Josef Vormoor

Leukocyte adhesion deficiency II syndrome, a generalized defect in fucose metabolism

Abstract Leukocyte adhesion deficiency II has been described in only 2 patients; herein we report extensive investigation of another patient. The physical stigmata were detected during prenatal ultrasonographic investigation. Sialyl-Lewis X (sLex) was absent from the surface of polymorphonuclear neutrophils, and cell binding to E- and P-selectin was severely impaired, causing an immunodeficiency. The elevation of peripheral neutrophil counts occurred within several days after birth. A severe hypofucosylation of glycoconjugates bearing fucose in different glycosidic links was present in all cell types investigated, demonstrating that leukocyte adhesion deficiency II is not only a disorder of leukocytes but a generalized inherited metabolic disease affecting the metabolism of fucose. (J Pediatr 1999;134:681-8)

Impact of Cranial Radiotherapy on Central Nervous System Prophylaxis in Children and Adolescents With Central Nervous System–Negative Stage III or IV Lymphoblastic Lymphoma

PURPOSE In the Non-Hodgkins Lymphoma-Berlin-Frankfurt-Munster (NHL-BFM) 95 trial, we tested, against the historical control of the combined trials NHL-BFM90 and NHL-BFM86, whether prophylactic cranial radiotherapy (PCRT) can be omitted for CNS-negative patients with stage III or IV lymphoblastic lymphoma (LBL) with sufficient early response. PATIENTS AND METHODS Apart from the removal of PCRT in NHL-BFM95, the chemotherapy of the three trials was identical except for the amount of l-asparaginase and daunorubicin during induction. The therapy in NHL-BFM95 was accepted to be noninferior when compared with trials NHL-BFM90/86 if the lower limit of the one-sided 95% CI for the difference in the 2-year probability of event-free-survival (pEFS) between target patients of NHL-BFM95 and the historical controls of NHL-BFM90/86 did not exceed -14%. The target patient group consisted of stage III and IV patients who were CNS negative and responded well to induction therapy. RESULTS The number of target patients was 156 in NHL-BFM95 (median age, 8.6 years; range, 0.2 to 19.5 years) and 163 in NHL-BFM90/86 (median age, 8.4 years; range, 0.6 to 16.6 years). For the target group, the pEFS rates at 2 and 5 years were 86% +/- 3% and 82% +/- 3%, respectively, in NHL-BFM95 (median follow-up time, 5.1 years; range, 2.1 to 9.1 years) compared with 91% +/- 2% and 88% +/- 3%, respectively in NHL-BFM90/86 (median follow-up time, 10.7 years; range, 5 to 15.4 years). The lower limit of the one-sided 95% CI for the difference in pEFS was -11% at 2 years and -13% at 5 years. In NHL-BFM95, one isolated and two combined CNS relapses occurred compared with one combined CNS relapse in NHL-BFM90/86. Five-year disease-free-survival rate was 88% +/- 3% in NHL-BFM95 compared with 91% +/- 2% in NHL-BFM90/86. CONCLUSION For CNS-negative patients with stage III or IV LBL and sufficient response to induction therapy, treatment without PCRT may be noninferior to treatment including PCRT.

Leukemic Stem Cells in Childhood High-Risk ALL/t(9;22) and t(4;11) Are Present in Primitive Lymphoid-Restricted CD34+CD19− Cells

Open questions in the pathogenesis of childhood acute lymphoblastic leukemia (ALL) are which hematopoietic cell is target of the malignant transformation and whether primitive stem cells contribute to the leukemic clone. Although good-prognosis ALL is thought to originate in a lymphoid progenitor, it is unclear if this applies to high-risk ALL. Therefore, immature CD34(+)CD19(-) bone marrow cells from 8 children with ALL/t(9;22) and 12 with ALL/t(4;11) were purified and analyzed by fluorescence in situ hybridization, reverse transcription-PCR (RT-PCR), and colony assays. Fifty-six percent (n = 8, SD 31%) and 68% (n = 12, SD 26%) of CD34(+)CD19(-) cells in ALL/t(9;22) and ALL/t(4;11), respectively, carried the translocation. In addition, 5 of 168 (3%) and 22 of 228 (10%) myeloerythroid colonies expressed BCR/ABL and MLL/AF4. RT-PCR results were confirmed by sequence analysis. Interestingly, in some patients with ALL/t(4;11), alternative splicing was seen in myeloid progenitors compared with the bulk leukemic population, suggesting that these myeloid colonies might be part of the leukemic cell clone. Fluorescence in situ hybridization analysis, however, shows that none of these myeloid colonies (0 of 41 RT-PCR-positive colonies) originated from a progenitor cell that carries the leukemia-specific translocation. Thus, leukemic, translocation-positive CD34(+)CD19(-) progenitor/stem cells that were copurified by cell sorting were able to survive in these colony assays for up to 28 days allowing amplification of the respective fusion transcripts by sensitive RT-PCR. In conclusion, we show that childhood high-risk ALL/t(9;22) and t(4;11) originate in a primitive CD34(+)CD19(-) progenitor/stem cell without a myeloerythroid developmental potential.

Haematopoetic stem cell transplantation for refractory autoimmune cytopenia.

This study describes the outcome of patients receiving haematopoietic stem cell transplantation (HSCT) to treat severe refractory autoimmune cytopenia. The registry of the European Group of Blood and Marrow Transplantation holds data on 36 patients receiving 38 transplants, the first transplant was autologous for 27 and allogeneic for nine patients. Patients had autoimmune haemolytic anaemia (autologous: 5; allogeneic: 2), Evanss syndrome (autologous: 2; allogeneic: 5); immune thrombocytopenia (autologous: 12), pure red cell aplasia (autologous: 4; allogeneic: 1), pure white cell aplasia (autologous: 1; allogeneic 1), or thrombotic thrombocytopenic purpura (autologous: 3). Patients had longstanding disease having failed multiple prior treatments. Among 26 evaluable patients mobilized for autologous HSCT, three died of treatment‐related causes, one died of disease progression, seven were non‐responders, six patients had transient responses and nine had continuous partial or complete remission. Of the seven evaluable patients receiving allogeneic HSCT, one died of treatment‐related complications, one with transient response died of progressive disease and five had a continuous response. Autologous and allogeneic HSCT may induce a response in a subset of patients with autoimmune cytopenia of long duration albeit at the price of considerable toxicity.

Human γδ T cells as mediators of chimaeric‐receptor redirected anti‐tumour immunity

Human peripheral blood γδ T cells (Vγ9+ Vδ2+) can be selectively expanded in vivo by the systemic administration of aminobisphosphonates without prior antigen priming. To assess the potential of human γδ T cells to serve as effector cells of specific anti‐tumour immunity, we expanded peripheral blood‐derived γδ T cells and transduced them with recombinant retrovirus encoding GD2‐ or CD19‐specific chimaeric receptors. Flow cytometric analysis of T cells from four individual donors cultured in the presence of zoledronate at day 14 of culture showed selective enrichment of the γδ T cell population (Vγ9+ Vδ2+ CD3+ CD4− CD8−) to 73–96% of total CD3+ T cells. Retroviral gene transfer resulted in chimaeric receptor surface expression in 73 ± 12% of the population. Transduced γδ T cells efficiently recognized antigen‐expressing tumour cell targets, as demonstrated by target‐specific upregulation of CD69 and secretion of interferon‐α. Moreover, transduced γδ T cells efficiently and specifically lysed the antigen‐expressing tumour targets. They could be efficiently expanded in vitro and maintained in culture for prolonged periods. Zoledronate‐activated human γδ T cells expressing chimaeric receptors may thus serve as potent and specific anti‐tumour effector cells. Their responsiveness to stimulation with aminobisphosphonates may enable the selective re‐expansion of adoptively transferred T cells in vivo, permitting long lasting anti‐tumour immune control.

Non-linkage of familial rhabdoid tumors to SMARCB1 implies a second locus for the rhabdoid tumor predisposition syndrome.

Rhabdoid tumors represent an independent entity among embryonal neoplasms. These tumors affect the kidney (RTK, rhabdoid tumor of kidney) and central nervous system (AT/RT, atypical teratoid, rhabdoid tumor), but may also be found in peripheral soft tissue. Unifying features include immunohistochemical characteristics and inactivation of the putative tumor suppressor gene SMARCB1 (hSNF5/INI1) in chromosome 22q11.2. Several familial cases have been published and summarized under the term rhabdoid tumor predisposition syndrome. In all of the published familial cases, inactivation of SMARCB1 was detected in tumor tissues.

Establishment of an In Vivo Model for Pediatric Ewing Tumors by Transplantation into NOD/ scid Mice

Ewing tumors are a clinically heterogeneous group of childhood sarcomas that represent a paradigm for understanding solid tumor biology, as they are the first group of sarcomas for which a chromosome translocation has been characterized at the molecular level. However, the biologic organization of the tumor, especially the processes that govern proliferation, differentiation, and metastasis of primitive tumor stem cells is poorly understood. Therefore, to develop a biologically relevant in vivo model, five different Ewing tumor cell lines and primary tumor cells from three patients were transplanted into immune-deficient mice via intravenous injection. NOD/ scid mice that carry a complex immune deficiency and thus nearly completely lack the ability to reject human cells were used as recipients. Overall, 26 of 52 mice (50%) transplanted with VH-64, WE-68, CADO-ES1, TC-71, and RM-82 cells and 4 of 10 mice (40%) transplanted with primary tumor cells engrafted. Moreover, primary cells that did not grow in vitro proliferated in mice. The pattern of metastasis was similar to that in patients with frequent metastases in lungs (62%), bone marrow (30%), and bone (23%). Using limiting dilution experiments, the frequency of the engraftment unit was estimated at 1 Ewing tumor-initiating cell in 3 × 105 VH-64 cells. These data demonstrate that we have been able to establish an in vivo model that recapitulates many aspects of growth and progression of human Ewing tumors. For the first time, this model provides the opportunity to identify and characterize primitive in vivo clonogenic solid tumor stem cells. This model will, therefore, be instrumental in studying many aspects of tumor cell biology, including organ-selective metastasis and tumor angiogenesis.

Children with myelodysplastic syndrome (MDS) and increasing mixed chimaerism after allogeneic stem cell transplantation have a poor outcome which can be improved by pre-emptive immunotherapy

We recently reported that virtually all children with acute leukaemia and myelodysplastic syndrome (MDS) who develop the phenotype of increasing mixed chimaerism (MC) after allogeneic stem cell transplantation (allo‐SCT) will relapse. We therefore performed a prospective, multi‐centre study focused on children with MDS (n = 65; advanced MDS = 44, refractory cytopenia = 21) after allo‐SCT in order to determine to what extent relapse can be prevented by pre‐emptive immunotherapy on the basis of increasing MC. Analyses of chimaerism in 44 patients with advanced MDS revealed 31 cases with complete chimaerism (CC)/low‐level MC/transient MC, 11 cases with increasing MC and two cases with decreasing MC. The same analyses in 21 MDS patients with refractory cytopenia revealed 17 cases with CC/low‐level MC, one case with increasing MC and three cases with decreasing MC. Pre‐emptive immunotherapy performed on each patient that showed increasing MC improved event‐free survival from 0%, as seen in prior studies, to 50%. We therefore conclude that pre‐emptive immunotherapy is an effective treatment option to prevent impending relapse in children with MDS after allo‐SCT.

PI3K/AKT is involved in mediating survival signals that rescue Ewing tumour cells from fibroblast growth factor 2-induced cell death.

While in vitro studies had shown that fibroblast growth factor 2 (FGF2) can induce cell death in Ewing tumours, it remained unclear how Ewing tumour cells survive in vivo within a FGF2-rich microenvironment. Serum- and integrin-mediated survival signals were, therefore, studied in adherent monolayer and anchorage-independent colony cell cultures. In a panel of Ewing tumour cell lines, either adhesion to collagen or exposure to serum alone only had a minor protective effect against FGF2. However, both combined led to complete resistance to 5 ng ml−1 FGF2 in three of four FGF2-sensitive cell lines (RD-ES, RM-82 and WE-68), and to an increased survival as compared to other culture conditions in TC-71 cells. Inhibition studies with LY294002 demonstrated that the serum signal is mediated via the phosphoinositide 3-OH kinase/AKT pathway. Thus, Ewing tumour cells escape FGF2-induced cell death by modulating FGF2 signalling. The tumour microenvironment provides the necessary survival signals by integrin-mediated adhesion and soluble serum factor(s). These survival signals warrant further investigation as a potential resistance mechanism to other apoptosis-inducing agents in vivo.

Leukemic fusion genes MLL/AF4 and AML1/MTG8 support leukemic self-renewal by controlling expression of the telomerase subunit TERT

MLL/AF4 and AML/MTG8 represent two leukemic fusion genes, which are most frequently found in infant acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), respectively. We examined the influence of MLL/AF4 and AML1/MTG8 fusion genes on the expression of TERT coding for the telomerase protein subunit, and subsequently telomerase activity in t(4;11)-positive ALL and t(8;21)-positive cell lines, respectively. MLL/AF4 suppression diminished telomerase activity and expression of TERT. Blocking pro-apoptotic caspase activation in conjunction with MLL/AF4 knockdown enhanced the inhibition of TERT gene expression, which suggests that MLL/AF4 depletion does not reduce TERT expression levels by inducing apoptosis. Knockdown of HOXA7, a direct transcriptional target of MLL/AF4 fusion gene, caused a reduction of telomerase and TERT to an extent similar to that observed with MLL/AF4 suppression. Chromatin immunoprecipitation of SEM cells, using ectopically expressed FLAG-tagged Hoxa7, indicates HOXA7 binding site in the TERT promoter region. Furthermore, suppression of the AML1/MTG8 fusion gene was associated with severely reduced clonogenicity, induction of replicative senescence, impaired TERT expression and accelerated telomere shortening. We thus present findings that show a mechanistic link between leukemic fusion proteins, essential for development and maintenance of leukemia, and telomerase, a key element of both normal and malignant self-renewal.