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Allogeneic natural killer cells may exert cytotoxic activity against HLA-nonidentical tumor cells. This effect may be exploited after T-cell depleted haploidentical hematopoetic stem cell transplantation (HSCT) by using donor NK-cell infusions (NK-DLI) as adoptive immunotherapy. In a prospective phase II study in 2 centers we treated 15 patients with NK-DLI preemptively with the goals to demonstrate feasibility and to explore whether such infusions would contribute to stabilize engraftment and exert graft versus leukemia activity without causing GvHD. Patients were adults (6) or children (8) receiving transplants from haploidentical donors to treat AML (7) ALL (5) Hodgkin lymphoma (2) or sarcoma (1). Donors were haplotype mismatched siblings in 3 and parents in 12. Donor NK cells were selected from unstimulated leukapheresis using immunomagnetic T cell depletion with anti-CD3 and NK cell enrichment with anti-CD56 coated microbeads on the CliniMACS ® device. Per protocol, patients in center A received NK-DLI on days +40 and +100 and in center B on days +3, +40 and +100. NK-DLI were either freshly infused or cryopreserved and thawed. Center A Center B N 8 7 Adults / Children 6/2 0/7 Disease: AML/ALL/HD/oth 6/1/1/0 1/4/1/1 Number of NK-DLI per patient 1/2/3 2/5/1 2/3/2 Median NK-cell recovery (%) 62 37 NK-cell dose (10e7/kg) (median, range) 1.2 (0.5–3.4) 1.4 (0.66–3.23) T-cell dose (10e4/kg) (median, range) 0.2 (0–7.2) 0.23 (0–5.3) Events: graft failure / progression / GvHD (III–IV) 2/2/1 1/2/1 Alive / Dead 3/5 5/2 Cause of death: graft failure/progression/GvHD 2/2/1 0/2/0 NK-cell collection and ex vivo purification was successful in all donors. Outcome is shown in the Table. Of note: One patient in each center developed severe (grade III / grade IV) GvHD respectively. These 2 patients had received the highest T-cell dose. The remaining patients tolerated NK-DLI well and did not develop GvHD. Two patients in Center A experienced graft failure after NK-DLI in spite of NK-alloreactivity in GvHD direction. In conclusion: highly purified NK cells may be infused in recipients of haploidentical HSCT recipients. None of the patients receiving < 10e4/kg CD3+ cells developed GvHD. Further studies are needed to determine the optimal dose and timing of NK-DLI for anti-tumor activity in this setting.

The optimum stem cell source for allogeneic hematopoietic stem cell transplantation (HSCT) remains highly controversial. A recent study in older children and adolescents receiving HSCT from HLA-identical sibling donors has revealed higher mortality after peripheral blood stem cell (PBSC) compared to bone marrow (BM) transplantation (Eapen et al, JCO, 2004). However, despite the increasing use of PBSC in pediatric HSCT from matched unrelated donors (MUD), comparative studies on the relative risks and benefits of both stem cell sources are lacking. We therefore analysed the outcome of unrelated PBSC (n=118) and BM (n=102) transplants reported to the German/Austrian Pediatric Registry for Stem Cell Transplantation (PRST) between 1998 and 2003. Patients with hematologic malignancies (ALL, AML, CML, or MDS), who had received unmanipulated HSCT from ≥ 5/6 HLA antigen-matched unrelated donors following myeloablative conditioning were included into the analysis. PBSC and BM groups were comparable with regard to sex, age, CMV serostatus, GvHD prophylaxis, disease status at transplant, prophylactic growth factor use and degree of HLA-matching, while differences were detected in disease category (more MDS patients in PBSC group, p=0.02), transplanted cell dose (higher CD34-cell graft content in PBSC group, p=0.001) and median year of transplant (PBSC transplantations were more recent, p=0.01). Engraftment was achieved significantly faster after PBSC compared to BM transplantation: 15 vs. 19 days for neutrophil engraftment (p=0.001) and 21 vs. 25 days for platelet engraftment (p

One aim of the AML-BFM 98 study was to evaluate prospectively the impact of matched sibling donor (MSD)-SCT in 1st complete remission (CR) in children with high-risk (HR)-AML. Patients: Between 7/1998 and 3/2004 494 children and adolescents were enrolled in the studies AML-BFM 98 and the AML-BFM 98 Interim 2003. Patients with favorable cytogenetics [t(8;21), inv(16), t(15;17)], corresponding morphology (AML FAB M1/2 with Auer rods, M4eo, M3) and < 5% blasts on day 15 were defined as standard risk (SR)-group (n=177). All others were stratified to HR-group (n=317) and eligible for SCT in 1st CR. Out of 275 patients achieving 1st CR (87%) 63 patients had a MSD and 188 had no MSD (intent-to-treat group, ITT) while 24 patients had to be excluded due to missing HLA typing or initial refusal to SCT. Thirty-six patients received MSD-SCT (=as-treated group), another two children were transplanted from a matched unrelated donor (MUD) although an MSD was available. The reasons not performing SCT in 1st CR in the remaining 25 children with a donor available were early relapse (n=7), death in CR (n=2), clinical reasons (n=8), or refusal of SCT (n=8). Chemotherapy (CHT) only was given in 192 patients (n=167 without MSD, n=25 with a MSD). Another 21 children were transplanted from an unrelated donor (n=18), family donor (n=1), haploidentical donor (n=2). The reasons were slow response, very poor cytogenetics or other clinical reasons. Treatment: All patients received double induction and consolidation. MSD-SCT in 1st was scheduled after 4 blocs of treatment. Children who were not transplanted in 1st CR received intensification and a 1-year maintenance therapy. The recommended conditioning regimen consisted in busulfan and cyclophosphamide. Results ITT analysis revealed no significant differences in of 5 year disease-free survival (DFS) and overall survival (OS) between children with or without a donor (no donor vs. donor: DFS 43±4% vs. 47±7% plog rank 0.52; OS 56±4% vs. 58±9%, plog rank 0.16). The as-treated analysis gave similar results: CHT only vs. MSD-SCT: DFS 35±11% vs. 59±9% p Mantel-byar 0.13; OS 59±13% vs. 62±12%, p Mantel-byar 0.51). The major reasons of treatment failures were relapses in all groups [CHT only n=100 (52%), MSD-SCT n=13 (34%), MUD-SCT n=9 (50%)]. A 2nd CR was achieved in 71% of the CHT only group (n=74). After SCT in 2nd CR, OS was 40±6%. By contrast, only two children (9%) of 22 children after relapse following SCT in 1st CR could be salvaged. Severe late adverse events (e.g. severe chronic GvHD CTC grade IV, destruction of big joints, intracerebral bleedings) were more frequent in children transplanted in 1st CR (15%) than in those with CHT only (including SCT in 2nd CR; 5%, p×2< 0.05). Conclusion: In the population-based, prospective multi-center study AML-BFM 98 allogeneic MSD-SCT in 1st CR showed no advantage in children and adolescents with HR-AML. Considering the higher toxicity of allogeneic SCT, in the ongoing trial AML-BFM 2004, this SCT (MSD or MUD) is restricted to patients in 2nd CR and refractory AML.

In the absence of an HLA identical donor, T-cell depleted haploidentical hematopoietic stem cell transplantation (Haplo-HSCT) is an alternative option to treat children with very high risk acute lymphoblastic leukemia (ALL). However, little data is available in children. We have analyzed 118 children (≤16 years old) with ALL transplanted with a myeloablative Haplo-HSCT from 1995 to 2004 in Europe. Only transplants with 2 or more HLA disparities out of 6 (A, B and DRB1) were included. The median age was 8.5 years and median follow-up 56 months (8–116). At transplant, 21 (18%) were in CR1, 72 (61%) in CR2 or CR3 and 25 (21%) in more advanced phase. The 3-year leukemia free survival was 32±10%, 28±5% and 0%, respectively. Therefore we restricted the analysis to patients in remission (n=93) transplanted in 30 EBMT centers. Thirty-four (37%) patients were treated in centres performing more than 10 Haplo-HSCT in the study period (3 centres). The median age of recipient and donor was 8.7 and 37 years, respectively. Fifty four (65%) received a full Haplo-HSCT and 95% of the donors were parental. Twenty seven (29%) had t(9;21) or t(4;11). The majority of patients did not receive drugs for GVHD prophylaxis, ATG/ALG was used in conditioning in 74%, and 73% received TBI. The Clinimacs® device was used in 74% of selections. The median number of CD34+ cells infused was 12.8 106/Kg. Cumulative incidence with competing risk and KM estimates were used to calculate outcome probabilities. The median days of neutrophil recovery was 15 days (8–55) and 90% of patients had signs of engraftment. Acute GVHD II–IV was observed in 24% of the patients. In univariate analysis for LFS there was a trend towards better results for patients receiving higher CD34 cell dose (p=0.08) and a significant difference for patients transplanted in centres performing more Haplo-HSCT (49% versus 17%, p=0.002). Relapse incidence (RI) and non relapse mortality NRM) tended to be different between the experienced and less experienced centres. In less experienced centres NRM was 41% vs 27% (p=0.13) and RI 41% vs. 24% (p=0.10). There were patient, donor and transplant related differences between less and more experienced centres, specifically related to donor sex, Philadelphia positivity, year of transplantation, use of TBI, ATG and DLI and previous autograft. In conclusion, Haplo-HSCT is an alternative option to treat children with very high risk ALL in the absence of HLA identical donor. Centres with more experience have better LFS.

Graft failure is a rare but life-threatening complication after transplantation of hematopoietic stem cells. Treatment comprises immunoablative reconditioning regimens and a second stem cell donation from the same or from a different donor as soon as possible to minimize the time of pancytopenia and its sequelae. We report a cohort of 11 pediatric patients with leukemias (acute lymphatic n=4, acute or chronic myeloic/MDS n=4) and severe aplastic anemia (n=3) who experienced graft rejection after TBI, busulphan or melphalan based myeloablative transplantation from mismatched related donors (MMRD) (n=6) or after cord blood/matched unrelated donor (MUD) transplantation (n=5) between 2000 and 2006. In the latter the original donor was not available a second time. Thus, all patients were re-transplanted with CD34+ positive selected or CD3/CD19 depleted stem cells from a second, haploidentical parental donor (MACS method, Miltenyi Biotec). Median time span from diagnosis of graft rejection to second donation was 16 days. The reconditioning regimens consisted of total lymphoid irradiation or cyclophosphamide, thiotepa (5mg/kg), fludarabine (120 mg/m2) and ATG/OKT3. A median number of 25×106/kg of body weight stem cells with 60.000/kg residual T cells were infused. Mofetilmycophenolat was given as GvHD prophylaxis, if residual T cells exceeded 25 000/kg bw. Sustained engraftment was achieved in all patients (ANC>500/μl: 9 (11–32) days). No GvHD > grade II was observed. T cell recovery was delayed, however no lethal viral infection occurred. Severe organ toxicity was observed in 2 patients (BOOP, hemorrhagic cystits) and moderate mucositis in 11 patients. 8/11 patients are disease free (median follow up 1.5 (0.3–6.6) years; 1 year EFS=73%). Causes of death were: BOOP (n=1), infection (n=1); only one patient with refractory AML relapsed. Thus, transplantation of stem cells from haploidentical donors represents a realistic option to rescue patients with graft failure within a short time span and for whom a second donation of the original donor is not available. The use of a different donor may help to avoid a second rejection.

Invasive fungal infections (IFI), in particular infections due to Aspergillus spp and Candida spp, still pose considerable problems in patients undergoing allogeneic stem cell transplantation (SCT). Despite the availability of new antifungal agents, morbidity and mortality of IFI are still unacceptable high. Although neutropenia is known as the single most important risk factor for IFI, there is a growing body of evidence that T cells play a major role in the defense against fungi. Therefore, adoptive immunotherapy with T cells against Candida spp. might be an interesting therapeutic option in patients undergoing allogeneic SCT. After overnight incubation of 1×108 peripheral blood mononuclear cells from 4 healthy individuals with cellular extracts of C.albicans, activated T cells were selected using the IFN-γ secretion-assay (Miltenyi Biotec, Bergisch Gladbach, Germany). After 14 days of culture, T cell clones were generated by limiting dilution and incubated for another 14 days. The median number of cells obtained was 2.6×107 (range, 0.85–5.75×107). Flow cytometry revealed a highly homogenous population of CD3+CD4+ cells (97.2% ± 2.6; n=6), of which an average of 8.6% (range, 4.8–58.2%) produced IFN-gamma on re-stimulation with C.albicans antigens, as assessed by intracellular cytokine staining assay. 20.5% (range, 5.8–72.4%) of the generated cells produced TNF-alpha, whereas no significant number of cells produced TH2 cytokines such as IL-4 and IL-10, indicating that the generated T cell clones were TH1 cells. The percentage of IFN-gamma producing T cells was significant upon stimulation with C.albicans and C.tropicalis, whereas less than 1% of cells produced IFN-gamma upon stimulation with antigens of other yeasts such as C.glabrata, Debaryomyces hansenii and Kluyveromyces lactis and molds such as A.fumigatus, Penicillium chrysogenum and Alternaria alternata. Compared to CD4+ T cells of the original fraction, the isolated and expanded anti-Candida T cells showed reduced alloreactivity, as assessed by means of CSFE. In addition, a strong proliferation of the generated anti-Candida T cells was seen after re-stimulation with C.albicans antigens. The potency of the generated T-cells to damage C.albicans was evaluated using the XTT assay. Compared to polymorphonucelar cells (PMNs), APCs and T-cells alone or to the combination of PMNs with T cells or APCs, respectively, the combination of PMNs, APCs and T-cells showed highest fungal damage (n=4). In conclusion, our data suggest that the isolation and expansion of anti-Candida T cells is possible and feasible. The generated T cells show low alloreactivity in vitro and increase the antimycotic potential of phagocytes. Thus, antimycotic T cells might become an important tool in the prophylaxis and therapy of IFI in patients after allogeneic SCT.

Invasive aspergillosis (IA) remains a major cause of morbidity and mortality in patients with hematological malignancies, in particular in patients who have undergone allogeneic hematopoietic stem cell transplantation (SCT). There is a growing body of evidence that T-cells play an important role in the immunological response to Aspergillus fumigatus. Using the Aspergillus fumigatus antigen extract EC SAB and the IFN-γ secretion assay (Miltenyi Biotec, Germany), we generated Aspergillus fumigatus specific T-cell clones by limiting dilution (n=4). Flow cytometry revealed a cell population of CD3+/CD4+ cells (mean±SEM, 98.2±1.2%). Functional assessment by ICC revealed that an average of 8.7% of these cells (range, 6.6%–18.5%) specifically secreted IFN-γ on stimulation with EC SAB, which supports the TH1 response of the generated cells to Aspergillus fumigatus antigens. The antigenic components of EC SAB are one or more proteins, since the addition of proteinase completely suppressed the stimulating effect of this preparation. The percentage of IFN-γ producing CD3+/CD4+ cells was less than 1% upon activation with antigen extracts from Aspergillus flavus, Aspergillus niger, Alternaria alternata, Mucor racemosus, Penicillium notatum and Candida albicans, indicating that the generated T-cell clones are specific for Aspergillus fumigatus. A strong proliferation of the generated Aspergillus fumigatus specific T-cells was seen after re-stimulation with EC SAB, whereas alloreactivity was reduced compared to CD4+ T-cells of the original fraction. Hyphal damage of Aspergillus fumigatus was assessed by means of an XTT assay. Polymorphonuclear leukocytes (PMNs) showed a similar hyphal damage when tested alone (mean±SEM, 14.2±2.1%), in combination with antigen presenting cells (APCs) (15.1±1.4%), or in combination with Aspergillus fumigatus specific T-cells (15.0±2.0%). A comparable hyphal damage was seen when Aspergillus fumigatus specific T-cells were co-incubated with APCs (14.2±1.7%). In contrast, the combination of APCs and Aspergillus fumigatus specific T-cells with PMNs resulted in a significantly higher hyphal damage compared to all other settings (23.3±2.8%; P

We examined the feasibility and efficacy of a salvage strategy of intensive re-induction chemotherapy followed by autologous or allogeneic hematopoetic stem cell transplantation (HSCT) for children and adolescents suffering from relapsed anaplastic large cell lymphoma after BFM front-line therapy. From 4/1990 to 2/2003, 75 evaluable patients, median age at relapse 9.8 (range 0.4–22.2) years, were enrolled. The median time from the start of first therapy to failure was 7.1 (range 1–76) months. 17 pts suffered from progressive disease during front-line chemotherapy. ALK staining was positive in 57 pts, negative in 5 pts and not available in 13 pts. CD3 reactivity was positive in 24, negative in 42 pts and not available in 7 pts. 2 pts had B-cell ALCL. Salvage therapy consisted of 2 or 3, 5-day chemotherapy courses based upon dexa, vindesine, cytarabine, etoposide, MTX 5g/m2, cyclophosphamide/ifosfamide, vincristine, doxorubicine, intrathecal therapy followed by HSCT. Conditioning regimen: total body irradiation (2x2 Gy day −7,−6,−5), etoposide 40 mg/kg day -4, cyclophosphamide 60 mg/kg/d days −3,−2. Data were updated as of April 1st, 2004. With a median follow-up of 6.9 [0.8–13.2] years, the probability of survival at 3 years after first relapse is 54+6%. 28 pts died of lymphoma, 6 pts of toxicity. 41 pts are alive in ≥CR2. 21 pts received no salvage or chemotherapy only (2 TRM, 18 death of disease, 1 CR). 39 pts received autologous HSCT (21 CR, 2 TRM, 16 re-relapses [6 >CR2]). 15 pts received allogeneic HSCT (10 CR, 3 TRM, 2 relapses). 3/17 pts with relapse during front-line survived compared to 37/57 pts who relapsed later (p We conclude that this strategy proved feasible and efficacious for pts who suffered a relapse after front-line therapy while outcome for pts with progressive disease during front-line treatment was poor. Autologous HSCT is an efficient consolidation for pts with relapses of CD3 negative ALCL but not efficacious for those with CD3 positive tumors. These patients may benefit from allogeneic HSCT.

Hemophilia B is an X-linked bleeding disorder. Substitution of recombinant (rFIX) or plasma derived (pdFIX) FIX preparations are required to restore coagulation. A rare but severe complication is the development of FIX inhibitors that are often accompanied by allergic reactions. Immune tolerance therapies (ITTs) for inhibitor elimination are difficult and can induce further allergic reactions. ITTs for FIX inhibitors are less established and less successful than for FVIII inhibitors. In this study, three phage displayed random peptide libraries were screened by biopanning to isolate small peptides that mimic the epitope of FIX inhibitors. In the patient, the inhibitor was eliminated by ITT but reappeared again. Plasma samples before ITT and at reappearance of inhibitor were used to isolate inhibitor specific phage clones. Phage clones showing specific reactivity with inhibitor positive plasma compared to control plasma by ELISA were amplified and DNA was sequenced to determine the peptide sequence displayed on the phage. Before ITT, 31 isolated phage clones specific for FIX inhibitors have been sequenced so far. 10/31 contain a common motif. After ITT, 24 clones specific for patient plasma have been sequenced. 10/24 contain a different common motif. Synthetic peptides have been generated according to the phage displayed sequence to demonstrate binding of inhibitors and test for cross reactivity. The surface of the model of FIX was screened for these motifs with a novel software tool. Different conformational epitopes could be identified on the surface of the molecule before and after ITT. Small peptides (mimotopes) have been identified that mimic epitopes for FIX inhibitors in a hemophilia B patient. The sequences can be aligned to different conformational epitopes on FIX. It appears that the epitope has changed in the course of the ITT. The mimotopes can also be used to target inhibitor specific B cells for a novel approach in inhibitor elimination in hemophilia B.

In allogeneic transplantation it is a major goal to maintain a stable long term engraftment. However, several factors like viral infections, drug toxicity or low stem cell numbers may hamper hematopoietic recovery or may even cause a late graft failure after successful initial engraftment. Hematopoietic growth factors or additional stem cell donations (boosts) may help to overcome this problem. In this retrospective analysis, the efficacy of boosts with highly purified peripheral stem cells either from closely matched unrelated or from haploidentical related donors was evaluated in pediatric patients with stagnant hematopoiesis (10x106 (unrelated donors) or >20x106/kg BW CD34+ cells, surplus stem cells were purified with an immunomagnetic selection method using antiCD34 or antiCD133 coated microbeads and cryopreserved. Three haploidentical donors were asked for a second donation. Our aim was to maintain stable and complete hematopoiesis after transplantation without inducing acute or chronic GvHD. A total of 18 boosts were given between 30 and 290 days after transplantation with a median number of 5 (0.6–34) x 106 CD34+/CD133+ progenitor cells/kg and only 2200 (100–25000) residual T-cells/kg. All patients had a complete donor chimerism and no reconditioning or posttransplant pharmacological immunosuppression was administered. Patients who received G-CSF were excluded. Due to the low number of T cells, acute GvHD >grade I and also chronic GvHD could be completely avoided even in haploidentical donors. Leukocyte counts, neutrophile counts, lymphocyte counts (and platelet counts in patients with thrombocytopenia) within the week before infusion of the boosts were compared with cell counts 4 and 8 weeks after infusion, using the paired T-test. A significant increase of all cell counts was observed (medina numbers pre boosting: 900/μl, 312/μl, 142/μl; 4 weeks post boosting: 2065/μl, 1264/μl, 317/μl; 8 weeks post boosting:2570/μl, 1780/μl, 385/μl; p Conclusions: Boosts with purified stem cells and low T-cell contamination (

Hemophilia A is an X-linked bleeding disorder. Mutations in the Factor VIII (FVIII) gene result in reduced or non-functional expression of FVIII. In hemophilia A recombinant (rFVIII) or plasma derived (pdFVIII) preparations are substituted to restore coagulation activity. However, a major problem of current treatment in hemophilia A is the development of an antibody response (inhibitors) to FVIIII. Various Immune Tolerance Therapies (ITTs) are being applied to overcome the FVIII specific immune response. In this study, phage displayed random peptide libraries were screened with various patients’ plasma samples. We recently reported about the identification of a conformational epitope in the A2 subunit of FVIII. Phages as well as corresponding synthetic peptides specifically block inhibitor binding and crossreact with heterologous plasma. Peptide ligands specifically stained primary B cells (CD19+/CD27+ double positive cells) of the inhibitor patient, but not of a healthy control by FACS analysis. To increase the potential of small synthetic peptide ligands as tools for the development of novel inhibitor elimination strategies, the peptide was expressed in a multimeric form in an eukaryotic expression system. In multimeric form the peptide ligand is highly stable and reveals better inhibitor blocking capacities than the synthetic peptide we sowed before. The multimer now serves as basis for the construction of LECs (Ligand Effector Conjugates) for the specific targeting and destruction of inhibitor specific B cells.

In the absence of an HLA identical donor, T-cell depleted haploidentical hematopoietic stem cell transplantation (HSCT) is an alternative option to treat children with high risk or relapsed acute leukaemia. However very few data is available in a large series of children. With the aim to study risk factors of outcomes we have analyzed 196 children ( Outcomes at two years CR1 (n=41) CR2 (n=74) Advanced (n=81) p value Transplant related mortality 32+/−8% 26+/−5% 33+/−5% 0.44 Relapse 32+/−8% 40+/−6% 51+/−6% 0.03 Leukaemia free survival 36+/−8% 34+/−6% 16+/−4% In fact, in a multivariate analysis for LFS and relapse only patients transplanted in remission had better LFS and decreased relapse incidence compared with non remission patients (p

Chromosomal rearrangements of the MLL gene are responsible for 5–10% of all acute leukemias, biphenotypic leukemias and myelodysplastic syndromes. 5–10% of these MLL aberrations are therapy-related leukemias with the frequent fusion partners ELL, MLLT3, MLLT10 and MLLT2. The large number of known and unknown MLL fusions (>80) renders a precise diagnosis a demanding task. Even though all MLL rearrangements are associated with high risk acute leukemias, the outcome (favorable or poor) depends on the partner gene. Thus, the identification of MLL gene fusions is necessary for rapid clinical de-ci-sions resulting in specific therapy regimens. After their cytogenetic identification, only the most common MLL fusions MLLT2, MLLT3, MLLT1, MLLT4, ELL and MLLT10 (80%) are investigated by RT-PCR analysis, whereas infrequent or unknown MLL rearrangements (20%) are more or less excluded from further analysis. Therefore we established a LDI-PCR (long-distance inverse PCR) based method that uses small amounts of genomic DNA to determine any type of MLL associated chromosomal abnormality. With this diagnostic tool more than 400 prescreened und unscreened adult and pediatric samples (AML/ALL) from different European diagnostic centers have been analyzed. During this study more than 200 MLL rearrangements were characterized for their precise localization of genomic break-points. The identified MLL rearrangements consist of 25 different fusion genes including the following eight novel MLL partner genes: ACACA, ARGHEF17, BCL9L, MAML2, SELB, SMAP1, and TIRAP. The identified MLL aberrations are basically reciprocal translocations, but also deletions, inversions, insertions and interstitial duplications have been determined. Combining the data of our study and data retrieved from the literature, a total of 88 partner genes can now be annotated. 52 fusion partners (60%) have been characterized on the molecular level whereas at least 36 (40%) remain to be identified. These results demonstrate that this new diagnostic tool in combination with split-signal FISH is qualified for the rapid analysis of known as well as unknown MLL partner genes. Furthermore, the determined patient specific fusion sequences are useful for minimal residual disease (MRD) monitoring of MLL associated acute leukemias. The use of these MRD markers will contribute to improve the treatment and outcome of acute leukemia patients. A first prospective study was already initiated by the German ALL study group and verified the reliability of these genomic markers for MRD monitoring.

In acquired hemophilia polyclonal autoantibodies (inhibitors) to coagulation factor VIII are generated. Inhibitors functionally interfere with interaction of molecules involved in the coagulation cascade for example the interaction of FVIII and the von Willebrand factor. Inhibitors in acquired hemophilia have been reported to bind either to the A2 or to C2 domain of FVIII. Random peptide phage display libraries were screened with patient plasma to identify small peptides mimicking inhibitor epitopes. Peptide sequences were used to map the inhibitor epitope to the surface of FVIII. The phage libraries included libraries with linear or cyclic 7mer or 12mer inserts. Biopanning was performed including positive selections and also negative selections to deplete irrelevant binders. Phages bound to inhibitors were eluted by pH shift or by competition with FVIII. Single phage clones were tested for their specificity for inhibitor positive plasma by ELISA. For specific binders, the sequence of the inserts was identified by DNA sequencing. For one patient 33 phage clones were analysed from three libraries and 28 peptide sequences mimicking the inhibitor epitopemimotopes - were determined. The peptide sequences map to two conformational epitopes on the A2 and A1 domain of FVIII. Clusters of six and four amino acids are the proposed binding regions on the A2 and A1 domain respectively. The matching conformational motifs contain PPLRQ and PPLS. Synthetic peptides corresponding to the sequence displayed on phage were generated and binding of inhibitors to peptides was confirmed by different ELISA formats. Currently, functional studies with synthetic peptides are being performed to further analyse the epitopes. Additionally, more patient samples are being screened to analyse further epitopes and understand immuno dominant regions of FVIII in acquired haemophilia and compare them to the immune response in hemophilia A. The mimotopes isolated could be a basis for the development of ligand effector conjugates to target inhibitor specific B cells.

Profound depletion of T- and B-cells is a fundamental prerequisite for haploidentical 1–3 loci mismatched stem cell transplantation. Here, we compare hematopoietic engraftment and occurrence of GvHD after positive selection procedures (with anti-CD34 coated or anti-CD133 coated beads, n=64 patients) and after a direct depletion procedure with anti-CD3/anti-CD19 coated microbeads (n=12) in children with leukemias (ALL, AML, CML), lymphomas and nonmalignant diseases. 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 methods resulted in 10 000 residual CD3+ cells/kg (7000–30 000). Patients with grafts depleted with anti-CD3/anti-CD19 coated microbeads, received 32 000 (7000–160 000) residual T-cells/kg. Those grafts comprised also remarkable amounts of effector cells such as NK-cells (median number: 86 × 106/kg), dendritic cells and monocytes/granulocytes (median number: 700 × 106/kg) with antileukemic activity in vitro. Primary engraftment occurred in 83% of patients with positive selected stem cells. After reconditioning, sustained engraftment was achieved in 98%. In patients with CD3+/CD19+ depleted grafts, primary engraftment was observed in 92% (11/12) and sustained engraftment (after reconditioning) in 100% (12/12). GvHD grade II occurred in 6% (positive selection) and 25% (CD3/CD19 depletion). Only one patient had severe GvHD grade III-IV (CD34+). T-cell recovery was significantly faster in patients with CD3/CD19 depleted grafts. In the group with positive selected grafts, 5 year EFS for patients with ALL CR1-3, myeloic leukemias and nonmalignant diseases was 45%, 22% and 60%, respectively. CD3+/CD19+ depleted grafts were given to patients with refractory disease (AML/MDS, ALL) or with high risk of graft failure. 4/10 patients with leukemias and 2/2 patients with SAA/PNH are disease free (median follow-up: 8 months). Conclusions: Haploidentical transplantation with standard CD34+ or CD133+ selected grafts provides stable long term survival rates. CD3+/CD19+ depletion resulted in better and more reliable engraftment but produced slightly more GvHD than positive selection methods. Our 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.

Attenuated androgens like danazol are the current treatment of choice for long-term prophylaxis in patients with hereditary angioedema (HAE), who may experience life-threatening acute attacks. Unfortunately, this group of drugs bears the risk of severe side effects (e.g. depression, weight gain, hirsutism, transaminase elevations, liver adenoma and carcinoma, headaches, hypertension, menstrual abnormalities). Objectives: We therefore explored the option to administer a pasteurized, plasma-derived C1-Inhibitor concentrate (C1-INH, Berinert®P) for individual replacement therapy (IRT) in patients with HAE in whom danazol induced severe side effects, was not effective or was contraindicated (e.g. children, pregnant women). Methods: Prospective, observational, intra-individual cross over study, comparing efficacy, safety and quality of life of danazol (prophylaxis) vs. C1-INH (IRT) in 23 patients suffering from severe HAE. Results: Mean annual attack frequency decreased from 48.8 ± 37.1 (danazol) to 8.1 ± 22.1 (C1-INH); p Conclusions: Compared to danazol, IRT with pasteurized C1-INH in patients with severe HAE significantly reduces the frequency of HAE attacks, especially of life-threatening attacks, and significantly improves the quality of life.

CML is a rare disease in childhood and hematopoietic stem cell transplantation (SCT) remains the only proven option for cure of young patients (pts). We here report the results of the GPOH-trial “CML-paed”. From December 1995 to June 2004 pts younger than 19 years (median age: 11.4 yrs) with Philadelphia-chromosome positive CML (n=193; 99 boys, 94 girls) were treated by hydroxyurea ± interferon and scheduled for SCT from an HLA-matched family donor within 6 months after diagnosis (Dx) and from an unrelated donor within 12 months. 85% of the pts were diagnosed in chronic phase (CP). Six pts (3%) died from disease without SCT with a median interval from Dx to death of 6.5 months (range 0.5–12 months) and 25 pts are still searching for a donor. 168 pts underwent SCT (n=50 HLA-matched related; n=69 HLA-matched unrelated (MUD); n=18 HLA-mismatched related; n=31 HLA mismached unrelated) in CP (n=139), in accelerated phase (AP, n=9), in blast crisis (BC, n=9), or in 2. CP (n=10). Probability of overall survival (OS) was 75 % if SCT was performed

Hereditary and acquired deficiencies in C1 inhibitor (C1-INH) function can result in potentially life-threatening attacks of hereditary angioedema (HAE). A highly purified and pasteurized C1-INH concentrate has been used effectively as prophylaxis against and treatment for angioedema attacks in patients with hereditary C1-INH deficiencies, but relatively little is known about its pharmacokinetic properties. Objective: To evaluate the pharmacokinetics and in vivo recovery (IVR) of C1-INH concentrate (Berinert®P) in two groups of patients with hereditary angioedema (HAE) receiving this preparation either as individual replacement therapy (IRT, regular, immediate treatment of first HAE symptoms in patients with frequent and severe attacks) or as on-demand treatment. Methods: Forty subjects (15 under IRT, 25 under on-demand treatment) with HAE received intravenous injections of C1-INH (542–1,617 U) in an attack-free interval in a prospective, open, uncontrolled, single-center study. Blood was sampled for determination of C1-INH with a commercially available functional chromogenic assay for up to 72 hours after dosing. Pharmacokinetic parameters were calculated using a single-compartment model and IVR was determined using standard methods. Results: The mean (± SD) time to maximum plasma concentration (Tmax) for C1-INH administered in patients under IRT was 1.3 ± 2.1 hours, the area under the time versus plasma concentration curve (AUC) was 20.5 ± 19.1 hour•U/mL, the elimination half-life (t½) was 33.3 ± 19.8 hours, mean residence time (MRT) was 48.0 ± 28.5 hours, total body clearance (Cl) was 1.1 ± 0.6 mL/kg/hour, and volume of distribution at steady state (Vss) was 39.5 ± 9.9 mL/kg. The respective values for patients treated on demand were 2.9 ± 6.5 hours, 20.0 ± 14.5 hour•U/mL, 43.9 ± 22.4 hours, 63.4 ± 32.3 hours, 1.2 ± 1.0 mL/kg•hour, and 51.4 ± 10.9 mL/kg. The mean IVRs for IRT and on-demand treatment were 108.2 ± 48.3% and 85.8 ± 28.3%, respectively. Children tended to have slightly lower half-life and a slightly higher Vss compared to adults. Conclusions: C1-INH concentrate has a short Tmax and a long, t½ and MRT consistent with the rapid onset of clinical efficacy for this preparation in subjects suffering angioedema attacks and the ability to effectively carry out IRT with injections administered every 2–5 days. This analysis provides to our knowledge the most comprehensive pharmacokinetic evaluation in subjects with HAE.

Purpose: Infectious complications remain a substantial cause of morbidity and mortality in patients with acute myeloid leukemia (AML). Candidate gene studies have identified common polymorphisms in genes of molecules of innate immunity as risk factors for susceptibility and/or outcome to a spectrum of pathogens in various populations. This study was performed to investigate whether common polymorphism in genes of innate immunity might influence the risk for serious infection during therapy for pediatric AML. Patients and Methods: Genotype analysis was performed in 168 children treated on clinical trial AML-BFM 93. Infectious events were categorized as microbiologically or clinically documented infections and as fever without an identifiable source. Candidate gene polymorphisms included tumor necrosis factor-alpha, interleukin 6 and 8 (IL6 and IL8), myeloperoxidase, chitotriosidase (CHIT), the low affinity Fc receptor 2A, and the toll like-receptors 2 and 4. Results: The 168 children treated according to clinical trial AML-BFM 93 experienced a total of 508 infectious episodes. Only three patients escaped significant infectious complication. One-hundred-and-ten patients had a least one microbiologically documented infection. Gram-positive bacteria were isolated in the bloodstream of 84 children, whereas Gram-negative bacteria were recovered from the bloodstream of 24 patients. There was an association between Gram-negative bacterial infection and each of the IL6 and CHIT genetic variants. The risk of bacteremia with Gram-negative organisms was significantly higher in patients with the G allele in the IL6 promoter at 174bp [22/123 (17.9%) vs 0/25 (0%); P=.022 (OR 11.31, 95% CI 1.41–90.6)] and in patients with the H allele (24-bp duplication in exon 10) of CHIT [13/56 (23.25) vs 11/112 (9.8%); P=.020 (OR 2.78; 95% CI 1.18–6.54)]. No significant association of the other variant genotypes with any of the clinical endpoints analyzed was observed. Conclusion: The data suggest that variant alleles of both IL6 and CHIT influence susceptibility to infection with Gram-negative bacteria in children undergoing therapy for AML. Further studies are warranted to confirm our findings and to investigate underlying mechanisms.

In order to further improve survival of children undergoing therapy for acute myeloid leukemia (AML), the multicenter clinical trial AML-BFM 98 intensified chemotherapy for standard risk (SR) patients (pts.). In addition, this randomized trial prospectively evaluated whether 2 short cycles of chemotherapy resulted in better prognosis than a 6-week consolidation. Patients and Methods: Between July 1998 and June 2003, 461 pts. < 18 years with de novo AML were enrolled in the trial AML-BFM 98. The SR group consisted of 170 (37%) pts. (FABM1/M2 with Auer rods or M4eo with ≤ 5 % blasts in the day 15 bone marrow; all pts. with FAB M3). All other pts. (n=291) were considered as high-risk (HR) pts.. In contrast to trial AML-BFM 93, a 2nd induction (HAM) was included in the treatment strategy for SR pts. (excluding FAB M3) which consisted of high-dose cytarabine (3g/m/12h x3 days) and mitoxantrone (10mg/m/d x2 days). Both SR and HR pts. were then randomly assigned to receive a 6-week consolidation or two short cycles of therapy. Compared to the 6-week consolidation, the short cycles contained higher doses of cytarabine, but the same cumulative dose of anthracylines. All other therapy elements [first induction (AIE; cytarabine, idarubicin and etoposide), intensification (HAE; high dose cytarabine, etoposide), and maintenance therapy] were identical in studies 93 and 98. Results: Overall, 407 out of 461 (88%) pts. achieved remission (CR). Five-year survival, event-free survival (EFS) and disease-free survival (DFS) were 59%±3%, 49%±3% and 55%±3%, respectively. Estimated survival and pEFS were similar to those of study 93 (58%±2% and 50%±2%, p logrank .09 and .80). Analysis of SR pts. (FAB M3 excluded) showed that the additional application of HAM did not improve the prognosis of SR pts. compared to AML-BFM 93 [CR rate 92% vs. 91%, p(chi)=.78; 5-year pEFS 58%±5% vs. 66%±4%; p logrank =.24]. However, when comparing HAM in study 98 and the 2nd chemotherapy cycle in study 93, significantly more severe infections (grade 3/4) occurred with HAM. Overall treatment related mortality in CR was 4% in both HR and SR pts., which was similar to trial AML-BFM 93. In addition, the outcome of pts. randomized for the 6-week consolidation was similar to that of the short cycles (p logrank .81). However, morbidity was lower in the short cycle arm (6 vs. 11 deaths in CCR, 2 vs. 4 in SR pts.). Conclusion: Our results indicate that in SR pts. with AML, a more intensive chemotherapy consisting of HAM does not result in improved survival. Therefore, new treatment options should be considered in this patient group. At the same time, optimizing treatment using the less toxic therapy with short cycles and improvement of supportive care strategies might help to reduce treatment related mortality and improve outcome in children with AML.

In primary immunodeficiencies the substitution of human immunoglobulins (Ig) is a major therapeutic concept. In Germany, Ig substitutions relied on various Ig formulations for intravenous (iv) substitutions. From December 2002 Ig preparations for subcutaneous (sc) home treatment were licensed in Germany. In the Frankfurt cohort several PID PTPs decided to switch from intravenous therapy (ivIg) in our outpatient clinic to subcutaneous infusions (scIg) at home. For PUPs current protocols suggest an iv loading dose prior to scIg or daily scIg infusions for one week. In a Pediatric cohort, daily infusions seem not feasible, therefore a novel approach was developed to rapidly achieve sufficient IgG levels sparing an iv loading dose and daily infusions. In a longitudinal analysis, immune parameters were monitored for PTPs changed to scIg and PUPs starting on scIg primarily. In a prospective study we report about 12 patients at the age of 14 to 58 years that have been switched to or were started on scIg. These are pediatric as well as adult patients. Their conditions include IgG2-IgA deficiencies, IgG3 deficiency, CVID, hypo- and agammaglobulinemia. Trough levels of total IgG, IgG subclasses, markers of infection and white blood cell counts were monitored under ivIg for at least one year prior to changing to scIg for PTPs retrospectively. Identical parameters have been monitored for 3 to 16 months since the start of scIg prospectively. Monitoring also included the history of any infections, general well-being and the body weight. For all patients the monthly scIg dose was identical to the respective ivIg dose at the time of switching therapy (200-400mg/kg bw). For PUPs a protocol of infusions on every other day was established changing to weekly intervals when sufficient IgG levels were achieved. The scIg infusions were well tolerated by all patients besides initial local swelling and slight pain on the following day. Before starting home therapy, patients were trained for a period of 4 weeks and were able to safely perform infusions. For PTPs, after switching to scIg the IgG trough levels increased. Also serum IgG subclasses increased and reached physiological levels in all subclasses. Patients that did not have a sufficient amount of IgG of any subclasses under ivIg reached normal levels after scIg was initiated within a maximum of 6 weeks (average of 3,5 weeks). We saw a tendency to a decrease in the frequency of infections. For PUPs, patients with subclass deficiencies reached normal IgG levels in total and in the respective subclasses after 2 to 3 infusions. A PUP with CVID needed 4 infusions (1 week) to achieve sufficient levels to change to weekly infusions. In summary at the Frankfurt immunodeficiency outpatient clinic switching from ivIg to self-administered scIg was well tolerated. In all PTPs higher IgG trough levels were reached with the same amount of substituted IgG. Patients with subclass deficiencies under ivIg reached physiological levels after changing to scIg. For PUPs a novel protocol was established with infusions on every second day for one week to achieve sufficient IgG levels without iv loading dose. In comparison to established protocols, this novel approach spares daily visits and infusions. Additionally this prevents injections into still painful tissues, which is highly relevant especially for the treatment and adherence of pediatric patients.

Acute leukemias are frequently associated with specific chromosomal translocations of the human MLL gene. In general, MLL translocations define a distinct disease entity that needs to be diagnosed with precision to facilitate rapid clinical decisions. Here we present data about a new PCR based method that uses patient genomic DNA to identify any MLL fusion. Fourty different MLL translocations were successfully analyzed. We will present three novel MLL translocation partner genes and a new MLL deletion. The benefits of this novel technique for diagnosis and MRD analyses will be discussed. Supported by grant 2001.061.1 from the Wilhelm Sander foundation.

Juvenile myelomonocytic leukemia (JMML) is a rare clonal disorder of early childhood. Currently, only allogeneic stem cell transplantation (SCT) offers long-term cure. Relapse remains the major cause of treatment failure. Although graft-versus-leukemia (GVL) effect most likely plays an important role in controlling JMML, the benefit of donor leukocyte infusion (DLI) following SCT in JMML is currently unknown. Patients and methods: Twenty-one patients with JMML who received DLI after SCT, including 4 patients after given a second SCT, were studied (BMT 14, PBSCT 6, CB 1). The median age at SCT was 15 (8–99) months. A normal karyotype, monosomy 7 or other aberrations were observed in 15, 2, and 4 patients, respectively. Six patients were transplanted from a matched sibling and 15 from an alternative donor. Chimerism analyses were performed by microsatellite PCR system or FISH for sex mismatch in all the patients. Response was defined as the achievement of complete chimerism (CC) and no evidence of hematological relapse. DLI was given either for the development of mixed chimerism (MC) in 7 patients (MC group) or for cytogenetic/hematological relapse in 14 patients (relapse group). Prior to DLI, cyclosporin A had been stopped in all patients, and no child had received chemotherapy. Results of DLI: Five of the 21 patients received a single DLI, 16 patients 2–6 infusions (median 3). The total T cell dose given ranged from 9x104 to 2.4 x108/kg. Six of 21 patients responded: 3 of 7 patients in the MC group and 3 of 14 patients in the relapse group. The infusion of at least 1x107/kg T cells was needed for durable response. Response was observed in all karyotype subgroups. None of the 6 patients receiving DLI from a matched sibling responded. Five patients developed acute GVHD following DLI and 4 of them responded to DLI. On the contrary, only 2 of the 16 children who did not show acute GVHD after DLI responded. Chronic GVHD developed in 2 responders. The outcome of even the responders was unfavorable. Only one of the responders is alive in remission, with severe chronic GVHD, 72 months after DLI. Two patients relapsed 26 days and 54 months after DLI (one as gastric chloroma), and 3 died of complications of DLI (acute GVHD, bone marrow aplasia, and hyper-eosinophilic syndrome). Four non-responders and one responder with subsequent relapse were rescued by a second SCT. Conclusion: This study shows that DLI can induce a GVL effect in some of the JMML patients. However, the benefit of DLI in our series of patients was limited because of severe complications in responders and lack of a durable effect. Some modification of DLI, with previous cytoreduction by chemotherapy or a novel drug such as E21R and concomitant administration of cytokines such as interferon alpha, can possibly improve the result of DLI.

Hemophilia A is an X-linked bleeding disorder. Mutations in the Factor VIII (FVIII) gene result in reduced or non-functional expression of FVIII. Patients are commonly treated by substitution of recombinant (rFVIII) or plasma derived (pdFVIII) FVIII preparations. However, a significant number of patients treated develop an antibody response (inhibitors) against substituted FVIII. Various Immune Tolerance Therapies (ITTs) are applied to overcome the FVIII specific immune response. We recently reported on a novel approach to eliminate the FVIII specific immune response by several rounds of rituximab (Mabthera®) infusions after several failed ITT approaches. In this study, phage displayed random peptide libraries were screened with sera from a patient before, under and after rituximab treatment. After several rounds of biopanning more than 100 phage clones specific for FVIII-inhibitors were identified and sequenced. One amino acid motif - EVPN - was most common in isolated clones. Computer based analysis identified a conformational epitope in the A2 domain of FVIII. The clones specifically inhibit binding of inhibitor to FVIII. Also sera from other inhibitor patients cross reacts with isolated phage clones. Inhibitor sera also bind to synthetic peptides, which sequences correspond to the peptide insert on isolated phage. Functional studies to target inhibitor specific B cells with isolated peptides are currently being performed. Specific ligands for FVIII inhibitors were identified. The epitope was mapped to the A2 domain. When inhibitor relapsed after Rituximab treatment, the same epitope was recognized. Small peptides representing the epitope on the phage (mimotopes) bound to inhibitor positive sera and even cross-reactivity with heterologous sera was observed. The inhibitor specific peptide ligands may serve as useful tools to develop novel approaches for inhibitor elimination therapies.

Profound depletion of T and B cells is a fundamental prerequisite for haploidentical transplantation. Here, we compare hematopoietic engraftment and occurrence of GvHD after two different positive selection procedures (with anti-CD34 coated or anti-CD133 coated beads) and after a direct depletion procedure with CD3/CD19 coated microbeads in children with leukemias (ALL, AML, CML) and lymphomas. 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 was better after CD34+ selection than after CD133+ selection (4.1log vs. 3.75log, p Primary engraftment occurred in 85% (44/52) of patients with CD34+ selected stem cells and in 78% (11/14) of patients with CD133+ selected cells. After reconditioning, sustained engraftment was achieved in 98% and 100%. All patients (10/10) with CD3+/CD19+ depleted grafts had primary engraftment. Recovery of platelets was rapid especially in patients with CD133+ selected grafts (median time to reach independence from substitution = 14.5 days). GvHD grade II occurred in 6%, 7% and 25%, respectively. Only one patient had severe GvHD grade III-IV (CD34+).Conclusions: CD34+ selection and CD133+ selection resulted in similar engraftment and GvHD rates. CD3+/CD19+ depletion resulted in better engraftment but produced slightly more GvHD. Thus, the methods may be individually used according to the patient’s requirements.

In order to assess the impact of age at first exposure to F VIII and therapy regimen on neutralizing FVIII-antibodies in previously untreated patients (PUP) with hemophilia A a prospective study was performed. Over a 23-years study period a total of 74 severely affected hemophilia A -PUPs have been consecutively recruited, treated with F VIII and investigated for inhibitor development. The patients were divided into two groups according to their treatment regimen: Group 1 (n=23) started prophylaxis at the age of 1 year (before or immediately after the first relevant bleed). Group 2 (n=43) was treated on-demand or prophylaxis was started after more than 2 bleeds. The following parameters were equally distributed among both groups: caucasian ethnicity, intron-22-inversion, age at 1st ED >0.5 years. Out of 74 hemophilia A patients 23 developed inhibitors (31%). Inhibitor incidence was 0% (1 transient inhibitor out of 23 patients) in those patients who received early prophylaxis (group 1) and 42% (18/43 patients) in case of delayed prophylaxis or on-demand treatment (group 2) (p=0.002). No linear correlation was found between the age at first exposure and inhibitor formation. However, patients treated before the age of 0.5 years showed a significantly higher inhibitor incidence (62%) than those treated at an more advanced age. Our data confirm that very early age at first exposure is a risk factor for inhibitor development in severe hemophilia A patients. Early prophylaxis might be protective against inhibitor development. To confirm the data a larger patient cohort has to be investigated.

Invasive aspergillosis is still a life-threatening complication, in particular in patients after allogeneic hematopoietic stem cell transplantation (HSCT). Whereas prolonged neutropenia is a well established risk factor for invasive fungal disease, there is a growing body of evidence that T-cells also play an important role in the immunological response to Aspergillus species. Since invasive aspergillosis often occurs during the phase of postengraftment, which is characterized by impaired cell-mediated immunity, Aspergillus-specific T-cells could be a potential therapeutic target in these patients. We therefore analyzed in a first step the response of T-cells to several potential antigens of Aspergillus fumigatus by means of 3H-thymidine incorporation assay. In order to generate Aspergillus-specific T-cells, the antigens with the highest proliferation indices (EC-SAB and 90 kDa catalase) were used to stimulate 1.0 x 108 mononuclear cells from healthy donors. The activated T-cells were isolated on the following day using the IFN-γ secretion assay (Miltenyi Biotec, Germany) and then expanded for 14 days. Intracellular cytokine analysis of EC-SAB generated cell lines (n=7) revealed a significant IFN-γ secretion by 13.6%±2.3 of CD4+ cells (seven out of seven tested cell lines) and an Aspergillus-specific IL-2 secretion by 6.5%±1.9 of CD4+ cells (three out of three tested cell lines), which supports the TH1 response of the generated cells to Aspergillus antigen. In contrast to EC-SAB generated T-cell populations, all three cell lines which were generated with 90 kDa catalase were not informative. Further analysis showed that restimulation with EC-SAB induced a strong proliferation of EC-SAB generated T-cell populations (all three populations tested), whereas alloreactivity was unaffected. The number of these cells could be expanded within 14 days up to 20fold using OKT-3, IL-2 and feeder cells. Currently, we investigate the impact of these Aspergillus-specific cell populations in the defense to different species of Aspergillus. Our preliminary results suggest that Aspergillus-specific T-cells could be an interesting option in prophylaxis and therapy of invasive aspergillosis in patients undergoing HSCT.

Ten children with stage III or IV neuroblastoma that had either relapsed or was refractory were treated with [131I]-metaiodobenzylguanidine (MIBG) from 1984 to 1986. The total dose ranged from 4,365 to 21,900 MBq and was given in one to five courses. Two patients achieved a complete remission (CR), two, a partial remission (PR), and three, an arrest of the disease. Pharmacological studies showed that 93% of detectable radioactivity was attributable to MIBG at the beginning of the infusion. However, by the end of the infusion this had decreased to 88%. The terminal half-life of MIBG was 37.0 h, whereas that of non-MIBG-bound iodine was 71.6 h. Therefore, the radioactivity-time product of non-MIBG-bound 131I was much higher than that of MIBG. Dosimetric studies showed a mean level of absorbed radiation for the total body of 160 microGy/MBq, a liver irradiation of 540 microGy/MBq and a mean tumour radiation of 10,500 microGy/MBq.

Between April 1984 and December 1985 we treated ten children suffering from neuroblastoma in a total of 25 metaiodobenzylguanidine (MIBG) courses. Five had had a relapse of neuroblastoma stage III or IV, three had never achieved a remission in spite of intensive chemotherapy, and two were treated with an unstable remission. The children were each administered from 1 to 5 courses with a dosage per course of between 1,295 and 9,065 MBq. The sum of the single doses during the whole course of therapy ranged between 3,145 and 21,904 MBq per child. Five of five children suffering from bone pain and fever became free of complaints during the first three treatment days. Six of eight children with manifest tumor at onset of therapy responded well to the treatment: response extended from transitory decrease in elevated catecholamine levels in serum and urine to complete disappearance of large abdominal tumor masses. We also observed a decrease in bone marrow involvement and a stabilization of osteolytic lesions. Seven of these eight children died in spite of a good response from 55 to 350 days after the first MIBG treatment course. The only side effect we witnessed was a reversible bone marrow depression. In three children we combined the MIBG therapy with bone marrow transplantation.

The long term ovariectomized adult rat was used to test the effects of exogenous estradiol, 4-hydroxyestradiol, and 2-hydroxyestradiol on LH secretion. To this end, different doses of estradiol 3-benzoate, 4-hydroxyestradiol 3,4-dibenzoate, and 2-hydroxyestradiol 2,3-dibenzoate were injected daily at 0800 h, and the LH serum levels were measured on 4 experimental days. At a dose of 1 micrograms/day, estradiol benzoate lowered LH secretion, beginning 48 h after the first injection (morning of day 2), and induced a characteristic LH surge 10 h later. 4-Hydroxyestradiol dibenzoate at the same dose produced less suppression of LH on the morning of day 3, but caused a comparable and highly significant surge on the same evening. Higher doses (3 and 10 micrograms/day) resulted in the same pattern seen with estradiol benzoate. 2-Hydroxyestradiol dibenzoate at comparable doses had no effect. Only extremely high doses (129 micrograms/day) caused slight suppression of tonic LH secretion 72 h after the first injection, and inconsistent LH elevations occurred on the same evening. It is concluded that in this model, catecholestrogens act as estrogens with respect to LH suppression and release, with 4-hydroxyestradiol being a potent estrogen and 2-hydroxyestradiol a weak estrogen.

The incidence of infection with penicillin-non-susceptible Streptococcus pneumoniae is increasing rapidly worldwide. Spain and France are highly affected, whereas the level of penicillin resistance in Germany, Italy, The Netherlands and Scandinavia is low. We report a lethal episode of meningitis due to penicillin- and cefotaxime-intermediate S. pneumoniae in a 7-year-old, allogene bone marrow transplanted German boy, 5 weeks after a holiday in Spain. Three days prior to the infection the patient showed good performance status. He was in complete remission without signs of graft-versus-host disease (GVHD). He died on day 341 post bone marrow transplant (BMT), 2 days after the onset of meningitis. Penicillin-non-susceptible S. pneumoniae should be regarded as a potential infectious agent even in countries with a low prevalence of resistance.

Rearrangements of the MLL (mixed lineage leukemia) gene are associated with pediatric, adult and therapy-related acute leukemias of both the myeloid and lymphoid lineage. Today, about 60 MLL fusion partner genes have been characterized at the molecular level involving almost all chromosomes. Nearly one-third of these partner genes have been identified at the DCAL (Diagnostic Center of Acute Leukemia) where more than 700 MLL rearrangements, including 36 different partner genes, have been analyzed so far. 85% of the analyzed MLL rearrangements account for the 6 most frequent partner genes, and 15% for less frequent partner genes including the 8 novel partner genes ACTN4, C2CD3, DCP1A, FLNA, LAMC3, LOC100128568, NRIP3, and TNRC18. Here, we present an overview of all current known translocation partner genes. This overview indicates all the specific introns of the TPGs found to be involved in MLL translocations, their chromosomal locations and the type of genetic abnormality that is leading to the MLL fusion. Additionally, the current breakpoint cluster region (bcr) is also presented for the 10 most frequent partner genes, namely AFF1 (AF4), MLLT3 (AF9), MLLT1 (ENL), MLLT10, MLLT4 (AF6), ELL, EPS15, MLLT6 (AF17), MLLT11 (AF1Q) and partial tandem duplications (PTDs). These bcrs have been used as basis for the development of an long range multiplex PCR. This is an additional tool for the identification of genomic MLL breakpoints to enhance the diagnostic efficiency. Approximately 20% of all MLL rearrangements are complex ones. In this study, we have identified 109 (15%) complex rearrangements. Within these complex rearrangements, the 5’ part of the MLL gene is generally fused in frame to one of the most frequent partner genes. But the 3’ part of the MLL gene is fused in 45 cases (41%) to a novel so-called “reciprocal MLL partner gene” like ADSS or ATG16L2. This heterogeneous group of novel translocation partner genes had not been identified as fusion partners to the 5’ part of the MLL gene before. Whether the pathobiology of complex rearrangements is different in comparison to their not complex counterparts has still to be proven. Also a novel “spliced MLL fusion” involving the AFF1 gene has been identified at the DCAL. In this case, the breakpoint is 90 kb up-stream of the AFF1 gene. Unfortunately, no cDNA/RNA was left to identify the chimeric fusion transcript. Nevertheless, the disease phenotype of a biphenotypic AL strengthens the involvement of the AFF1 gene. With these results, the number of genes involved in „ spliced MLL fusions“ has increased from seven to eight. For five patients, no partner gene could be identified at the molecular level. Also the “spliced MLL fusions” which is a mechanism to generate functional chimeric fusion transcripts with neighbouring genes described already for 8 partner genes of the MLL gene, could not be identified for these patients by RACE and RT-PCR. Furthermore, the determined patient-specific fusion sequences are succesfully used worldwide for minimal residual disease (MRD) monitoring to improve the treatment and outcome of acute leukemia patients.

Background Cytokine-induced-killer (CIK) cells are a promising immunotherapeutic approach for impending relapse following hematopoietic stem cell transplantation (HSCT). However, there is a high risk for treatment failure associated with severe graft versus host disease (GvHD) necessitating pharmaceutical intervention post-transplant. Whether immunosuppression with mycophenolate mofetil (MMF) or Ciclosporin A (CsA) influences the cytotoxic effect of CIK cell immunotherapy is still an open issue. Methods CIK cells were generated from PBMC as previously described followed by co-incubation with mycophenolic acid (MPA) or CsA. Proliferation, cytotoxicity and receptor expression were investigated following short- (24 h), intermediate- (3 days) and long-term (7 days) MPA incubation with the intention to simulate the in vivo situation when CIK cells were given to a patient with relevant MPA/CsA plasma levels. Results Short-term MPA treatment led to unchanged proliferation capacity and barely had any effect on viability and cytotoxic capability in vitro. The composition of CIK cells with respect to T-, NK-like T- and NK cells remained stable. Intermediate MPA treatment lacked effects on NKG2D, FasL and TRAIL receptor expression, while an influence on proliferation and viability was detectable. Furthermore, long-term treatment significantly impaired proliferation, restricted viability and drastically reduced migration-relevant receptors accompanied by an alteration in the CD4/CD8 ratio. CD3+CD56+ cells upregulated receptors relevant for CIK cell killing and migration, whereas T cells showed the most interference through significant reductions in receptor expression. Interestingly, CsA treatment had no significant influence on CIK cell viability and the cytotoxic potential against K562. Conclusions Our data indicate that if immunosuppressant therapy is indispensable, efficacy of CIK cells is maintained at least short-term, although more frequent dosing might be necessary. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-1024-4) contains supplementary material, which is available to authorized users.

Background: Emerging evidence indicates that mesenchymal stromal cells (MSCs) isolated from different tissue sources may be used in vivo as tissue restorative agents. To date, there is no evidence, however, on migration and proliferation ("wound healing") potential of different subsets of MSCs. The main goal of this study was therefore to compare the in vitro "wound healing" capacity of MSCs generated from positively selected CD271+ bone marrow mononuclear cells (CD271-MSCs) and MSCs generated by plastic adherence (PA-MSCs). Methods: The in vitro model of wound healing (CytoSelect™ 24-Well Wound Healing Assay) was used in order to compare the migration and proliferation potential of CD271-MSCs and PA-MSCs of passage 2 and 4 cultured in presence or absence of growth factors or cytokines. Results: CD271-MSCs of both passages when compared to PA-MSCs demonstrated a significantly higher potential to close the wound 12 and 24 h after initiation of the wound healing assay (P < 0.003 and P < 0.002, respectively). Noteworthy, the migration capacity of PA-MSCs of second passage was significantly improved after stimulation with FGF-2 (P < 0.02), PDGF-BB (P < 0.006), MCP-1 (P < 0.002) and IL-6 (P < 0.03), whereas only TGF-β enhanced significantly migration process of PA-MSCs of P4 12 h after the treatment (P < 0.02). Interestingly, treatment of CD271-MSCs of both passages with growth factors or cytokines did not affect their migratory potential. Conclusions: Our in vitro data provide the first evidence that CD271-MSCs are significantly more potent in "wound healing" than their counterparts PA-MSCs.