Stem cell transplantation.

Stem cell transplantation in pediatric patients with leukemia has been a matter of debate since the first successful transplantation in a child with aplastic anemia in Germany in 1975. Since then, there has been a long way to implement fully stem cell transplantation strategies into the treatment studies organized within BFM Groups for first line or second line treatment of acute and chronic leukemias and myelodysplastic syndrome. The role of different risk groups, different donors, stem cell sources and alternative approaches as haploidentical transplantation as well have been studied as the impact of chimerism and minimal residual disease. Finally, for all leukemias and myelodysplastic syndromes integrated treatment pathways have been developed, which integrate stem cell transplantation on an evidence base into the treatment of the patient.

Identification and selective depletion of alloreactive T-cells for adoptive immunotherapy.

BACKGROUND: T-cell-depleted allografts may exhibit delayed T-cell recovery, severe infections, and relapse after haploidentical hematopoietic stem cell transplantation (HSCT). Required donor lymphocyte infusions containing nonalloreactive cells may transfer immune function without causing graft-versus-host disease. METHODS: We developed an ex vivo approach for the immunomagnetic depletion of alloreactive CD25+, CD69+, and HLA-DR+ T-cells. To achieve highest rates of alloantigen expression, we cocultured peripheral blood mononuclear cells (PBMNCs) with PBMNCs (A/B*), dendritic cells (A/B* DCs), or cytokine-pretreated PBMNCs (A/B* cyt cells). Functional analyses were performed after depletion. RESULTS: After coculture with PBMNCs (A/B* cells), 29% of T-cells became CD25+, CD69+, and HLA-DR+. In modified mixed lymphocyte reactions (MLR) (A/B* cyt cells and A/B* DCs), 35% and 37% of T-cells became CD25+, CD69+, and HLA-DR+. Alloactivation was confirmed by interferon gamma release and proliferation. Immuno-magnetic depletion produced <1% alloactivated cells. Furthermore, this depletion strategy was allospecific and hardly impaired the immune function of the retained cells. DISCUSSION: The efficiency of immunomagnetic depletion depended on the stimulatory capacity of stimulator cells and was improved by using cytokine-pretreated PBMNCs for alloactivation. Overall, this approach might be a promising strategy for restoration of the immune system, particularly after haploidentical HSCT.

Long-term results of five consecutive trials in childhood acute lymphoblastic leukemia performed by the ALL-BFM study group from 1981 to 2000.

Between 1981 and 2000, 6609 children (<18 years of age) were treated in five consecutive trials of the Berlin-Frankfurt-Münster (BFM) study group for childhood acute lymphoblastic leukemia (ALL). Patients were treated in up to 82 centers in Germany, Austria and Switzerland. Probability of 10-year event-free survival (EFS) (survival) improved from 65% (77%) in study ALL-BFM 81 to 78% (85%) in ALL-BFM 95. In parallel to relapse reduction, major efforts focused on reducing acute and late toxicity through advanced risk adaptation of treatment. The major findings derived from these ALL-BFM trials were as follows: (1) preventive cranial radiotherapy could be safely reduced to 12 Gy in T-ALL and high-risk (HR) ALL patients, and eliminated in non- HR non-T-ALL patients, if it was replaced by high-dose and intrathecal (IT) MTX; (2) omission of delayed re-intensification severely impaired outcome of low-risk patients; (3) 6-month-less maintenance therapy caused an increase in systemic relapses; (4) slow response to an initial 7-day prednisone window was identified as adverse prognostic factor; (5) condensed induction therapy resulted in significant improvement of outcome; (6) the daunorubicin dose in induction could be safely reduced in low-risk patients and (7) intensification of consolidation/re-intensification treatment led to considerable improvement of outcome in HR patients.

Efficient in vitro generation of adult multipotent cells from mobilized peripheral blood CD133+ cells.

OBJECTIVES: To generate non-haematopoietic tissues from mobilized haematopoietic CD133(+) stem cells. MATERIALS AND METHODS: Mobilized peripheral blood CD133(+) cells from adult healthy donors were used. In vitro ability of highly enriched CD133(+) cells from mobilized peripheral blood to generate multipotent cells, and their potential to give rise to cells with characteristics of neuroectoderm, endoderm and mesoderm layers was investigated. RESULTS: We found that a recently identified population of CD45(+) adherent cells generated in vitro after culture of highly purified CD133(+) cells for 3-5 weeks with Flt3/Flk2 ligand and interleukin-6 can, in presence of the appropriate microenvironmental cues, differentiate into neural progenitor-like cells (NPLCs), hepatocyte-like cells and skeletal muscle-like cells. We have termed them to be adult multipotent haematopoietic cells (AMHCs). AMHC-derived NPLCs expressed morphological, phenotypic and molecular markers associated with primary neural progenitor cells. They can differentiate into astrocyte-like cells, neuronal-like cells and oligodendrocyte-like cells. Moreover, AMHC-derived NPLCs produced 3,4-dihydrophenylalanine and dopamine and expressed voltage-activated ion channels, suggesting their functional maturation. In addition, AMHC-derived hepatocyte-like cells and skeletal muscle-like cells, showed typical morphological features and expressed primary tissue-associated proteins. CONCLUSION: Our data demonstrate that AMHCs may therefore serve as a novel source of adult multipotent cells for autologous replacement cell therapies.

Haematopoietic stem cell transplantation trends in children over the last three decades: a survey by the paediatric diseases working party of the European Group for Blood and Marrow Transplantation.

This paper describes the trends in haematopoietic stem cell transplantation (HSCT) activity for children in Europe over the last three decades. We analysed 31,713 consecutive paediatric HSCTs reported by the European Group for Blood and Marrow Transplantation (EBMT) centres between 1970 and 2002. Data were taken from the EBMT registry and were compared according to period and centre category (paediatric or combined). Since 1996, there has been a significant increase in the number of HSCTs performed exclusively by paediatric centres, as well as in the number of alternative donor HSCTs, and in the use of peripheral blood stem cells (P<0.0001). The number of allogeneic HSCTs (allo-HSCTs) for acute lymphoblastic leukaemia, acute myeloblastic leukaemia and chronic myeloid leukaemia remained stable, whereas it increased for myelodysplastic syndromes and lymphomas, and decreased significantly for non-malignant diseases (P<0.0001). Multivariate analysis showed that younger age, human leukocyte antigen genoidentical donors, HSCT performed after 1996 and transplant centres performing more than 10 allo-HSCT/year were all associated with decreased transplant-related mortality (TRM) (P<0.0001). The number of autologus HSCTs (auto-HSCTs) for acute leukaemia decreased significantly, whereas it increased for solid tumours (P<0.0001). Multivariate analysis showed that both auto-HSCT performed before 1996 and paediatric solid tumours (P<0.0001) had higher TRM. Indications for paediatric HSCT have changed considerably during the last seven years. These changes provide tools for decision making in health-care planning and counselling.

Adenoviral infections after transplantation of positive selected stem cells from haploidentical donors in children: an update.

UNLABELLED: We present updated results of stem cell transplantation with highly purified stem cells from haploidentical parental donors and infection with human adenovirus (HAdV) post stem cell transplantation (SCT). Survival post SCT is primarily determined by relapse, infections and far less by GvHD or other transplant related mortality. During the immune reconstitution the host is at significant risk for severe viral infections. HAdV infection is especially in children an important complication post SCT, with significant morbidity and mortality despite new antiviral treatment strategies. Although control of infection seems to require T-cells, the characterization of HAdV-specific T-cells post SCT has not been introduced in surveillance and treatment decisions. METHODS: Therefore we evaluated the impact of HAdV-infections on the survival between 1995 and 2004 (n = 63) and studied the occurrence of adenovirus-specific T-cells in children with (n = 9) and without (n = 9) HAdV-infection post allogeneic SCT and in healthy donors (n = 53). After stimulation ex-vivo with HAdV-antigen IFN-gamma secreting T-cells were analyzed by flowcytometry and defined as HAdV-specific T-cells. RESULTS: Until day 180 post SCT the cumulative incidence of all lethal viral infections (HAdV n = 5, cytomegalovirus n = 3, herpes simplex virus n = 1) was 16 % for the whole cohort of patients. Cumulative incidence of HAdV-associated mortality was 8.5 %. Cumulative incidence of all lethal viral infections could be now reduced from 16 % to 8 % in conjunction with new surveillance- and therapeutic-strategies. Children with HAdV-associated mortality all had no specific T-cells, although reconstitution of absolute lymphocyte counts exceeded 300/microl within 30 days post transplant. Patients who cleared HAdV infection had normal frequencies of HAdV-specific T-cells until day 200 post SCT. CONCLUSION: 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.

A comparison between three graft manipulation methods for haploidentical stem cell transplantation in pediatric patients: preliminary results of a pilot study.

Transplantation of hematopoietic stem cells from mismatched related donors makes a potential donor available for every child in need of stem cell transplantation. Here, we compare three different graft manipulation methods in patients with leukemias and lymphomas: positive selection of stem cells with either CD34 (n = 39) or CD133-coated magnetic microbeads (n = 14) and a new strategy which depletes T- and B-cells through the use of CD3- and CD19-coated microbeads (n = 11). Median purity of stem cells was comparable after CD34 (+)-selection and CD133 (+)-selection, whereas stem cells were only slightly enriched after CD3 (+)/CD19 (+)-depletion (97.5 %, 93.4 % and 1.02 %). Indirect depletion of T-cells by positive selection resulted in 1 x 10 (4) (median) residual CD3 (+)-cells/kg (0.7-3 x 10 (4)). Patients with CD3/CD19-depleted grafts received 3.2 x 10 (4) (median) (0.7-16 x 10 (4)) residual T-cells/kg. Those grafts also comprised NK-cells (median number: 86 x 10 (6)/kg), dendritic cells and monocytes/granulocytes. Primary engraftment of the stem cell products was comparable after CD34- and CD133-selection (85 and 72 %). In the CD3/CD19 group, 91 % had a primary engraftment. After reconditioning, all patients (64/64) were finally engrafted. Patients with CD34-selected or CD133-selected grafts had similar incidences of a GvHD II-IV (3 and 7 %), whereas a GvHD was slightly increased in patients receiving CD3/CD19-depleted cells (27 %). Reconstitution of CD3 (+) T-cells was faster in the CD3/CD19 group than in the CD34 or CD133 group. These preliminary results indicate, that CD3/CD19-selected grafts may be advantageous regarding engraftment and immunoreconstitution. Since effector cell with potential antileukemic activity are cotransfused, such grafts may be suited in particular for patients with insufficient remission.

Rituximab mediates in vitro antileukemic activity in pediatric patients after allogeneic transplantation.

Relapse is a major problem after allogeneic transplantation in children with acute B-lineage lymphoblastic leukemias (ALL) and lymphomas and additional therapeutic strategies are needed to increase graft versus leukemia effects without inducing graft versus host disease (GvHD). Several studies have shown the efficacy of a humanized CD20 antibody (rituximab) for treatment of CD20+ malignancies together with conventional chemotherapy but less is known about its post transplant usefulness. We studied the ability of rituximab to mediate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) with effector cells and complement from patients who were transplanted with T-cell-depleted grafts from unrelated or mismatched related donors. Highest lytic activity (ADCC) was observed against leukemia-derived MHH4 cells and Burkitt's lymphoma-derived Raji cells in the first months after transplantation, corresponding to the high percentage of regenerating CD56+ CD16+ cells. Moreover, primary cryopreserved ALL-blasts from a pediatric patient were also efficiently lysed. Increased lysis was obtained after stimulation with interleukin-2. Combination of ADCC and CDC had additive effects. These findings encourage clinical trials on the use of rituximab for improving minimal residual disease control and relapse prevention after allogeneic high-risk transplantation in the small group of pediatric patients with CD20+ leukemias/lymphomas.

Survey on haematopoietic stem cell transplantation for children in Europe.

A recent report, prepared in March 2003, regarding the paediatric transplantation activity registered between 1970 and 2002 in the European Bone Marrow Transplantation (EBMT) database showed a decrease in the number of registrations in 2001 and in 2002. In order to validate this observation, the Paediatric Diseases Working Party (PDsWP) secretariat distributed a questionnaire to 395 institutions participating in the EBMT Registry. Each institution was requested to check the number of transplants they reported and to confirm or to correct the figures. As of 15 March 2004, replies had been received from 135 centres reporting a median of 48 transplants per centre over the study period, total 17 891 (58% of the total number). Among them, 55 confirmed their original figures, while 80 corrected the numbers. The overall number of autologous and allogeneic SCTs performed and not reported were 461 and 692, respectively. Most of the teams that corrected their figures stated that their data managers could provide missing data to the EBMT; 260 other teams, each reporting a median of 15 transplants during the study period, total 12 866 (42% of the total number) chose not to reply. A report prepared in March 2004, following the PDsWP survey, showed an increasing number of transplants performed on patients below 18 years of age between 1973 and 2002 and reported to the EBMT Registry (328 autologous and 628 allogeneic) as compared to the 2003 report. This first PDsWP survey, reaching more than 50% of activity in the field, illustrates that the decrease in activity we observed in the 2003 report does not correspond to a decrease in the number of transplants that were actually performed. It demonstrates the compliance of most major paediatric institutions and confirms the important role of cooperation between National Registries and EBMT Registries.

Late effects after stem cell transplantation (SCT) in children--growth and hormones.

Stem cell transplantation (SCT) has established itself as a very successful therapy in often otherwise unbeatable disorders. In a subset of children and adolescents there are, however, late effects, often as a combination of the underlying disorder, its primary treatment and subsequent SCT. In children and adolescents, disorders of growth and the endocrine system have been observed to occur frequently. The assurance of normal growth, puberty, fertility and thyroid function--including the prevention of secondary malignancies--is of utmost importance for the overall success of treatment and the maintenance of quality of life. This, however, requires a systematic and structured follow-up programme for patients after SCT. Patients and their families need to be made familiar with this concept early and physicians need to understand that such a system must be implemented as part of a comprehensive care.

Infants with stage 4 neuroblastoma: the impact of the chimeric anti-GD2-antibody ch14.18 consolidation therapy.

BACKGROUND: Antibody treatment is considered tolerable and potentially effective in the therapy of neuroblastoma. We have analyzed the clinical data of infants < 1 year with stage 4 neuroblastoma with regard to the consolidation treatment. PATIENTS AND METHODS: Infants < 1 year with stage 4 neuroblastoma who completed initial treatment (6-8 chemotherapy cycles followed either by 4 cycles low dose oral chemotherapy or high dose chemotherapy with stem cell transplantation) without event were eligible for this trial. Consolidation therapy consisted of 6 cycles of antibody ch14.18 (20 mg/m(2) x d ch14.18 for 5 days every 2 months) or 12 months oral maintenance chemotherapy (MT). RESULTS: Of 59 evaluable patients, 31 received a total of 159 ch14.18 cycles, 16 received MT instead, and 12 had no further treatment. Fever (47 % of cycles), abnormal CRP without infection (25 %), rash (23 %), cough (16 %), and pain (8 %) were the main side effects. Univariate analysis found no difference in event free survival (3-year-EFS 80.5 +/- 7.1 %, 87.5 +/- 8.3 %, and 75.0 +/- 12.5 % for patients treated with antibody ch14.18, MT, and no further therapy, p = 0.433) and overall survival (3-year-OS 90.1 +/- 5.4 %, 93.8 +/- 6.0 %, and 91.7 +/- 8.0 % for patients treated with antibody ch14.18, MT, and no further therapy, p = 0.931). Multivariate analysis failed to demonstrate an advantage of antibody treatment. CONCLUSION: The outcome of infants with stage 4 neuroblastoma is good. Consolidation treatment with ch14.18 was tolerable but associated with fever, elevated CRP, rash, cough, and pain as side effects. Compared to oral maintenance chemotherapy and no consolidation treatment, ch14.18 treatment had no impact on the patients' outcome which confirms the results found in children > 1 year.

How and when should we monitor chimerism after allogeneic stem cell transplantation?

SUMMARY: Chimerism analysis has become an important tool for the peri-transplant surveillance of engraftment. It offers the possibility to realize impending graft rejection and can serve as an indicator for the recurrence of the underlying malignant or nonmalignant disease. Most recently, these investigations have become the basis for treatment intervention, for example, to avoid graft rejection, to maintain engraftment and to treat imminent relapse by pre-emptive immunotherapy. This invited review focuses on the clinical implications of characterization of hematopoietic chimerism in stem cell transplantation.

Antiviral activity against CMV-infected fibroblasts in pediatric patients transplanted with CD34(+)-selected allografts from alternative donors.

Human cytomegalovirus (HCMV) remains a cause of serious infectious complications after allogeneic transplantation of hematopoietic stem cells, especially in recipients of T-cell-depleted grafts. Here we investigated the antiviral activity of natural killer (NK) cells from healthy donors (n = 8) as well as of mononuclear cells (MNC) from transplanted pediatric patients (n = 11) who had received CD34(+) selected (and thus T-cell-depleted) stem cells from unrelated and mismatched related donors. Allogeneic human fibroblasts infected with HCMV laboratory strain AD169 for 5 days were used as targets in a 2-h cytotoxicity assay. Downregulation of human leukocyte antigen class I and upregulation of the adhesion molecules CD54 (ICAM-1) and CD58 (LFA-3) were observed after infection. In this experimental setting, NK cells from healthy donors exerted no specific lysis. However, antibody-dependent cellular cytotoxicity (ADCC) mediated by human anti-CMV IgG (cytoglobin) as well as stimulation with low-dose interleukin-(IL)-2 or IL-15 enhanced lysis markedly. MNC from two thirds of the patients (7/11) were capable of lysing infected targets without stimulation. Here also, lytic activity was significantly increased by IL-2 or IL-15, used in combination with ADCC. In contrast, 4/11 patients exerted no lysis. The observed antiviral activity may contribute to the low incidence of CMV DNAemia (29% at day 100, detected by polymerase chain reaction) in the whole group of our patients who have been transplanted with CD34(+)-selected allografts since 1995. Furthermore, our data suggest a potential benefit of using low-dose IL-2 or IL-15, also combined with anti-CMV immunoglobulinG, for immune modulation in CMV disease.

Increasing mixed chimerism defines a high-risk group of childhood acute myelogenous leukemia patients after allogeneic stem cell transplantation where pre-emptive immunotherapy may be effective.

Children with leukemias and increasing mixed chimerism (increasing MC) after allogeneic stem cell transplantation have the highest risk to relapse. Early immunological intervention was found to be effective in these cases. To substantiate this on a defined group of pediatric acute myelogenous leukemia (AML) patients, we performed serial analysis of post transplant chimerism and pre-emptive immunotherapy in patients with increasing MC. In total, 81 children were monitored, 62 patients revealed complete chimerism (CC), low-level MC or decreasing MC. Increasing MC was detected in 19 cases. Despite early immunological intervention relapse was still significantly more frequent in patients with increasing MC (9/19) than in patients with CC, low-level or decreasing MC (8/62, P<0.005). The probability of 3-year event-free survival (EFS) was 52% for all patients (n=81), 59% for patients with CC, low-level MC, 60% for patients with decreasing MC (n=62), and 28% for patients with increasing MC (n=19, P<0.005). Patients with increasing MC who received early immunological intervention showed a significantly enhanced probability for event-free survival (pEFS 36%, n=15) compared to patients with increasing MC without intervention (pEFS 0%, n=4, P<0.05). These results prove that pediatric AML patients with increasing MC are at highest risk for relapse and that early immunological intervention can prevent relapse in these patients.

Transplantation of highly purified peripheral-blood CD34+ progenitor cells from related and unrelated donors in children with nonmalignant diseases.

Transplantation of allogeneic stem cells is currently the only curative treatment for some nonmalignant pediatric diseases. We investigated whether transplantation of purified CD34(+) stem cells prevents acute and chronic GvHD and reduces transplant-related mortality. A total of 25 pediatric patients with nonmalignant diseases underwent allogeneic transplantation from 26 donors (matched related n=4, matched or partially matched unrelated n=14, mismatched related n=8). All grafts were purified peripheral-blood CD34(+) stem cells mobilized with G-CSF. Patients received a median of 12.9 x 10(6) CD34(+) progenitor cells with a median of 6.1 x 10(3) contaminating T-lymphocytes per kilogram of body weight. No post transplant immunosuppressive drugs were given for prophylaxis of GvHD. Engraftment was seen in 21 patients. Three patients engrafted after a second transplant and one patient failed to engraft. Two patients had autologous reconstitution 1.5 years post transplant and one of them was successfully retransplanted. No acute GvHD >grade II was seen, and only two patients developed limited, chronic GvHD. In all, 22 patients (88%) are alive with a median follow-up of 3.7 years. In total, 19 patients (76%) are free of disease or of progression. Transplantation of highly purified peripheral-blood CD34(+) stem cells is associated with low toxicity in patients with nonmalignant diseases.

Cloning of a human antibody directed against human neuroblastoma cells and specific for human translation elongation factor 1alpha.

Human sera have shown antitumor effects mediated by tumor-specific immunoglobulin M (IgM) antibodies. Most people who have cytotoxic serum are in good health and show no evidence of exposure to tumor antigens. We characterized the serum of a healthy female adult that was highly lytic to a neuroblastoma cell line via IgM-activated complement (>60% of malignant cells were killed during the 60-min assay). Complement-dependent lysis was not mediated by other classes of serum antibodies (data not shown) which is consistent with the findings of Ollert et al. To identify the target antigen on neuroblastoma cells, we fractionated neuroblastoma cell lysates by ion-exchange chromatography. In the fraction that showed maximal IgM binding, the dominant protein was identified as the 47-kDa translational elongation factor 1alpha (eEF1alpha). We used the donor's B-cells to create hybridomas producing the antibody (B12.6.22) that bound to neuroblastoma cells and mediated cytotoxicity. This antibody recognized eEF1alpha in a specific manner. Sequence analysis of the heavy chain of B12.6.22 showed usage of VH3-23 and JH6 gene segments, with no somatic mutation. The structural similarity of B12.6.22 to antibodies of the innate immune system supports the assumption that natural antibodies are a potential source of therapeutic antibodies.

Clinical relevance of serial quantitative analysis of hematopoietic chimerism after allogeneic stem cell transplantation in children for severe aplastic anemia.

Allogeneic stem cell transplantation (allo-SCT) is a well-established treatment modality for children with severe aplastic anemia (SAA). Treatment failures are rare and mostly caused by graft rejection. Increasing mixed chimerism represents a stage at the very beginning of graft rejection, where immunological intervention might be an effective prophylactic approach. To substantiate this, we: (1) monitored peripheral blood cells from children with SAA after allo-SCT and performed pre-emptive immunotherapy in patients with increasing MC. In all, 23/34 courses of 32 children with SAA after allo-SCT showed a complete chimerism (CC) throughout and 10/34 developed different types of mixed chimerism (MC). Altogether, 4/10 with MC spontaneously developed decreasing MC, 2/10 courses persisted with low proportions of autologous cells below 30% (stable-MC), 4/10 developed increasing MC and one patient showed an autologous recovery. All patients with CC, decreasing MC or stable MC remained in continuous complete remission (CCR). In all, 2/4 patients with increasing MC developed graft rejection. Based on these observations, 2/4 new patients with increasing MC received low-dose DLIs prophylactically, and remained in CCR without any GVHD. These results substantiate that low-dose DLI in children with SAA and increasing MC can prevent graft rejection with a calculable risk to induce severe GVHD.