[Germline mutations in children with cancer - leopoldina meeting report, september 26, 2014, jesuit monastery merzhausen]. / Keimbahnmutationen bei krebskranken Kindern - Symposium der Leopoldina am 26.09.2014 im Jesuitenkloster Merzhausen bei Freiburg.

Under the umbrella of the Leopoldina, National Academy of Science Germany, a one-day workshop took place with experts from Pediatric Oncology, Human Genetics, Jurisprudence and Science Ethics. Professor Dr. Matthias Brandis, former head of the Clinic of Pediatrics and Adolescent Medicine, Albert-Ludwigs-University Freiburg, encouraged the authors to organize this workshop near Freiburg and provided professional and logistic support. Professor Dr. Matthias Brandis serves as the chairmen of our pediatric-gynecological section within the Leopoldina.

[Research in pediatric hematology [Germany, 1970-2000]]. / Forschung in der Pädiatrischen Hämatologie [Deutschland, 1970-2000].

Professor Riehm was not only an excellent pediatric oncologist, but also an excellent hematologist. He was a mentor also for young scientists who were interested in hematological research as his/her main focus. In the 1970s he has promoted research in coagulation disorders, during 1980s he supported the investigation on hematopoetic growth factors. During the 1990 s he was one of the first in Europe who have used hematopoietic growth factors as a supportive therapy in the protocol ALL-BFM and has also helped to set up the International Registry for Severe Chronic Neutropenias [SCNIR] in his Department. The sense of the top research directions of the time distinguishes him not only as a pioneer of the leukaemia therapy studies, but also as a pioneer in hematology. He created the environment in which research could blossom.

[The West-Berlin therapy study of acute lymphoblastic leukemia in childhood--report after 6 years]. / Die West-Berliner Studie zur Behandlung der akuten lymphoblastischen Leukämie des Kindes--Erfahrungsbericht nach 6 Jahren.

During the last 6 years 73 previously untreated children and adolescents with acute lymphoblastic leukemia were enrolled on a non-randomized therapy protocol. In this longitudinal study the specific accent was put on the intensified induction treatment of 8 weeks' duration which was thought to achieve a higher remission quality. In this phase 8 effective drugs were applied up to the patient's tolerance limits; in addition prophylactic irradiation to the central nervous system was given in two modifications. After 4 weeks all patients were in complete remission. During the first 4 months 6 children died due to complications for which therapy must be at least partially responsible. 17 out of 67 patients relapsed between 4 and 59 months after diagnosis, which corresponds to a remission rate according to the life table analysis of 62% (50 out of 67 patients in first remission). The majority of patients with relapse is characterized at diagnosis by a specific pattern of clinical findings. 2 therapy groups, differently treated in respect to central nervous system irradiation and duration of continuation therapy, do not at present statistically differ from each other. According to statistics 8 more children may be expected to suffer from relapse. The improved results are due to a lower incidence of bone marrow relapses; it seems that there is a direct relation between intensity of treatment during the induction phase and the occurrence of bone marrow relapses. The specific problems of the presented study take place during the induction phase, which, due to toxicity, regularly results in a number of side effects and severe complications. In order to realize the induction therapy, the use of prophylactic and supportive procedures is of utmost importance. Profound knowledge of possible side effects and complications is most essential as well as the knowledge of how to cope with these problems. It is the authors' opinion that the induction phase can only be performed in specialized institutions, because only at these places enough information may be obtained from an adequate number of patients and only there pediatric oncologists may share in the decisions and responsibilities. Only by using every kind of medical service this method of therapy may be justified at present, according to the results of this study, for the benefit of children with leukemia.

Key treatment questions in childhood acute lymphoblastic leukemia: results in 5 consecutive trials performed by the ALL-BFM study group from 1981 to 2000.

Between 1981 and 2000, 6 609 children (<18 years of age) were treated in 5 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 (survival) improved from 65% (77%) in study ALL-BFM 81-78% (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 ALL patients and eliminated in non-high-risk non-T-ALL patients, if it was replaced by high-dose and intrathecal methotrexate; 2) omission of delayed reintensification severely impaired outcome of low-risk patients; 3) 6 months 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 a significant improvement of outcome; 6) the daunorubicin dose in induction could be safely reduced in low-risk patients; 7) intensification of consolidation/reintensification treatment led to considerable improvement of outcome in high-risk patients.

Human endothelial cells regulate survival and proliferation of human mast cells.

Mast cells (MCs) are immunoregulatory and inflammatory tissue cells preferentially located around blood vessels. Since endothelial cells have been suggested to regulate MC functions, we analyzed MC-endothelial cell interactions in vitro by performing coculture experiments with purified human intestinal MCs and human umbilical vein endothelial cells (HUVECs). We found that HUVECs provide signals allowing MCs to survive for at least 3 wk and to proliferate without addition of cytokines; otherwise all MCs died. HUVEC-dependent MC proliferation was more pronounced than that induced by stem cell factor (SCF), known to act as an MC growth factor both in vitro and in vivo. After coculture with HUVECs, most MCs were of the tryptase and chymase double-positive phenotype (MC(TC)). Transwell experiments suggested that the HUVECs' effects on MCs are not mediated by soluble factors. HUVEC-dependent MC adhesion and proliferation were inhibited by neutralizing antibodies directed against SCF and vascular cell adhesion molecule (VCAM)-1 expressed on HUVECs, and c-kit and very late antigen 4 (VLA-4) on MCs. The data suggest that two mechanisms (membrane-bound SCF/c-kit and VCAM-1/VLA-4) are involved in human MC-endothelial cell interactions. In conclusion, our study provides evidence that endothelial cells regulate MC survival and preferentially support human MC(TC) development.

Purification of human interleukin 2 to apparent homogeneity and its molecular heterogeneity.

Interleukin 2 (IL-2), produced with and without co-stimulation by the Burkitt's lymphoma line Daudi, was purified 37,000-fold to apparent homogeneity from lymphocyte conditioned medium by (NH4)2SO4 precipitation, DEAE-cellulose ion-exchange chromatography, gel filtration, and chromatography on blue agarose and on Procion-red agarose. The purified IL-2 showed a 10(6) U/mg protein sp act. IL-2 produced in the absence of Daudi cells had a mol wt of 26,000 as measured by gel filtration and an isoelectric point of 6.7. This IL-2 showed a 16,000 and 17,000 mol wt in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). IL-2, produced in the presence of Daudi cells (10(6)/ml), showed a mol wt of approximately 14,000, as measured by both gel filtration and SDS-PAGE, and an isoelectric point of 8.1. The purified IL-2 lacked detectable interferon (alpha and gamma), granulocyte-macrophage colony-stimulating factor, B cell growth factor, T cell-replacing factor, and thymocyte-differentiating activity and was free of any contaminating proteins as judged by silver staining in SDS-PAGE. All three molecular forms of IL-2 were biologically active at concentrations of 10(-11) - 10(-10) M, supporting the growth of human and murine cytotoxic T cell lines.

Interleukin 2 regulates the expression of Tac antigen on peripheral blood T lymphocytes.

We investigated the effect of OKT3 antibody and interleukin 2 (IL-2) on Tac antigen expression and the proliferation of human peripheral blood mononuclear leukocytes. OKT3 monoclonal antibody at low, nonmitogenic concentrations (25 pg/ml) or IL-2 alone at optimal concentrations (20 U/ml) did not induce IL-2 receptor expression, as measured by Tac antibody or by T cell proliferation. However, costimulation with these concentrations of OKT3 antibody and IL-2 led to Tac antigen expression and T cell proliferation. These data suggest that the T cells are activated in two steps: OKT3 antibody at 25 pg/ml does not induce Tac antigen expression, but preactivates T cells to become responsive to IL-2. The addition of exogenous IL-2 then leads to expression of the IL-2 receptor, as recognized by Tac antibody, and to subsequent proliferation.

Biological activities of a human pluripotent hemopoietic colony stimulating factor on normal and leukemic cells.

We studied the biological effects of pluripoietin, a human pluripotent hemopoietic colony-stimulating factor (CSF) purified from the 5637 bladder carcinoma cell line. We found that this human CSF appears to be a unique hemopoietic growth factor, differing from interleukin 3 (IL-3) by virtue of its leukemia differentiating activity in mouse and man, and from mouse granulocyte CSF, which does have differentiation-inducing activity, but lacks pluripoietic activity. In addition, differences from IL-3 were observed in cross-species activity on normal and leukemic cells.

Recombinant human granulocyte colony-stimulating factor. Effects on hematopoiesis in normal and cyclophosphamide-treated primates.

We examined the in vivo effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in primates (cynomolgus monkeys) treated with subcutaneous doses of rhG-CSF for 14-28 d. A dose-dependent increase in the peripheral white blood cells (WBC) was seen, reaching a plateau after 1 wk of rhG-CSF treatment. The elevation of WBC was due to an increase in the absolute neutrophil count. These results demonstrate that rhG-CSF is a potent granulopoietic growth and differentiation factor in vivo. In cyclophosphamide (CY)-induced myelosuppression, rhG-CSF was able to shorten the time period of WBC recovery in two treated monkeys to 1 wk, as compared to more than 4 wk for the control monkey. Its ability to significantly shorten the period of chemotherapy-induced bone marrow hypoplasia may allow clinicians to increase the frequency or dosage of chemotherapeutic agents. In addition, the increase in absolute numbers of functionally active neutrophils may have a profound effect in the rate and severity of neutropenia-related sepsis. Furthermore, the activities reported here indicate a potential role for rhG-CSF in the treatment of patients with myelodysplastic syndrome, congenital agranulocytosis, radiation-induced myelosuppression, and bone marrow transplantation.

Activation of human macrophages. Comparison of other cytokines with interferon-gamma.

Cytokines affecting mononuclear phagocytes were screened for activation of human macrophages to secrete H2O2 and kill toxoplasmas. In contrast to recombinant interferon-gamma (rIFN gamma), the following factors, tested in partially or highly purified form and over a wide range of concentrations, did not augment these functions: native interferon-alpha (nIFN alpha), rIFN alpha A, rIFN alpha D, rIFN beta, colony stimulating factor (type 1) (CSF-1), CSF for granulocytes and macrophages (GM-CSF), pluripotent CSF (p-CSF), tumor necrosis factor (TNF), native interleukin 2 (nIL-2), and rIL-2. Partially purified migration inhibitory factor (MIF) enhanced H2O2-releasing capacity submaximally without inducing antitoxoplasma activity, and warrants further study.

Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells.

Experiments were conducted to isolate and characterize the gene and gene product of a human hematopoietic colony-stimulating factor with pluripotent biological activities. This factor has the ability to induce differentiation of a murine myelomonocytic leukemia cell line WEHI-3B(D+) and cells from patients with newly diagnosed acute nonlymphocytic leukemia (ANLL). A complementary DNA copy of the gene encoding a pluripotent human granulocyte colony-stimulating factor (hG-CSF) was cloned and expressed in Escherichia coli. The recombinant form of hG-CSF is capable of supporting neutrophil proliferation in a CFU-GM assay. In addition, recombinant hG-CSF can support early erythroid colonies and mixed colony formation. Competitive binding studies done with 125I-labeled hG-CSF and cell samples from two patients with newly diagnosed human leukemias as well as WEHI-3B(D+) cells showed that one of the human leukemias (ANLL, classified as M4) and the WEHI-3B(D+) cells have receptors for hG-CSF. Furthermore, the murine WEHI-3B(D+) cells and human leukemic cells classified as M2, M3, and M4 were induced by recombinant hG-CSF to undergo terminal differentiation to macrophages and granulocytes. The secreted form of the protein produced by the bladder carcinoma cell line 5637 was found to be O-glycosylated and to have a molecular weight of 19,600.

Pharmacokinetic monitoring of intravenous cyclosporine A in pediatric stem-cell transplant recipients. The trough level is not enough.

In order to monitor CsA serum levels after SCT, trough levels (C0) are widely used. The aim of this study was to estimate the population and individual PK parameters for patients receiving intravenous CsA after SCT. In 27 pediatric patients after SCT receiving CsA (3 mg/kg/day) every 12 h, a total of 289 CsA concentrations was obtained. To describe the PK parameters of CsA, a two-compartment model with first order elimination was used. Covariate analysis identified body weight, age, and the co-administration with itraconazole and tobramycine as factors influencing the Cl. The statistical comparison of AUC, trough level, and C2 indicates a correlation between AUC and C2, but no correlation between the AUC and C0, r = 0.24 (p = 0.146) vs. r = 0.526 (p = 0.000692), respectively. Our results underscore the fact that CsA trough levels do not reflect the drug exposure in patients receiving intravenous CsA after SCT. By contrast, CsA blood levels measured 2-6 h after CsA infusion showed a better correlation with the AUC. Our data provide new information to optimize the balancing act between GvHD-prophylaxis, graft vs. leukemia effect, and CsA side-effects after SCT.

Multiple independent second-site mutations in two siblings with somatic mosaicism for Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disorder associated with microthrombocytopenia, eczema, autoimmunity and predisposition to malignant lymphoma. Although rare, few cases of somatic mosaicism have been published in WAS patients to date. We here report on two Ukrainian siblings who were referred to us at the age of 3 and 4 years, respectively. Both patients suffered from severe WAS caused by a nonsense mutation in exon 1 of the WAS gene. In both siblings, flow cytometric analysis revealed the presence of Wiskott-Aldrich syndrome protein (WASp)-positive and WASp-negative cell populations among T and B lymphocytes as well as natural killer (NK) cells. In contrast to previously described cases of revertant mosaicism in WAS, molecular analyses in both children showed that the WASp-positive T cells, B cells, and NK cells carried multiple different second-site mutations, resulting in different missense mutations. To our knowledge, this is the first report describing somatic mosaicism in WAS patients caused by several independent second-site mutations in the WAS gene.

Inhibitor-Immunology-Study. Different HLA-types seem to be involved in the inhibitor development in haemophilia A.

UNLABELLED: The development of inhibitors is one of the most important complications of replacement therapy in haemophilia, affecting mortality and morbidity. Inhibitor development is based on complex immunological factors. Cytokines and their receptors, T-cell receptors, and the Major Histocompatibility Complex may play important roles in the development of inhibitors. Earlier studies showed non significant associations between HLA class and inhibitor development. Later studies found an increased risk of inhibitor development if there was a combination between certain factor VIII mutations and HLA antigens. We performed HLA typing in 50 patients with haemophilia A in an effort to find associations with inhibitor development. RESULTS: 25 patients had developed an inhibitor (11 low titre, 14 high titre), and 25 never had. In logistic regression analysis, HLA-A 34, DRB1 0405, DRB1 1301 seemed to be involved in inhibitor development and HLA-A 30, B 13, B15, B 57, Cw 12, DQB1 0303, DPB1 0201 protection against inhibitor development. In our patients, the HLA-associations with inhibitor development were different from those in previous publications.

Analysis of prostaglandin E2 effect on T lymphocyte activation. Abrogation of prostaglandin E2 inhibitory effect by the tumor promotor 12.0 tetradecanoyl phorbol-13 acetate.

We have investigated the inhibitory potential of prostaglandin E2 (PGE2) with respect to intracellular messengers implicated in the signaling system of T-lymphocyte activation pathway. Using the fluorescent indicator Quin 2, it is demonstrated that PGE2 inhibits the increase in cytosolic-free calcium concentration [Ca2+]i. Reconstitution of calcium mobilization in the presence of PGE2 by the calcium ionophore A23187 results in a partial restoration of both interleukin 2 (IL2) production and cell proliferation and has no effect on the inhibition of transferrin receptor expression. In contrast, the treatment of cell cultures with the tumor promotor 12.0 tetra decanoyl phorbol-13-acetate (TPA) abrogates the suppressor activity of PGE2. When T lymphocyte stimulation is provided by the combination of A23187 and TPA, the PGE2 inhibitory effect does not occur. These data also indicate that the down regulation of transferrin receptor by PGE2 is proximal to protein kinase C activation and is not associated with decreased expression of the functional IL2 receptor.

Production of and in vitro response to interleukin 2 in the acquired immunodeficiency syndrome.

To test the hypothesis that deficient interleukin 2 (IL-2) secretion may underlie the impaired capacity of T cells from patients with Acquired Immunodeficiency Syndrome (AIDS) and the AIDS-related complex (ARC) to generate the macrophage-activating lymphokine, gamma interferon (IFN-gamma), we used five specific microbial antigens to examine IL-2 production. Mononuclear cells from only one of 32 (3%) AIDS patients secreted normal levels of IL-2, and 21 (66%) failed to produce any detectable IL-2. For 36 ARC patients, IL-2 generation was normal in nine (25%) and absent in 11 (31%). Given these results, recombinant (r) IL-2 was tested for its capacity to stimulate or enhance IFN-gamma production. rIL-2 (10 U/ml) alone stimulated cells from controls, ARC, and AIDS patients to secrete 93 +/- 25, 99 +/- 33, and 7 +/- 3 U/ml of IFN-gamma, respectively. rIL 2 (10 U/ml) plus antigen induced no change in mean IFN-gamma levels for controls, a 4.4-fold increase for 17 AIDS patients (16 +/- 16 vs. 71 +/- 21 U/ml), and a 7.2-fold increase (18 +/- 5 vs. 130 +/- 27 U/ml) for 19 ARC patients with abnormal IFN-gamma generation to antigen alone. Individual responses indicated that six of the 17 (35%) AIDS patients with opportunistic infections and 12 of the 19 (63%) with ARC were apparent responders to 10-100 U/ml of rIL-2. These results (a) document profound impairment in antigen-induced IL-2 secretion by AIDS and ARC T cells, (b) indicate that, in vitro, mononuclear cells from certain patients can respond to rIL-2 with enhanced IFN-gamma production, and thus (c) suggest that in selected patients rIL-2 might have a potentially beneficial therapeutic (AIDS) or prophylactic (ARC) effect against opportunistic infections.

Substitution of cyclophosphamide and busulfan by fludarabine, treosulfan and melphalan in a preparative regimen for children and adolescents with Shwachman-Diamond syndrome.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only definitive treatment for severe bone marrow dysfunction and clonal disorders in patients diagnosed with Shwachman-Diamond syndrome (SDS). In an attempt to minimize regimen-related toxicity (RRT), we have initiated a fludarabine/treosulfan/melphalan-based pilot protocol avoiding the combination of busulfan and cyclophosphamide. Median age at transplantation was 9.6 years (range 1.5-17 years). All three patients received conditioning with fludarabine (30 mg/m2/day x 6), treosulfan (12 g/m2/day x 3) and melphalan (140 mg/m2/day x 1). CAMPATH-1H (0.1 mg/kg x 2) was added in two cases, while rabbit ATG (Genzyme; 3 x 2.5 mg/kg) was given to the cord blood recipient. One patient was transplanted with a non-manipulated marrow graft from an HLA-identical sibling, one with a marrow graft from a 10/10 matched unrelated donor, and one with a 9/10 matched unrelated umbilical cord blood (UCB) unit. Mean cell doses given were 3.6 x 10(8) nucleated cells/kg BW for the bone marrow recipients and 4.2 x 10(7) nucleated cells/kg BW for UCB recipient. Overall, two of three patients are alive and display 100% donor chimerism. Acute graft-versus-host disease grade II was seen in one patient, while no GVHD exceeding grade I occurred in the remaining two.

Relapse, not regimen-related toxicity, was the major cause of treatment failure in 11 children with Down syndrome undergoing haematopoietic stem cell transplantation for acute leukaemia.

We report a retrospective analysis of 11 children with Down syndrome (DS) treated by SCT in eight German/Austrian SCT centres. Indications for transplantation were acute lymphoblastic leukaemia (N=8) and acute myeloid leukaemia (N=3). A reduced intensity conditioning (RIC) containing 2 Gy TBI was given to two patients, another five received a myeloablative regimen with 12 Gy TBI. Treosulphan or busulphan was used in the remaining four children. Four of eleven (36%) patients are alive. All of them were treated with a myeloablative regimen. One of the four surviving children relapsed 9 months after SCT and is currently receiving palliative outpatient treatment. The main cause of death was relapse (5/11). Two children died of regimen-related toxicity (RRT), one from severe exfoliative dermatitis and multiorgan failure after a treosulphan-containing regimen, the other from GvHD-related infections after RIC. Acute GvHD of the skin was observed in 10 of 10 evaluable patients, and chronic GvHD in 4 of 8. Our data show that DS patients can tolerate commonly used, fully myeloablative preparative regimens. The major cause of death is relapse rather than RRT resulting in an event-free survival of 18% and over all survival of 36%.

Chromosomal aberrations in congenital bone marrow failure disorders--an early indicator for leukemogenesis?

As chromosomal instability may contribute to leukemogenesis in patients with congenital bone marrow failure (CBMF) disorders, it was the aim of this study to characterize chromosomally aberrant clones that arise during the clinical course of disease by means of R-banding and fluorescence in situ hybridization (FISH) analyses. In addition, multicolor-FISH and array-comparative genomic hybridization (CGH) were applied to characterize clonal chromosome aberrations in more detail. Between January 2004 and December 2005, we prospectively analyzed 90 samples of 73 patients with proven or suspected CBMF disorders enrolled in a German Study Network of CBMF diseases. Clonal aberrations could be identified in four of 73 patients examined. In one child with congenital thrombocytopenia, Jacobsen syndrome [del(11)(q24)c] was diagnosed, and thus a CBMF could be excluded. In a girl with Shwachman-Diamond syndrome, two independent clones, one with an isochromosome i(7)(q10), another with a complex aberrant karyotype, were identified. Simultaneously, transition into a myelodysplastic syndrome (MDS) occurred. The brother, who was also afflicted with Shwachman-Diamond syndrome, showed an isochromosome i(7q) as a single aberration. In the fourth patient with severe congenital neutropenia, an add(21)(q22) marker containing a low-level amplification of the AML1 gene was identified at the time point of transition into acute myelogenous leukemia (AML). In summary, we suggest that follow-up of patients with CBMF using chromosome and FISH analyses will be helpful for the early detection of transition into MDS or AML and thus should be an integral part of the clinical management of these patients.

Unsupervised proteome analysis of human leukaemia cells identifies the Valosin-containing protein as a putative marker for glucocorticoid resistance.

The response to initial glucocorticoid therapy in childhood acute lymphoblastic leukaemia (ALL) reliably predicts the response to multiagent chemotherapy. Patients resistant to glucocorticoids (prednisone poor responders (PPR)) have a poorer event-free survival compared to glucocorticoid-sensitive patients (prednisone good responders (PGR)). A case-control study was performed to investigate differential protein expression in leukaemic blasts from PGR and PPR childhood ALL patients. Two-dimensional gel electrophoresis (2-DE) was used for an unsupervised screening and surface enhanced laser desorption/ionisation-time of flight mass spectrometry (SELDI-TOF MS) for the characterisation of protein spots. In difference maps of average gels for the proteomes of each responder group, differentially expressed proteins were identified after tryptic digestion and spotting onto H4-SELDI-TOF-MS chips. Proteins overexpressed in PPR were Catalase, RING finger protein 22 alpha, Valosin-containing protein (VCP) and a G-protein-coupled receptor. Proteins overexpressed in PGR were protein kinase C and malate dehydrogenase. Valosin-containing protein was chosen for validation and quantification by Western blot analysis in a second case-control group of ALL patients. In this second independent cohort, median VCP expression (P25-P75) was 0.15 (0.11-0.28) in PGR and 0.34 (0.14-0.99) in PPR patients (P = 0.04). We conclude that high VCP expression is associated with poor prednisone response in childhood ALL patients.