In summary, the results demonstrate that serial and quantitative chimerism analysis at short time intervals by PCR provides a reliable and rapid screening method for the early detection of recurrence of underlying dis- ease and is therefore a prognostic tool to identify patients at highest risk of relapse.

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We report the long-term follow-up of children transplanted with Treosulfan (TREO)-based conditioning in Germany and Austria. Nine centres reported a total of 109 transplantations. Patients were stratified according to the paediatric TRM risk score derived from the paediatric BMT registry (PRST) and compared with the historical transplant population of this registry. Underlying diseases were malignancies, immunodeficiencies, and haematologic and metabolic disorders. TREO total dose ranged from 21-42 g/m 2 . Additional conditioning drugs included fludarabine, thiotepa, melphalan, CY and/or TBI. EFS at 3 years for non-malignant and malignant diseases was 88% and 49%, respectively. Leukaemia patients in remission had a survival of 51% at 3 years; nonremission patients relapsed and died within 18 months. TRM and OS in the low-risk groups 0 and 1 were similar to PRST controls. TRM in the high-risk groups 2 and 3 was markedly lower (9% vs 28% and 13% vs 53%, respectively) than in the PRST group, but OS was similar.In conclusion, TREO-based conditioning regimens in children resulted in excellent engraftment and long-term survival in nonmalignant disease. In high-risk malignancy, low acute toxicity was followed by low TRM but it did not translate into increased survival.

Summary:Case reports Patient No. 1 The success of allogeneic BMT (allo-BMT) in children with acute leukemias is mainly affected by relapse.The first patient, a 2-year-old boy with MDS (CMML and There is evidence that these patients have only a little monosomy 7), was referred for BMT. After conditioning or no benefit from additional immunotherapy if the with busulfan (BU) 20 mg/kg, etoposide (VP16) 40 mg/kg treatment is started in frank hematological relapse.and cyclophosphamide (CY) 120 mg/kg, the patient was Recently we were able to demonstrate that pediatric grafted with the marrow of an HLA-identical family donor patients with acute leukemias and increasing mixed (cousin). The MLC reaction was negative. GVHD prophychimerism (MC) post-transplant have a significantly laxis consisting of cyclosporin A (CsA) 3 mg/kg/day was enhanced risk of developing relapse. We asked whether started on day −3. Unmanipulated bone marrow comprising there is a possibility of preventing relapse, eg by with-3.7 × 10 8 /kg nucleated cells was transfused on day 0. After drawal of post-tranplant immunosuppression or by engraftment chimeric analysis on day +24 revealed increasadministration of donor lymphocytes in an early phase ing autologous patterns until no donor hematopoiesis was of the development of relapse. We present the case detectable on day +70 (Figure 1) when typical dysplastic reports of two children (MDS and AML) with rapidly cells and leukemic blasts with recurrence of monosomy 7 increasing MC in whom withdrawal of post-transplant were detected in the peripheral blood. Thereafter the disimmunosuppression or donor lymphocyte infusion ease was controlled by treatment with thioguanine until 10 (DLI) did prevent relapse. months after BMT. At that time leukocytes increased and Keywords: acute leukemia; hematopoietic chimerism; additional therapy with cytarabine (Ara-C) 20 mg/day for adoptive immunotherapy 5 days and hydroxyurea 250 mg/day was initiated. One year after the first transplantation, the patient was reconditioned with fractionated total body irradiation (TBI) (6 × 2 Gy), thiotepa 10 mg/kg, CY 120 mg/kg and antilymphocyte Although allogeneic BMT (allo-BMT) has been performed globulin (ALG) 30 mg/kg/day for 3 days. The boy was rewith increasing success in the situation of particular unfavinfused with 2.5 × 10 8 /kg nucleated bone marrow cells from orable acute leukemias in children over the last decades, the same donor on day 0. CsA was started at 3 mg/kg/day the outcome is mainly affected by relapse in this cohort of patients. 1 Treatment by which long-term remissions could be re-achieved is mainly restricted to additional marrow transplantation with a significantly increased rate of complications. 2,3 Recently we were able to demonstrate that patients with increasing amounts of autologous marrow repopulation (mixed chimerism MC) after allo-BMT have an increased risk (P = 0.0078) of developing relapse or graft rejection. 4 We subsequently asked whether treatment modalities, eg withdrawal of post-transplant immunos...

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In summary adenovirus specific T-cell reconstitution should be monitored in patients after SCT to limit the use of anti viral chemotherapy and help to identify those patients that would benefit from new therapeutic strategies like adoptive transfer of virus specific T-cells.

Natural killer (NK) cell activity has been shown to have potential activity against Ewing's sarcoma (EWS) especially in tumors with low HLA I expression and high NKG2D expression. Two patients with metastatic relapsed and primary metastatic stage IV EWS who had received two courses of high dose chemotherapy with autologous stem cell rescue were transplanted from a haploidentical parental stem cell donor. Patients are alive in ongoing CR for 10.2 and 3.4 years now. Post transplant local second and first relapses were treated successfully in both patients. In vivo IL-2 stimulation not only increased the number and activity of effector cells in one patient but was also associated with severe GvHD. In vitro studies demonstrated high NK cell activity against K562 and relevant activity against EWS cell line A673 post transplant. NK activity was enhanced by cytokine prestimulation as well as by EWS targeting anti-GD2 Ab. Haploidentical hematopoietic stem cell transplantation (HSCT) might contribute to long-term survival by NK cellmediated effect exerted by donor-derived NK cells. Local tumor recurrence was manageable in both high-risk patients indicating systemic immune control preventing subsequent metastasizing. The efficacy of haploidentical HSCT, cytokine application and tumor targeting antibodies for the use of Ab-dependent cellular cytotoxicity needs evaluation in clinical trials.

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